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CHARACTERIZATION OF TIGHT-BINDING NONNUCLEOSIDE

INHffiITORS OF HIV-l REVERSE TRANSCRIPTASE

Dimitrios Motakis

A thesis submitted to the Faculty of Graduate Studies and Research,

McGill University, in partial fulfillment of the requirement for the degree of

Doctor ofPhilosophy

Department of Medicine

Division of Experimental Medicine

McGill University, Montreal, Canada

Submitted in August 2002

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Canada ABSTRACT

5-chloro-3-(phenylsulfonyl) indole-2-carboxamide (CSIC) and clinically used efavirenz

(EFV, Sustiva®) are nonnucleoside inhibitors of HIV-l reverse transcriptase (RT) polymerization. They have unusuaHy low ICso values, which are in the range of the enzyme concentration used in the assay. Their binding constants (KjCSIC = 0.1 nM, KjEFV

= 8.5 nM) indicate that like UC781 (Ki = 2.4 nM) (Barnard et al., 1997), CSIC and EFV

(Maga et al., 2000) are also tight-binding nonnucleoside inhibitors (TBNNI) of HIV-I

4 3 RT. As expected, TBNNI dissociate from HIV-I RI slowly (6.4 x 10- - 2.2 X 10- S-I).

On the other hand, while we have confirmed that UC781 binds to RT rapidly (kon = 2.1 x

105 s-IM-I)(Bamard et al., 1997), CSIC and EFV undergo a slow conformational change

3 3 upon initial binding to HIV-l RT (k isomerisation Jorward = 3.4 X 10- S-I and 5.4 x 10- s-I, respectively), which leads to a tighter enzyme-inhibitor (E-I) complex. Once bound, aH

TBNNI are able to completely block polymerization ('dead-end' inhibition), a property that is not observed with other non-tight-binding NNRTI. 'Dead-end' inhibition may be due to the ability of IBNNI to block the conformational step preceding dNTP incorporation, unlike non-tight-binding NNRTI that slow down the ensuing chemistry step (Spence et al., 1995).

The development of resistance to UC781 is delayed when compared to other NNRTI such as UC84 and nevirapine (NVP) and several different genotypes (K103TN106A,

E138KJVI79D or V179D/YI8IC) appear in UC781-resistant HIV-l. Based on molecular modeling studies, the 'muIti-genotypic' nature of UC781-resistance may resuIt From the fact that UC781 assumes both a trans and a cis conformation, either one of which can

2 bind to mV-I RT. On the contrary, only a single pair of mutations appears with resistance to either CSIC (LlOOIIK103N) or EFV (LlOOIIK103N) consistent with the fact these two TBNNI assume only one conformation. Recombinant HIV -1 R T experiments showed that significant level of resistance (> 200-fold) to TBNNI could be attained only with combinations of at least two mutations. UC781 in specifie is able to bind to single mutant RTs (KI03T or V106A) as tightly as to the wt type enzyme. Nevertheless, the efficiency of UC781 inhibition is compromised in these enzymes since 'dead-end' inhibition is lost and a discernable level of polymerization is observed (- 3%) under maximal inhibitory conditions. In the K103TN106A double mutant RT both the tight binding and 'dead-end' inhibitory properties of UC781 are abolished thus resulting in high-Ievel resistance.

Cell culture experiments showed that EFV and CSIC completely inactivate isolated mv-

1 virus upon short exposure and subsequent removal of the drug (Motakis et al., 2002), as previously shown with UC781 (Borkow et al., 1997). Non-tight-binding NNRTI such as

NVP, UC84 and delavirdine (DL V) are completely ineffective in that respect.

Furthermore, TBNNI-pre-treated chronically infected H9 ceUs (H9+) become non infectious even after the exogenous drug has been removed (Motakis et al., 2002).

Interestingly, MT2 cells that are treated with TBNNI are refractory to subsequent infection by HIV-l, even after the complete removal of the exogenous drug. These results confirm that TBNNI are promising microbicidal candidates that may protect the genital epithelia from HlV-1 infection.

3 RESUME

5-chloro-3-(phenylsulfonyl) indole-2-carboxamide (CSIC) et efavirenz (EPV, Sustiva®), déjà utilisé en clinique, sont des inhibiteurs nonnucléosides (NNRTI) de la transcriptase inverse (TI) du VIH-1. Leur valeur ICso sont basses et sont dans la portée de la

CS1C concentration d'enzyme utilisée dans l'essai. De plus, leur constant de reluire (Ki = 0.1

EFV nM, Kj = 8.5 nM) démontre que CSIC et EPV (Maga et al., 2000) sont, tout comme

UC781 (Ki = 2.4 nM) (Barnard et al., 1997), des nonnucléosides de type 'tight-binding'

(TBNNI) du TI VIH-l. Comme prévu, TBNNI se dissocient du TI VIH-1 lentement (6.4

3 x 10-4 - 2.2 X 10- S-I). D'autre part, alors que nous avons confirmé que UC781 se lie à TI rapidement (leon = 2.1 x lOs s-IMI)(Bamard et al., 1997), CSIC et EPV subissent un

3 3 changement de conformation (k isomerisation forward = 3.4 X 10- s-1 et 5.4 x 10- s-l, respectivement) qui amène la formation d'un complexe enzyme-inhibiteur (E-I) plus solide. Une fois lié à TI, tout TBNNI peut complètement bloquer la polymerisation, soit l'inhibition 'dead-end', une propriété qui n'est pas observée avec d'autres NNRTI de type

'non-tight-binding'. L'inhibition 'dead-end' peut être due à la capacité des TBNNI de bloquer l'étape de conformation qui précède l'incorporation de dNTP contrairement aux

'non-tight-binding' NNRTI qui ralentissent l'étape de chimie (Spence et al., 1995).

Le développement de résistance à UC781 est plus lente en comparaison avec d'autres

NNRTI tels UC84 et nevirapine (NVP) , et plusieurs génotypes (K103N106A,

8K1V179D ou V179D/Y181) apparaissent dans la souche du VIH-I résistante au UC781.

Selon les modèles moléculaires, la nature 'multi-genotypic' de la résistance au UC781 peut être le résultat de la capacité de UC781 de prendre une conformation trans ou cis,

4 chaque conformation pouvant se lier à l'enzyme. Au contraire, une seule paIre de mutations apparaît avec la résistance au CSIC (LlOOI!K103N) ou EFV (LlOOI!K103N).

Ceci confirme que ces deux TBNNI supposent une seule conformation. Des expériences avec le TI recombinant ont démontré qu'un niveau de résistance significatif à TBNNI (>

200 fold) pourrait être atteint uniquement avec les combinaisons d'au moins deux mutations. UC781, spécifiquement, peut se lier solidement à TI avec une seule mutation

(K103T ou V106A) comme à TI sauvage. Néanmoins, l'efficacité de l'inhibition UC781 est compromise dans ces enzymes puisque l'inhibition 'dead-end' est perdue. Donc un niveau discernable de polymérisation est observé (~ 3%) sous des conditions de polymérisation maximales. Dans le mutant double KI03TN106A les deux propriétés de

UC781, 'tight-binding' et 'dead-end' sont abolies et un haut niveau de résistance en résulte.

Des expériences de culture cellulaire ont démontré que EFV et CSIC inactivent complètement le virus VIH-l isolé après une courte exposition et enlèvement subséquent du médicament (Motakis et al., 2002), démontré auparavant avec UC781 (Borkowet al.,

1997). Des NNRTI 'non-tight-binding' tels NVP, UC84 et delavirdine (DLV) sont complètement inefficaces à cet égard. De plus, les cellules H9 chroniquement infectées et

(H9 +) traitées avec TBNNI deviennent non contagieuses même après le retrait du médicament exogène (Motakis et al., 2002). Des cellules MT2 traitées avec TBNNI sont réfractaires à une infection subséquente avec le VIH-l, même après le retrait complet du médicament. Ces résultats démontrent que les TBNNI sont des candidats microbicides pouvant apportés une protection contre l'infection au VIH-l.

5 AKNOWLEDGEMENTS

1 want to express my gratitude and respect to my supervisor Dr. M. A. Pamiak without whose intellect, multitude of scientific resources and faith in my abilities this thesis would not have been possible. 1 would also like to express my gratitude to my colleagues and friends Dr. D. Arion and Dr. N. Sluis-Cremer whose intelligence and scientific excellence have been truly inspirational. Dr. N. Sluis-Cremer has been directly involved in my project and has provided me with valuable suggestions throughout my doctoral study. He has been a key component in carrying out aIl the molecular modeling studies involved in my thesis. He has also provided me with valuable reagents such as large quantities of purified wt heterodimeric R T for aIl my presteady-state experiments. 1 want to thank Dr. Gadi Borkow for his encouragement and positivity during our discussions and collaborations. His work set the ground for my own work in this thesis. Dr. Borkow together with Ms. Thuy Minh Nguyen were part of the development of resistance and initial identification of resistant residues in cell culture experiments. 1 am also grateful to

Ms Eva Nagi for helping me in the sequencing of several HIV-l resistant clones. Lastly, 1 want to express my gratitude to the Research Council of Canada for awarding me a doctoral feIlowship thus motivating me to excel in my research field.

6 LIST OF ABBREVIATIONS kp: pre-steady state rate constant of polymerization AIDS: acquired immunodeficiency kDa: kilodalton syndrome K.i: equilibrium dissociation AZTTP: 3'-azido-3'-deoxythymidine constant ([E][S]/[ES)) triphosphate Ki: inhibition constant bp: base pair ([E][I]/[EI]) BP: binding pocket kan: binding rate constant CA: capsid protein Km: Michaelis constant (see d4T: 2',3'-didehydro-2',3'- glossary) dideoxythymidine kaff: dissociation rate constant ddCTP: 2',3'-dideoxycytidine LTR: long terminal repeat triphosphate MA: matrix protein DDDP: DNA-dependent DNA NC: nucleocapsid protein polymerization NRTI: nucleoside HIV-l RT ddI: 2',3'-dideoxyinosine inhibitor triphosphate NNRTI: nonnucleoside HIV-l RT ddNTP: 2',3'-dideoxynucleotide inhibitor triphosphate NNIBP: NNRTI binding pocket dNTP: 2'-deoxynucleotide nt: nucleotide triphosphate PBMC: peripheral blood ds: double-stranded mononuclear cell E: enzyme PBS: primer binding site El: enzyme-inhibitor complex PCR: polymerase chain reaction ES: enzyme-substrate complex PI: protease inhibitor ECso: effective concentration for pol: polymerase gene 50% inhibition ofviral PPT: polypurine track infectivity PR: protease env(Env): envelope gene (prote in) RDDP: RNA-dependent DNA gag(Gag): gag gene (protein) polymerization gp: glycoprotein RNaseH: ribonuclease H HIV: human immunodeficiency RT: reverse transcriptase VIruS ss: single-stranded ICso: 50% inhibitory concentration DNA for HIV-1 R T polymerase TBI: tight-binding inhibitor activity TBNNI: tight-binding NNRTI IN: integrase TIP: template/primer 1: inhibitor v: velocity kapi apparent rate constant Va: initial velocity kcat: steady state rate constant of wt: wild type catalysis

7 GLOSSARY

CCso: cytotoxic concentration 50% is the concentration at which 50% of the cells die due to the toxicity of the drug.

Classical NNR TI this term refers to non-tight-binding NNR TI also known as rapid equilibrium inhibitors.

Chemistry step: the step of the polymerization mechanism (RDDP and DDDP) that follows the proper positioning of the TIP and the dNTP (dNTP conformational step) and during which the actual covalent phosphodiester bond forms (catalysis). This is the fastest step of the dNTP polyrnerization by HIV-l R T and, consequently, it cannot be measured (kChemistIy« 12s-1) (Spence et al., 1995).

Cytopathic effect: cell death due to the fusion of HIV-1 infected cells in multinuc1eated formations (syncytia, see definition below) that eventually burst thus resulting in cell death.

'Dead-end' inhibition: The abolishrnent of the polymerase activity of HIV- 1 R T polyrnerase activity upon binding of the inhibitor (completely inactive enzyme-inhibitor complex). This distinction is made with respect to non-competitive inhibitors that can theoretically bind to an allosteric site and result in a partially active enzyrne-inhibitor (E-I) complex.

ECso: effective concentration 50% is the concentration of drug at which 50% of the viral replication is inhibited in cell culture.

Efficiency: the maximal level of inhibition attainable (%) un der drug saturating conditions.

ICso: inhibitory concentration 50% is the concentration of the drug at which 50% of recombinant HIV-l RT RDDP activity is inhibited in vitro.

Michaelis constant (Km): the steady state equilibrium constant which under rapid equilibrium assumptions is defined as Km=(k2+k3)/kJ where kJ• k2• k3 are the rate constants below:

8 k2 k3 E+ S ~ ES ~ E+P k] Microbicide: a compound that is applied extemally/topically and is able to completely inactivate an invading pathogen (viral or bacterial) thus, preventing the establishment of infection.

M.O.I.: multiplicity of infection refers to the ratio of the infectious dose used (TCID50) over the number of cells infected.

Nonnucleoside inhibitor: a non-competItIve inhibitor of HIV-l polymerization that binds to an allosteric site of the enzyme.

Non-'tight-binding' inhibitor: a compound that shows classical Michaelis-Menten kinetics of inhibition with an equilibrium constant (Ki) that is significantly higher than the enzyme concentration in the assay.

Nucleoside analogue: a competitive inhibitor of the polymerase activity of HIV-1 R T that mimi cs the natural nucleotide substrates.

Potency: the term used to de scribe a drug with high ICso, ECso or both.

Syncytia: the large multinucleate formations that appear with infection of lymphocytes with HIV -1 due to the presence of both CD4 receptors and the surface viral antigens on the surface of these cells.

Tight-binding inhibitor: a drug (1) that binds to saturation, and thus completely inhibits, an enzyme (E) when the latter is present at a near equal concentration to the former (E/I ~ 1).

TCID50: tissue culture infectious dose 50% refers to the dilution of the viral stock, which results in infection of only half the cultures inoculated. In this thesis the amount of virus used is given in terms of TCIDso.

TI: Therapeutic index is the ratio of the ECso over the CCso of a specifie drug.

9 Virucide: a compound that is applied topically and is able to completely inactivate an invading virus thus, preventing the establishment of infection.

10 TABLE OF CONTENTS ABSTRA CT...... 2 RESUME ...... 4 LIST OF ABBREVIA TIONS ...... 7 GLOSSARY ...... 8 CHAPTER 1: LITERATURE REVIEW ...... 14 PREFACE ...... 15 1.1. RETROVIRUSES ...... 16 1.2. HIV-l LIFE CYCLE ...... 17 1.3. HIV-l REVERSE TRANSCRIPTASE ...... 18 1.4.1. NUCLEOSIDE INHIBITORS OF RT ...... 21 1.4.2. NONNUCLEOSIDE INHIBITORS OF HIV-l RT (NNRTI) ...... 24 1.4.3. IMPORTANT NNRTI ...... 26 1.5. MECHANISMS OF NNRTI INHIBITION ...... 36 1.6. MECHANISM OF NNRTI RESISTANCE ...... 41 1.7. TIGHT-BINDING INHIBITION ...... 43 1.8. MICROBICIDES ...... 47 1.8.1. THEMECHANISMOF HIV-l SEXUALTRANSMISSION ...... 48 1.8.2. CLASSIFICATION OF MICROBICIDES ...... 52 1.9. RATIONALE FOR THE PRESENT STUDIES ...... 60 CHAPTER 2: MECHANISM OF INHIBITION OF HIV-l REVERSE TRANSCRIPTASE BY TIGHT-BINDING NONNUCLEOSIDE INHffiITORS .... 61 2.1. INTRODUCTION ...... 62 2.2. MATERIAL AND METHODS ...... 64 2.3. RESULTS ...... 68 2.4. DISCUSSION ...... 79 CHAPTER 3: DEVELOPMENT OF RESISTANCE TO TIGHT-BINDING NNRTI ...... 86 3.1. INTRODUCTION ...... 87 3.2. MATERIALS AND METHODS ...... 88 3.3. RESULTS ...... 92 3.4. DISCUSSION ...... 103 CHAPTER 4: MECHANISM OF HIV-l RESISTANCE TO TIGHT-BINDING NNRTI: STUDIES WITH UC781 ...... 110 4.1. INTRODUCTION ...... 111 4.2. MATERIALS AND METHODS ...... 112 4.3. RESULTS ...... 116 4.4. DISCUSSION ...... 123 CHAPTER 5: MICROBICIDAL (VIRUCIDAL) ACTMTY OF TIGHT- BINDING NNRTI ...... 127 5.2. MATERIAL AND METHODS ...... 130 5.3. RESULTS ...... 133 5.4. DISCUSSION ...... 142 CHAPTER 6: GENERAL DISCUSSION ...... 146 CONTRIBUTIONS TO ORIGINAL KNOWLEDGE ...... 153 REFERENCES ...... 157

11 TABLE OF FIGURES AND TABLES Figure 1.1. The structure of mv-1 reverse transcriptase ...... 22 Figure 1.2. Structures ofvarious NNRTI ...... 37 Table 1.1. Pharmacologie characteristics of representative NNRTI ...... 38 Table 1.2. Representative HIV-l microbicides ...... 59 Table 2.1. Inhibition ofHIV-l RT by selected NNRTI ...... 75

Figure 2.1. Dependence of Kapp on the concentration of EFV (A), CSIC (B) or UC781 (C) ...... 76 Table 2.2. Pre-steady state kinetic parameters calculated for mV-l RT in the absence and presence of various NNRTI ...... 77 Table 2.3. Rate of incorporation of dCTP and dCTP(aS) by NNRTI-inhibited RT-TIP complexes ...... 78 Figure 3.1. Development ofresistance to UC84, UC38 and UC781 ...... 97 Table 3.2. Cross-resistance profiles of UC781, CSIC and EFV with UC781 and CSIC- resistant viruses ...... 98 Figure 3.3. Interactions of CÎs-UC781 with the residues of the NNRTI binding pocket..99 Figure 3.4. Interactions of trans-UC781 with the residues of the NNRTI binding pocket ...... 100 Figure 3.5. Interactions ofCSIC with the residues of the NNRTI binding pocket ...... 101 Figure 3.6. Interactions ofEFV with the residues ofthe NNRTI binding pocket...... 102 Table 4.1. Rate constants for binding and dissociation ofUC781 with wt and mutant RT determined from kinetic analysis of 'tight-binding' inhibition ...... 120 Table 4.2. Dissociation constants for binding of UC781 to different RT mechanistic forms determined from steady state fluorescence measurements ...... 121 Table 4.3. RT RDDP activity of wt and mutant RT in the presence of saturating concentrations ofUC781 as compared to UC38 ...... 122 Table 5.1. Summary of sorne properties of the NNRTI used in this study ...... 136 Figure 5.1. Inactivation of isolated mV-l virus particles following exposure to different concentrations ofNNRTI ...... 137 Figure 5.2. Inactivation of HIV-1 produced by TBNNI-pre-treated chronically infected H9 ceUs ...... 138 Figure 5.3. Effect ofNNRTI treatment ofH9+ celIs on ceIl-to-celI transmission ofHIV-1 in the absence of extracellular drug ...... 139 Figure 5.4. Effect of "transient" exposure of uninfected MT2 celIs to various concentrations of NNRTI on subsequent viral infection ...... 140

12 Figure 5.5. Effect of viral load on infection of MT2 cells pre-treated with various concentrations oftight-binding NNRTI...... 141

13 CHAPTER 1: LITERA TURE REVIEW

14 PREFACE The basis for this thesis was the novel finding that UC781 is a 'tight-binding' nonnucleoside inhibitor (TBNNI) of HIV-l reverse transcriptase (RT) (Barnard et al.,

1997). UC781 has several pharmacological properties such as exceptionaI in vitro potency, delayed development of resistance and microbicidal activity that distinguish it from other typical NNRTI (Borkow et al., 1997). Therefore, UC781 has potentiaI uses as both a systemic antiretroviral and as a preventative regimen. Recently, the identification oftwo additional 'tight-binding' NNRTI, CSIC and efavirenz (EFV) (Maga et al., 2000), has enabled us to test the hypothesis that these distinguishing properties of UC781 are most likely the result of its high affinity to HIV-1 R T.

The identification and characterization of other 'tight-binding' inhibitors ofHIV-l RT is important for two main reasons. First, NNRTI are notorious for their rapid development of HIV-l resistance (De Clercq, 1999). Compounds which are able to inhibit HIV-l repli cation at the Iowest nanomolar or even picomolar range may aIso prove to be effective against viral strains that carry multiple mutations and which are resistant to most other NNRTI. Second, UC781-like NNRTI may be able to effectively prevent, rather than just contain, HIV-l infection. There is a great need for microbicides that would block the pre-integrational steps of the HIV-l life cycle (see section 1.2) and which would thus, prevent the transmission ofHIV-l to healthy individuals.

In order to determine the selection criteria for 'tight-binding' NNRTI seve rai questions have to be addressed. For instance, are there other NNRTI, which are able to block polymerization in the same manner as UC781? Does the mechanism of NNR Tf binding

15 (tight-binding versus non-tight-binding) and inhibition correlate with a delay in the development of resistance? Is the identification of 'tight-binding' inhibitors the key to the development of effective microbicides? Overall, this thesis aims to identify the distinguishing parameters that set TBNNI apart from other NNRTI as improved systemic therapies and/or as novel microbicides against HIV-l.

The following reVlew will serve as an introduction to fields that are immediately pertinent to the interpretation of our findings. This includes a discussion of HIV-I RT,

NNRTI and 'tight-binding' inhibition. An overview of the mechanism of HIV-l transmission and the microbicides, which have recently been developed, is also presented.

1.1. RETROVIRUSES Retroviruses differ from other RNA viruses in that they replicate through a double stranded (ds) DNA intermediate that becomes integrated into the infected cell's genome.

The conversion ofretroviral (+) single stranded (ss) RNA into ds DNA is carried out by the viral enzyme reverse transcriptase (RT) (Baltimore, 1970; Temin and Mizutani,

1970). Once the proviral DNA has been integrated into the host genomic DNA, viral

RNA is regenerated using the cell's mRNA processing machinery. Retroviruses are divided into three main groups: oncoviruses, lentiviruses and spumaviruses (Coffin,

1997). Lentiviruses include the human immunodeficiency virus type 1 (HIV -1) which is the causative agent of acquired immunodeficiency syndrome or AIDS. HIV -1 is a complex retrovirus because, apart from its basic gag, pol and env genes found in other retroviruses, it also contains auxiliary genes that code for accessory proteins.

16 1.2. HIV-1 LIFE CYCLE Pre-integrational events. The first events of cellular infection by HIV -1 involve the binding and subsequent fusion of the membrane envelope with the membrane of the host cells (Coffin, 1997). Initial binding of HIV -1 requires the presence of specifie CD4 receptors found mainly in lymphocytes and macrophages. Binding of the virus to the cell is mediated via the surface glycoprotein gp120 (SU) that binds to the CD4 receptor. The transmembrane glycoprotein gp41 (TM) is involved in the subsequent fusion of the viral and cellular membranes. However, a coreceptor, CXCR4 or CCR5, is required in addition to CD4 for viral entry (Alkhatib et al., 1996; Choe et al., 1996; Feng et al., 1996). CCR5 is present in macrophages, which are the cells that are infected during the early stages of

HIV-l infection (M-tropic virus). CXCR4 is involved in T-cell infection mainly during the later stages of the disease (T-tropic virus). Upon entry, reverse transcription results in the conversion of the viral ss RNA to double stranded (ds) DNA (Coffin, 1997). This blunt-end linear DNA, complexed with several viral and host proteins including HIV-l integrase (IN), is transported into the nucleus where it is integrated in the host DNA.

Post-integrational events. The integrated proviral DNA is transcribed by the cellular

RNA polymerase II (Coffin, 1997). This process is dependent on the coordinated interaction of cellular transcription factors, cis-acting viral nucleic acids as weIl as viral proteins. Upon integration of the provirus the cellular transcription machinery enables the synthesis of a series of viral mRNA that play a role in the synthesis of transcription regulators, structural proteins and enzymes. One of the products of full length RNA

8a8 translation is the viral polyprotein Gag (gp55 ). Processing of Gag by HIV -1 protease

(PR) results in the matrix (MA; p 17), capsid (CA; p24) and nucleocapsid (NC; p7)

17 proteins. The pol gene is expressed as part of a larger Gag-Pol polyprotein. Cleavage of

Gag-Pol results in the production of the Gag proteins (described above) as weIl as protease (PR), reverse transcriptase (RT) and integrase (IN). Env RNA codes for the synthesis of the Env prote in, which becomes proteolytically cleaved by a cellular protease to give the surface viral proteins ofHIV, that is, the transmembrane (TM; gp41) and surface (SU; gp120) proteins.

During HIV-l viral assembly aIl viral protein precursors (Gag and Gag-Pol) move to the membrane of the ho st cell (Coffin, 1997). Two full-Iength RNA molecules are selectively packaged into the newly synthesized viral particles and serve as their genome. Once aIl polyproteins and RNA are bound to the internaI cell surface, they bud out forming an immature particle. Eventually, processing of Gag and Gag-Pol by HIV -1 PR gives rise to mature, infectious virus.

1.3. HIV-l REVERSE TRANSCRIPTASE HIV -1 R T is able to carry out three independent, but equally essential activities (Coffin,

1997). These are (1) the RNA-dependent DNA polymerase activity (RDDP) that uses

RNA as a template to synthesize DNA, (2) the RNase H activity that degrades the RNA component of the RNADNA duplex resulting in a (-) ss DNA and (3) the ONA- dependent ONA polymerase activity (DODP) that uses the (-) ss DNA as a template to synthesize the complementary (+) DNA strand (Coffin, 1997). This thesis deals with the polymerase activities of HIV -1 R T only. The structure of the heterodimeric HIV -1 R T based on a published crystal structure is shown in Figure 1.1 (Ren et al., 1998).

18 HIV-l RT is a heterodimer consisting of66 kDa (p66) and 51 kDa (pSI) subunits. Since both subunits are produced from the same precursor (c1eavage of p66 produces p51), the conformation of the individual subdomains in the two subunits is the same. However, the overall spatial organization of subdomains in the p66 and pSI differs substantially

(Kohlstaedt et al., 1992b; Jacobo-Molina et al., 1993). In pSI, the polymerase site is buried under the connection subdomain and is not exposed, (Jacobo-Molina et al., 1993;

Huang and Jeang, 1993; Hsiou et al., 1996; Ding et al., 1998) while the RNase H domain is absent (Tisdale et al., 1988; Schatz et al., 1989). The active polymerase (RDDP,

DDDP) and the nuc1ease catalytic sites of HIV-1 RT are found in the p66 subunit

(Prasad, 1993; Le Grice, 1993).

The polyrnerase domain resembles the shape of the human right hand (Kohlstaedt et al.,

1992b; Jacobo-Molina et al., 1993). The catalytic residues, Asp-185, Asp-186 and Asp-

110, are found on the palm (Larder et al., 1987; Boyer et al., 1992a; Hostomsky et al.,

1992a). The first two residues are part of a highly conserved YXDD motif found in aU

RTs (Scalka and Goff, 1993). The thumb together with the palm and the fingers result in what is known as the template/primer (T IP) grip that, together with other interactions along the polymerase-RNase groove, hold the TIP in place. The incoming nuc1eotide anneals to the complementary base of the TIP, adjacent to the 3' OH of the primer.

Further, the fingers, together with two Mg+2 ions and the catalytic aspartates Dll0 and

D 185, interact with and coordinate the phosphate moiety of the dNTP substrate (Huang et al., 1998). In this catalytically poised temary complex, RT appears to be in a 'closed' confonnation where the fingers are bent inward toward the palm. In the structure of free

19 RT a much wider binding cleft is observed ('open' conformation). In the 'closed' conformation the 3' OH of the primer is in place to attack the a-phosphate of the incoming dNTP by a 'two-metal ion mechanism' as seen with other polymerases (Huang et al., 1998).

RT-catalyzed DNA synthesis follows an ordered 'bi-bi' mechanism (Kati et al., 1992;

Hsieh et al., 1993). Reverse transcriptase binds template primer tirst to form a binary complex (RT-TIP). Upon template-primer binding RT undergoes a conformational change (Kati et al., 1992). In the crystal structure this change is seen as a rotation of the thumb that 'grabs' the TIP (Jacobo-Molina et al., 1993; Ding et al., 1998). Subsequently, the dNTP binds to RT to form a temary complex (RT-TIP-dNTP). In its RT-TIP-dNTP form the enzyme undergoes another conformational change (dNTP conformational step) resulting in a second temary complex (RT*-TIP-dNTP) (Rittinger et al., 1995). In a recent crystal of the temary complex the latter structural change is observed as the clamping of the fingers that bring the dNTP closer to the area of the catalytic site (Huang et al., 1998). A phosphodiester bond is formed while pyrophosphate is released.

Subsequently, the extended TIP can either dissociate from the enzyme (distributive pathway) or it can remain bound and undergo a translocation event to allow incorporation of the next complementary dNTP (processive pathway).

In the overall reaction mechanism, dissociation of the template-primer constitutes the rate-limiting step of this process (kcat (steady state) ~ 0.012-0.26 S-I) (Reardon et al.,

1992; Kati et al., 1992; Spence et al., 1995). While the TIP remains bound, two rapid

20 steps take place: (1) a conformational change (dNTP conformational step) and (2) catalysis, which is the actual bond formation. However, even though the conformational step that occurs upon binding of the dNTP is rapid, it is still much slower (kp (pre-steady state) - 12-74 S-I) than the following event ofphosphodiester bond formation (chemistry or catalytic step). The actual nucleophilic attack (chemistry step) that follows this conformational step is very rapid and cannot be measured directly (Spence et al., 1995).

In order to measure the dNTP conformational step rapid kinetics (time scale: 2ms - Isec) are necessary using a pre-steady state instrument. Biochemical assays which measure product production in minutes to ho urs are a measure of the steady state rate of polymerization, which is determined by the slowest step of the overall reaction (TIP dissociation).

l.4.INHffiITORS OF THE POLYMERSE ACTIVITY OF HIV-l RT

1.4.1. NUCLEOSIDE INHIBITORS OF RT

The first types of HIV-1 antivirals characterized were nucleoside inhibitors of HIV -1 RT polymerase such as AZT (zidovudine). Presently, there are five NRTI used clinically apart from zidovudine: didanosine, zalcitabine, stavudine, lamivudine and abacavir (De

Clercq, 2000). NRTI are modified analogues of deoxyribonucleotides which lack or have a modified 3'OH group. One important aspect ofNRTI inhibition is that they have to be phosphorylated in order to be active (Furman et al., 1986; Dahlberg et al., 1987). This modification is carried out by cellular kinases. Since NRTI are analogues of natural dNTP substrates they compete for the dNTP binding site. However, once bound, the

21 Figure 1.1. The structure ofHIV-1 reverse transcriptase

Polymerase domain

p66

p51

HIV-1 R T is a heterodimeric enzyme that consists of the p66 and the p51 subunits. In the p66 subunit there is one polymerase catalytic site and one RNase H catalytic site (C). NNRTI bind to a site that is distinct but proximal to the polymerase active site, the nonnucleoside HIV-1 RT inhibitor binding pocket (NNIBP). Aiso shown are the 'fingers', 'pahn', 'thumb' and 'connection' subdomains (modified from the crystal structure by Ren et al., 1998).

22 absence or the modification of the 3' OH in the nucleoside analogue, prevents any further incorporation resulting in 'chain termination'.

However, one of the major problems with NRTIs is the development ofresistant forms of

RT, where in most cases, the affinity ofNRTI for the mutant enzyme is much lower than its affinity for wt R T. There are a number of single and multiple mutations that appear in

RT in correlation with resistance to NRTI, aIl of which are found in the polymerase domain of the enzyme. Even though they appear to be scattered, they can be grouped as mutations of the fingers (M41L, 150T, A62V, K65R, D67N, T69D, K70RlE, L74V, V751 and F77L) and as mutations of the palm (Y115F, A116F, Q151M, M184V, L21OW,

T215YIF and K219Q) subdomains (Tantillo et al., 1994). There are two mechanisms of resistance to NRTI. Resistance to most NRTI is due to decreased binding affinity and thus decreased 'chain termination' (Mechanism 1). With the exception of abacavir

(ABC), single mutations are able to confer at least a moderate-Ievel ofresistance to NRTI

(Tisdale et al., 1997). AZT has a distinct mechanism of resistance that involves an increase in pyrophosphorolysis (Mechanism 2), the reverse reaction of polymerization, that results in the removal of already incorporated chain terminators (Arion et al., 1998;

Meyer et al., 1998; Meyer et al., 1999; Arion et al., 2000). Since resistance to NRTI monotherapy occurs rapidly, combination therapies of NRTI with protease inhibitors and/or NNRTI are currently being used for the treatment ofHIV-l infection (Ghani et al.,

2002; Nunez et al., 2002; King et al., 2002; Mocroft et al., 2002; Manfredi et al., 2002;

Plana et al., 2002; Engelhom et al., 2002; Barreiro et al., 2002).

23 1.4.2. NONNUCLEOSIDE INHIBITORS OF HIV-l RT (NNRTI)

Nonnucleosides are noncompetitive inhibitors of the polymerase activity of HIV-l RT.

The antivirals that are examined in this thesis belong to this broad group of compounds.

Presently, there are three NNRTI used clinically (NVP or Viramune®, DLV or

Rescriptor® and EFV or Sustiva®) while several others are in the preclinical stages (De

Clercq, 2001).

One of the first NNRTI to be discovered was HEPT, a nucleotide analogue which inhibits polymerization in a noncompetitive manner (Wu et al., 1991; Dueweke et al., 1992).

Subsequently, screenings for compounds with anti-I-llV-l activity resulted in several

NNRTI leads. Since the discovery of NNRTI, kinetic and crystallographic studies have confirmed that their mechanism of inhibition is allosteric. The residues involved in

NNRTI binding were deduced, before a crystal structure was available, by mutational analysis and cross-linking experiments (Jacobo-Molina et al., 1993; Tantillo et al., 1994).

The crystal structure of HIV -1 R T complexed with NVP subsequently confirmed that the nonnucleoside inhibitor binding pocket (NNIBP) is a largely hydrophobie domain that is distinct from the polymerase catalytic site, but is buried under it (Kohlstaedt et al.,

1992b).

Even though aIl NNRTI bind to the same pocket in HIV-l RT, they compnse a structurally diverse group of compounds. Nevertheless, NNRTI are similar in that they aIl contain several aromatic and lipophilic moieties. Unfortunately, the rapid development of resistance to most NNRTI has been somewhat disappointing (De Clercq, 1999). For

24 instance, substitution ofresidue Y181 (to a cysteine), which has been shown to have very strong interactions with several NNRTI, is the most common substitution seen with NVP both in vitro and in patients and results in high-Ievel resistance (Richman et al., 1994).

Resistance to NVP monotherapy in patients develops as early as one week after initiation of therapy. Nevertheless, continuous interest in the area of NNRTI has led to sorne promising compounds. Of particular interest are highly potent NNR TI with microbicidal properties such as UC781, EFV and CSIC (Table 1.1) which are discussed in the following chapters.

Moreover, several recent studies have also shown that NNRTI may have an even more promising role than initially thought. Such studies have shown that HIV-l isolates from patients who are resistant to NRTI are actually 'hypersusceptible' to NNRTI treatment.

Shulman et al. (2001) demonstrated that isolates from NR TI resistant patients were

'hypersusceptible' (2-fold increase in IC5o) to EFV, DEL and NEV in 50%, 45% and

36% of the cases, respectively. With EFV this enhancement in susceptibility was associated with an improved outcome after 24 weeks of therapy. In a second study

(Whitcomb et al., 2002), in which 17,000 plasma samples were screened against EFV,

DEL and NEV, 'hypersusceptibility' to these compounds was found in 10.7, 10.8 and 8% ofthese NRTI-resistant samples, respectively. An inverse correlation between the number of NRTI mutations and the level of 'supersusceptibility' was also reported in the same study. Lastly, in a third report by Haubrich et al. (2002), where 'hypersusceptibility' was defined as a >60% improvement in IC 5o, NNRTI such as EFV showed increased potency in 29% of the cases. OveraIl, these results indicate that NNRTI may be able to play a

25 strong role as salvage or second line therapy. In other words, the proper timing ofNNRTI use in the treatment of HIV-1 patients may significantly contribute in prolonged antiviral efficacy and reduced drug resistance in the future.

1.4.3. IMPORTANT NNRTI

Over thirty NNRTI groups exist inc1uding calanolide A, imidazodipyrido diazepinones, imidazopyridazines and DABO derivatives, to mention a few (De Clercq, 1999). The

NNRTI discussed below constitute the most widely studied categories. They inc1ude derivatives of HEPT, TIEO, dipyridodiazepinones, BHAP, pyridinones, TSAO, APA,

PETT and quinoxalinethiones. Furthermore, the arylsulfonylindoles, quinazolinones thiocarboxanilides, which are the three types of NNRTI studied in this thesis, are discussed in detail at the end of this section.

HEPT The discovery of HEPT as an NNRTI (Miyasaka et al., 1989; Debyser et al., 1992a;

Debyser et al., 1992b) presented an alternative way of effectively inhibiting HIV-l RT polymerization (ECSOHEPT ~ hl.M), compared to the NRTI c1ass ofinhibitors (Miyasaka et al., 1989). Unfortunately, resistance to HEPT developed readily in cell culture and the

Y188C mutation was found in HEPT-resistant HIV-l (Balzarini et al., 1993a). Similarly, resistance to two newer and more potent HEPT derivatives, E-EPU and E-EPU-dM with

ICsos of 20 nM and 2 nM, respectively (Tanaka et al., 1992), developed through mutations Y188H, Y181C and V106A (Baba et al., 1992). In contrast, the HEPT derivative 6-benzyl-l-ethanoxymethyl-5-isopropyluracil known as MKC-442 or emivirine (EMY) (Table 1.1) has been shown to be very potent against laboratory-

26 adapted HIV-1 (ECso ~ 1.6-19 nM) and clinical isolates (Baba, M. et al 1994, Brennan, T.

M., 1995, Seki, M., 1995), while, to the best of our knowledge, there are no reports of viral resistance to this compound. Emivirine has low toxicity to the human bone marrow

(CCso ~ 30-50 /J.M) and mitochondria while animal studies show no genotoxic, reproductive or other adverse effects (Szczech, GM., 2000). Furthermore, EMY is better absorbed orally in rats (68%) than previous HEPT compounds such as E-EPUdM « 1% in mice) (Sato et al., 1995). As a consequence of its favourable pharmacokinetic profile,

EMV is currently under clinical development (De Clercq, E., 2001).

TIBO Concurrently with the discovery of HEPT, another group of compounds containing the tetrahydroimidazo [4,5,I-j,k]-benzodiazepin-2(lH)one or (TIBO) moiety was found to inhibit HIV-l in cell culture (Pauwels et al., 1990; Kukla et al., 1991a). Further modifications led to two isomers of CI-TIBO (R82913 and R86183) with IC so values at the lowest nM range (30 and 4.3, respectively) (Kukla et al., 1991b). However, the development of 9-CI TIBO (R82913) was terminated after the first clinical trial (phase 1) due to its poor oral bioavailability (plasma levels were below effective concentration) and the fact that multiple resistance mutations arose in cell culture (LlOOI, K103N, E138K,

Y181C and Y188H) (Balzarini et al., 1993b). Despite the development of resistance mutations (Table 1.1), 8-CI TIBO (R86183), known as tivirapine, has been under clinical development (Ho et al., 1995a, De Clercq, 1999).

27 DIPYRIDODIAZEPINONES NVP (Table 1.1), one of the first NNR TI available, belongs to a group of compounds known as dipyridodiazepinenones (Hargrave et al., 1991). NVP is one of the most studied

NNRTI in terms of biochemical mechanism of inhibition and resistance (Merluzzi et al.,

1990; Richman et al., 1991a; Richman et al., 1991b; Grob et al., 1992; Mellors et al.,

1992; Balzarini et al., 1993a; Richman et al., 1994; Larder, 1994; Spence et al., 1996;

Balzarini et al., 1996a). It is quite effective in vitro (IC5o ~ 175 nM, EC95 ~ 400 nM) has good bioavailability (~ 92%) (Lamson et al., 1999) and was the first NNRTI to be approved for clinical use. Nevertheless, resistance to NVP alone develops rapidly both in cell culture and in vivo (1-2 weeks) (Richman et al., 1994; Havlir et al., 1996). Currently, numerous combinations of NVP with protease inhibitors (PI) and/or NRTI are used clinically with success (Tashima et al., 2000). Data on the combination ofNVP with two other NR TI suggest that this regimen is as effective as triple therapy with the PI indinavir

(Moyle, 2001). This is clinically important in light of the fact that PI cause metabolic disturbances, while NVP is weIl tolerated. Because of the leading role of NVP in the clinical development of NNRTIs, we have used this drug as a control NNR TI in many of the experiments described here.

BHAP Several modifications of bis heteroaryl piperazines (BHAP) yielded two clinical candidates: atevirdine and DLV (Romero et al., 1994). DL V (Table 1.1) was effective against numerous primary HIV-l isolates (EC50 > 9 nM) and had good bioavailability in vivo (Dueweke et al., 1993a; Romero et al., 1994; Darey et al., 1996). It also proved mildly effective against the NNRTI resistant mutant Y181C and was approved as the

28 second NNRTI to be used c1inically. Although neither the Y181C nor the K103N mutations appear with resistance to DL V, a novel P236L mutation is present (Romero et al., 1994). However, this mutant virus is still sensitive to other NNRTI. In patients, resistance to DL V develops within 12 weeks, even when zidovudine is co-administered

(Joly et al., 2000). For this reason, DL V is administered in combinations with at Ieast two other HIV -1 antivirals. In vitro, atevirdine was shown to inhibit a series of clinical isolates at concentrations that varied from 60 nM to 1.6/.!M (Campbell et al., 1993).

However, the compound failed to show significant antiviral activity in phase llIl clinical trials (Demeter et al., 1998) and its pharmacokinetic assessment showed variable levels of dose availability between patients (Morse et al., 2000).

PYRIDINONES Pyridinone derivatives were found to inhibit HIV-l in screenmgs by Merck

Pharmaceuticals (Saari et al., 1991). After several modifications a benzoxazole substitution gave NNRTIs L-697,639 and L-697,661 with increased stability and potency

(IC so ~ 20-800 nM, EC9S ~ 12-200 nM) (Coldman et al. 1991). High-level resistance

(lOOO-fold) to these NNRTI is conferred by substitutions K103N and Y181C (Nunberg et al., 1991; Sardana et al., 1992). Consequently, in phase 1 clinical trials patients became resistant to L-697,661 rapidly (Schooley et al., 1996), but combinations of L-697,661 with AZT showed no development of resistance or increase in viremia during the six- month period of the study (Perrin et al., 1996). Recently, the trifluromethyl and benzyl analogues of pyridinones have shown remarkable inhibition in vitro (IC90 > 32 nM and

IC50 ~ 0.2-6 nM, respectively) (DolIe et al., 2000; Corbett et al., 2001). It is noteworthy that one of the benzyl analogues inhibits NVP resistant virus (ICso ~ 40 nM).

29 TSAO Similarly to HEPT, the thymidine derivative TSAO-T (ICso ~ 59 nM, Figure 1.7.) was initiaHy synthesized as a nucleoside analogue and was later found to be an NNRTI

(Balzarini et al., 1992). The appearance of the E138K mutant further supported the premise that TSAO-T binds to the NNIBP on RT. Apart from this pyrimidine analogue, purine analogues such as TSAO-m3T have been synthesized with similar inhibitory potency in ceU culture (ECso ~ 34 nM) (Balzarini et al., 1993c). Further modification of

TSAO has not resulted in significant improvements in antiviral potency. Dimers [ddN]-

[TSAO-T] and of [TSAO-T]-chelating moiety have similar inhibitory values to TSAO-T but are less toxic (Chamorro et al. 1998; Velazquez et al., 1999). Recent data indicate that the E138A resistant mutation is a normal variant ofwild-type RT in HIV-l patients and may thus become dominant upon treatment with the TSAO compounds (Van Laethem et al., 2000; Pelemans et al., 2001).

APA Thea-AFA or a-anilinophenylacetamide derivative loviride or R89439 (ECso ~ 13 nM)

(Pauwels et al., 1993) has been used in several clinical trials, mostly in combination with

NRTI (Quattro Steering Committee, 1999; CateH et al., 1999). Monotherapy with loviride has resulted in the appearance of KI 03N and Y 181 C HIV-l, which is cross-resistant to other clinically used NNRTI such as NVP, EFV and DLV (Miller et al., 1998).

PETT Lilly Research Laboratories have developed a class of NNRTI based on phenylethylthiazolylthiourea (PETT). Compound L Y300046.HCI or trovirdine was significantly more potent (ICso ~ 7-15 nM, ECso ~ 20 nM) than the lead compound

30 LY73497 (ICso ~ 900 nM) (Bell et al., 1995; Zhang et al., 1995). However, trovirdine was 147-fold less potent against mutant Y181C in vitro. More recently, structural modifications of PETT have led to compounds HI-236, HI-346, HI-445 and HI-51 1 with

ECso in the range 3-5 nM (Mao et al., 1999; Uckun et al., 1999; Uckun et al., 2000;

Venkatachalam et al., 2000). AIl the compounds above inhibit the Y181C and multidrug resistant (MOR) strains of HIV-1 at much lower concentrations (50 to 1O,000-fold) than

NVP, DL V and trovirdine. In particular, HI-511 was able to inhibit the highly resistant

HIV-l strain carrying the mutations Y181C and K103N (ECso ~ 2.7J.lM) unlike trovirdine, DL V and NVP (ECso > 100J.lM).

QUINOXALINETHIONES Interestingly, the potent quinoxalinethione S-2720 (ECso ~ 14 nM, ICso - 39 nM, Table

1.1) develops resistance via the G 190E mutant, which was shown to 'cripple' HIV -1 by slowing down viral growth (Kleim et al., 1993; Balzarini et al., 1994). However, mutants

VI06A and P225H also appeared when lower concentrations of S-2720 were used, mutants which decreased sensitivity to aIl NNRTI, but not to DL V (Pelemans et al.,

1997; Pelemans et al., 1998). The quinoxaline HBY 097 inhibited numerous viral strains at very low effective concentrations (ECso ~ 2-7 nM) while its cytotoxicity was comparable to that of S-2720 (CCso > 200J.lM) (Kleim et al., 1995). The bioavailability of this compound was 48-54% in animal models, an improvement compared to the bioavailability of S-2720, which was only 10%. Although mutations G 190E (high concentration) and V106I1L+G190T appeared in cell culture with HBY 097 (Kleim et al.,

1997), in phase II clinical trials the KI03N was observed instead within 12 weeks (Kleim et al., 1999). At higher levels of resistance (24 wt;eks) the P225H mutation appeared in

31 addition to K103N. A new generation quinoxaline GW420867X has recently been tested in combination with other NNRTI and/or NRTI (Balzarini et al., 2000).

ARYLSULFONYLINDOLES Screening for NNRTI at Merck Research Laboratories led to the arylsulfonylindole

NNRTI 2-[(phenylsulfonyl) methyl]-3-phenylthioindole with an in vitro IC50 of approximately 63 nM and EC95 of 400 nM (Williams et al., 1993). Further modifications resulted in an even more effective compound, 5-chloro-3-(phenylsulfonyl) indole-2- carboxamide (L-737, 126) or CSIC (IC50 ~ 3 nM, EC95 ~ 3 nM) (Table 1.1). At about the same time, the metabolism of another arysulfonylindole L-734,005 was examined in

Rhesus monkeys. L-737,126 (CSIC), one of its metabolites in plasma, which was found to be more bioactive than its precursor (IC95 ~ 3 nM) (Balani et al., 1993). CSIC is an effective inhibitor of the Y181C and K103N RT mutants (IC50: 71 nM and 116 nM, respectively), which are normally resistant to most other NNRTI (Williams et al., 1993).

The availability of CSIC in plasma is modest (45%) in Rhesus monkeys attaining a peak level of 2 J..lM when dissolved in methocel. However, solid formulations of CSIC such as piUs have been reported to have lower oral bioavailability, presumably due to the high melting point of CSIC (~ 253°C) and its low solubility (Tucker et al., 1996).

Unfortunately, the effort to overcome these therapeutic limitations by further altering the structure of arylsufonyl indoles has so far proven unsuccessful (Silvestry et al., 1997;

Silvestry et al., 1998). Nevertheless, our work with CSIC indicates that this NNRTI may serve as an exceptional microbicide for the prevention of HIV-1 infection. With respect to this application its low bioavailability may actually be a favourable characteristic, allowing the compound to act only topically and resulting in lower systemic toxicity.

32 QUINAZOLINONES Even though several quinazolinones were found to be effective inhibitors ofHIV-l (ICsos

~ 6-56 nM, ECso ~ 25-110 nM), they were either chemically unstable or easily metabolized to an inactive derivative or lacked acceptable bioavailability (Lyle et al.,

1993; Sanders et al., 1993; Tucker et al., 1994). Second generation quinazolinones proved to be worse inhibitors overall, with serious bioavailability problems, and decreased antiviral potency, and were quite inactive against several NNRTI-resistant mutants such as K103T, Y181C. Substitution of a pyridyl group resulted in a highly potent NNRTI L-738,372 (ICso ~ 12 nM, EC9S ~ 25 nM) that had excellent bioavailability

(80%) as determined in animal studies with Rhesus monkeys (Huffman et al., 1994).

Nevertheless, L-738,372 showed a 2 to 45-fold loss of inhibitory potency against the

A98G, KlOOl, KI01E and K103N viruses (Lyle et al., 1993). L-738,372, however, remained active against the classical NNRTI mutant Y181C.

Recent development of a fourth generation quinazolinone has resulted in a remarkably effective compound (EC50 ~ 1.5 nM), known as DMP266, L-743, 726 or EFV (Table l.1)

(Young et al., 1995). EFV possesses acceptable oral bioavailability and the maximallevel attained in the serum of rats and monkeys was 16% and 42% of the administered dose, respectively (Balani et al., 1999). EFV is the third NNRTI to be used clinicalIy (Young et al., 1995). It is active against the K103N, Y18lC and K103N/Y181C mutants, among several other common NNRTI mutants, showing complete inhibition (ICys ) at < 1.5~M in aIl cases. However, high-Ievel resistance to EFV do es develop, although more than two substitutions are required. In celI culture, the LI 00l and KI 03N combination has been observed and it confers more than 10,000 fold resistance (Young et al., 1995). In patients,

33 the appearance of substitutions K103N, Y188L and G190SIE led to a decreased susceptibility to EFV, which is further enhanced by other secondary substitutions

(V106A, V108I, Y181C, Y188H, P225H and F227L) (Bacheler et al., 2001). The presence of K 103N and Y 188L substitutions conferred resistance both to NVP and DL V, whereas individuals lacking these substitutions showed a sustained response. EFV is one of the three potent NNRTI whose mechanism of inhibition and antiviral properties are examined in detail in this thesis. Recently, a newer generation of quinazolinones

(DPC082, DPC083, DPC96 1, DPC963) has showed slightly improved inhibitory potency against wild type HIV-l (EC90 ~ l.3-2.1 nM) (Corbett et al., 1999; Corbett et al., 2000).

More importantly, the se compounds are more effective against the K103N and LlOOI

HIV-l mutants (IC9o ~10-13 nM) when compared to EFV (IC90 ~ 64-77 nM), DLV (IC9o

~ 1 !-lM) and NVP (IC9o ~ 5 !-lM). They also show an increase in bioavailability in chimpanzees (:::;6.2!-lM) and Rhesus monkeys (:::;6.8 J..lM) and a decrease in protein binding

(~3% free drug) when compared to EFV (2.7 J..lM, 0.4 J..lM and ~0.5% free drug, respectively).

THIOCARBOXANILIDES A structural relative of the fungicide vitavax, the oxathiin carboxanilide UC84 was found to inhibit HIV-l in ceIl culture in submicromolar concentrations (EC50 ~ O.5J..lM) (Bader et al., 1991). UC84 and its thiocarboxanilide derivative UC38 were actuaIly found to inhibit HIV-l RT polymerase activity noncompetitively (Fletcher et al., 1995a).

Competition experiments with a NVP analogue supported the idea that both UC38 and

UC84 bind to the NNRTI binding pocket. Interestingly, UC84 and UC38 interacted with

34 different mechanistic forms of R T and were found to inhibit polymerization synergisticaIly (Fletcher et al., 1995b).

The E138K mutation appeared with resistance in cell culture experiments to UC NNRTI

(Balzarini et al., 1995a) while cross-resistance was found with mutant viruses LI00I,

VI06A, and Y181C (Buckheit, Jr. et al., 1995, Balzarini et al., 1995b). When UC84 underwent an extensive series of structural modifications two of the compounds formed,

UC82 and UC781, were effective against aIl above mutants apart from Y188L (100 to

200-fold resistant) (Balzarini et al., 1996b). Both inhibitors were stable in human serum for at least 24 hours while the decrease in their activity due to protein binding was negligible. UC781 had acceptable oral bioavailability in mice (31 %) and had a remarkably high therapeutic index (TI > 62,000), while UC82 was less orally bioavailable (16%) (Buckheit, Jr. et al., 1997a; Buckheit, Jr. et al., 1997b). Development of resistance to UC781 and UC82 proved difficult with only a 15-fold resistant virus arising (Balzarini et al., 1996c). However, resistance to UC781 eventuaIly did develop, even though it was quite slow when compared to other NNRTI (Buckheit et al., 1997;

Balzarini et al., 1998). Although the Y181C mutation was seen with low-Ievel resistance, the addition of two extra substitutions, VI08I and KI01E, resulted in 500 to 600-fold resistance.

In experiments with UC781, the in vitro ICso concentration (~ 2 nM) and the binding constant (Ki ~ 2 nM) were comparable to the concentration ofHIV-l RT (~2 nM) in the assay mixture (Barnard et al., 1997). This is a unique characteristic of 'tight-binding' inhibition and UC781 was the first 'tight-binding' inhibitor ofRT reported. Furthermore,

35 unlike other UC analogues that bind to only sorne of the mechanistic forms of RT,

UC781 binds to aU three mechanistic forms of the enzyme (see section 1.3). Lastly,

UC781 was found to act as an HIV-l microbicide (Borkow et al., 1997), properties that are described later in this thesis.

1.5. MECHANISMS OF NNRTI INHffiITION Several crystal structures of NNRTI complexed with HIV -1 R T have been published

(Kohlstaedt et al., 1992; Smerdon et al., 1994; Esnouf et al., 1995; Ren et al., 1995a; Ren et al., 1995b; Ding et al., 1995a; Ding et al., 1995b; Hopkins et al., 1996; Esnouf et al.,

1997; Hsiou et al., 1998; Ren et al., 1998; Ding et al., 1998; Ren et al., 2000; Hsiou et al.,

2001; Lindberg et al., 2002). Such crystals facilitate interpretation of the structural elements that may be involved in the inhibition mechanism of NNRTI. As se en in these structures the nonnuc1eoside hydrophobie pocket is formed by the following beta sheet structural elements of HIV -1 R T: the f35-f36 loop and f36 (aa 97-108), the f39-f31 0 (aa 179-

192) and the f312-f313 (primer grip, aa 224-236) hairpins and 1315 ofp66 (aa 319). The f37-138 100p (aa 135-139) ofp51 is also part of the NNIBP. The primer grip is involved in the correct positioning of the 3' OH of the TIP in the catalytic site. The RT catalytic site lies approximately 10 Â above the NNIBP. Inhibition of polymerization is due to structural changes that occur in the catalytic site upon NNRTI binding in this

36 CSIC Efavirenz (EFV) 8-CI TIBO L-737,126 DMP266, Sustiva ® Tivirapine

MKC-442 Nevirapine (NVP) L-697,661 E-EBU Viramune ®

I 0 c~ X)lof ~ l

Delavirdine (DLV) a-APA Rescriptor ® Loviride

"Yi P ~NÀ~:lS

PETT (L Y 300046) UC781 HBY 097 Trovirdine

Figure 1.2. Structures ofvarious NNRTI

37 Table 1.1. Pharmacologic characteristics of representative NNRTI

NNRTI Drug aICso bECso CCCso dOB "TI Resistance References category (nM) (nM) (/lM) (%) HEPT E-EPUdM 2 <2 >0.01 <1 >10 Y188H, Baba et al. et al., 1991 Y181C, Baba et al., 1992 VlO6A Tanaka et al., 1992 Sato et al., 1995 Ernivirine 8 2 40 68 20,000 Y181C, Baba et al., 1994 KI03N Yusa et al., 1995 Szczech et al., 2000 TIBO 8-Cl TIBO <50 5 138 NA 27,600 LIOOI, Larder et al., 1992 KI03N, Balzarini et al., 1993 VI06A, Bymes et al., 1993 E138K, Pauwels et al., 1994 V179N, Vandame et al., 1994 Y181C, Das et al., 1995 Y188HIL Boyer et al., 1999

Dipyrido- NVP >84 >48 <50 92 -1041 KI03N, Merluzzi et al., 1992 diazepinones VI06A, Koup et al., 1991 Y181C, Richman et al., 1994 Y188H, De Clercq et al., 1996 b G190E Havlvir et al., 1996 Lamson et al., 1999 BHAP DLV 260 9 >100 85 9,444 KI03N, Dueweke et al., 1993a VI08I, Romero et al., 1994 Y181C, Darcy et al., 1996 P236L Pyridinones L-697,661 20 <12 >60 NA >5000 KI03N, Goldman et al., 1991 Y181C Nunbergetal.,1991 Sardana et al., 1992 Kilby et al., 1996 TSAO TSAO-m'>T 4700 34 139 NA 30 E138K Balzarini et al. 1992 a-APA Loviride 200 13 710 . <0.3 54,000 KI03N, Pauwels et al., 1993 Y181C Miller et al., 1998 PETT Trovirdine >7 20 87 NA 4,350 LIOOI, Bell et al., 1995 Y181C Ahgren et al., 1995 Zhang et al., 1995 Cantrell et al., 1996 Quinoxaline- S-2720 39 14 200 10 14,286 VI06A, Kleim et al., 1993 thiones G190E, Balzarini et al., 1994 P225H HBY097 80 >2 200 -50 100,000 KI03N, Kleim et al., 1997 VI06VL, Kleim et al., 1999 G190T, P225H Indole CSIC 3 1 >30 145 > LIOOI, Balani et al., 1993 carboxamide 30,000 K103N Williams et al., 1993 Motakis et al., unpublished data Quinazolino- L-738,372 12 <25 80 <94 >3200 A98G, Lyle et al., 1993 nes KIOOI, Hoffman et al., 1994 KIOIE, Prueksaritanont et al., K103N 1995

38 NNRTI Drug aICso "ECso cCCso dOB "TI Resistance References category (nM) (nM) (J.l.M) (%) EFV 3 1 80 <42 80,000 LlOO!, Young et al., 1995 K103N, Balami et al., 1999 Y188L, Bacheler et al., 2001 G190S/E Carboxanili- UC84 <280 200 >40 <1 200 LlOO!, Baderet al., 1991 des V106A, Buckheit et al., 1995 E138K, Balzarini et al., 1995b Y181C UC38 <150 <90 >20 45 ~222 G90E, Buckheit et al., 1995b LlOO!, Balzarini et al., 1995 KIOIG Balzarini et al., 1996b Y181C

UC781 <2 2 >100 31 > KI0IE, Balzarini et al., 1995a 50,000 K103T, Buckheit et al., 1997b V106A, Parniak et al., 1997 V108I, Balzarini et al., 1998 Y181C, Motakis et al., F227L unpublished data

In order to compare the data presented above, the fact that the experimental conditions (substrates, animal models, cell lines etc.) used in each case differed greatly has to be taken into consideration. a ICso is the 50% inhibitory concentration determined in vitro in a recombinant HIV-l RT polymerisation assay. b ECso is the 50% effective concentration determined in cell culture experiments. C CCso is the 50% cytotoxic concentration as determined in cell culture experiments. d OB is the oral bioavailability expressed as the % of drug dose that is absorbed orally in the plasma. e TI is the therapeutic index of the drug, which is calculated as the ratio of the CCso value over the ECso f This bioavailability corresponds to solutions of CSIC in methocel. Solid formulations (e.g. pills) ofCSIC have a much lower bioavailability. The actual values are not available in the literature (Tucker et al., 1996) NA: data not available

39 proximal hydrophobic pocket. However, as yet, there are no structures of the temary RT

TIP-dNTP complex with an NNRTI have been published.

However, biochemical data suggest that there are three structural changes that seem to occur with NNRTI binding (Sarafianos et al., 1998; Pamiak and Sluis-Cremer, 2000).

When the TIP is bound to HIV-l RT the enzyme undergoes a structural change where the thumb folds over the palm in a 'closed' conformation. In the presence of NNRTI the thumb does not fold over the TIP, but remains in its 'open' conformation, similar to what is observed in absence of TIP (free RT). The NNRTI can therefore be perceived as 'a stone in the gears of a machine' with respect to the thump movement (Kohlstaedt et al.,

1992), since it may be unable to translocate the TIP (Zapp et al., 1991; Patel et al., 1995;

Hsiou et al., 1998), thus slowing down polymerization. This hypothesis, known as

'molecular arthritis model' (Model 1), appears inadequate by itself to explain the extensive levels of inhibition seen with sorne NNRTIs. The second hypothesis suggests that binding of NNR TI may cause the primer grip to be displaced, which may affect the correct orientation of the primer, specifically of its 3' OH, and thus catalysis may be slowed down (Model 2). Thirdly, upon NNRTI binding, Y181 and Y188 are pushed closer to the catalytic site causing the catalytic aspartic to become displaced by 2 Â. It is likely that this distortion of the catalytic site plays a key role in NNR TI inhibition. Even though the latter structural alterations are less dramatic than changes in the thumb and the primer-grip, they occur directly in the catalytic site and may be highly debilitating

(Model 3). The data available suggest that all three structural changes may be involved in the mechanism ofNNRTI inhibition.

40 Steady state kinetic analyses, as well as fluorescence studies, have provided information on the mechanism of NNR TI binding to HIV -1 RT and the effects of NNR TI on processivity. SpecificaIly, binding to aIl three mechanistic forms (see section 1.3) seems to result in higher antiviral potency (Fletcher et al., 1995b; Barnard et al., 1997; Maga et al., 2000). There is no decrease in the affinity of TIP or dNTPs for the enzyme in the presence of NNRTI. On the contrary, a small increase in the affinity of RT to TIP has been shown (Divita et al., 1993). The use of single-nucleotide incorporation kinetics has been pivotaI in identifying the exact step in HIV -1 R T polymerization, which is blocked by NNRTI. The available pre-steady state reports have shown that NVP and TIBO block the chemistry step of HIV-l RT catalysis (Spence et al., 1995). Based on this data, the consensus today is that aIl NNR TI inhibit HIV -1 R T by bloc king the chemistry ( catalytic) step of polymerization. Obviously, this is a generalization that may overlook possible differences between different NNR TIs and in this thesis we present evidence that not aU

NNRTI inhibit polymerization by this mechanism.

1.6. MECHANISM OF NNRTI RESISTANCE Resistance to NNRTI correlates with the appearance of amino acid substitutions in the

NNIBP. A number of classical NNRTI cross-resistant mutations such as YI81C and

Y188L exist that render most of these inhibitors ineffective. UC781, described in this thesis, demonstrates a delayed progression towards a highly resistant phenotype, compared to a classical NNRTI, such as NVP, where resistance can develop as rapidly as one week (Richman et al., 1994; Carr et al., 1996). This delay in resistance with UC781 has been attributed to the requirement for more than one substitution for a significant

41 level of resistance to occur [Borkowet al., unpublished data, Motakis et al., unpublished data].

In most cases, resistant substitutions in the NNRTI pocket, such as Y181C and Y188L, do not affect the replication competency ofHIV-l (Loya et al., 1994; Spence et al., 1995;

Maga et al., 1997). A slight catalytic disadvantage appears with substitutions LlOOl and

Y1811 (Maga et al., 1997). In contrast, mutation G190E, which is observed with resistance to S-2720, decreases the infectivity of HIV-l (Balzarini et al., 1994).

Similarly, HIV-l RT cannot tolerate substitutions at residue W229 (Pelemans et al.,

2000). It has been suggested that particular emphasis should be placed on NNRTI that interact with residues that are intollerent to substitution. Compounds that interact strongly with residues such as W229 would be expected to show a delayed development of resistance since substitution of these residues appears to be incompatible with viral function.

Our present understanding of how substitutions in the NNIBP lead to NNR TI resistance cornes from pre-steady state kinetics with NVP. Substitution Y181C results in a 500-fold decrease in the affinity of RT for NVP (Spence et al., 1996). Surprisingly, when saturating concentrations ofNVP are used in order to promote binding, YI81C-RT is 10 times more catalytically active than the wt RT-NVP complex. Most importantly, the conformational step is the rate-limiting step of polymerization by YI81C-RT-NVP, in contrast to the wt RT-NVP complex where the chemistry step is the rate limiting component. According to another group, cross-resistant substitutions LI 001 and V 106A

42 also increase the rate of NVP dissociation (kaff) while K103N decreases the rate of drug binding (kan) (Maga et al., 1997). Y181C and Y188L affect both the kaffand kan ofNVP.

Therefore, resistance to NVP may be due either to decreased interactions of the drug with the NNIBP or to steric hindrance caused by the side chain in the NNIBP. Overall, resistance to most NNR TI is thought to be mai nI y due to the decreased binding of

NNRTI to the mutated NNIBP.

1.7. TIGHT-BINDING INHffiITION NNRTI UC781 is a 'tight-binding' inhibitor of HIV -1 RT (Barnard et al., 1997), the first

RT inhibitor ofthis type to be reported. Therefore, it serves as a lead for the identification and characterization of other tight-binding nonnucleoside inhibitors (TBNNI). TBNNI are of great importance since they are able to completely saturate their enzyme-target at the lowest concentrations of drug possible. Furthermore, inhibition at low nanomolar or subnanomolar concentrations may play a role in diminishing the toxic effects of drugs and in overcoming viral resistance.

In kinetic studies the concentration of inhibitor used is in the range of its affinity constant

(Ki) for the enzyme. In addition, inhibitors are categorized based on the total concentration of the inhibitor (It) used in the assay. When the affinity of the inhibitor for the enzyme is low, the concentration of inhibitor required experimentally is much higher

than the enzyme concentration (Et « It). Also, the affinity constant for such an inhibitor

(KD is large. Therefore, when Et « Ki or EtfK j « 1 the inhibitor is considered a non- tight-binding or rapid equilibrium inhibitor. In contrast, when the total concentration of inhibitor (It) required to saturate the enzyme (E) approximates the concentration of E in

43 the assay (Et = lt), the interaction between the two molecules is tight. EII - 1 molar ratio can thus be used as an initial criterion for 'tight-binding' inhibition (Morrison and Walsh,

1988). Furthermore, the Ki value for such an inhibitor would be expected to be in the same range as both the E and l concentrations. Different groups have proposed different lower limits for this ElKj ratio in order to define 'tight-binding' inhibition (ElKi > 0.1-1)

(Straus and Goldstein, 1943; Szedlacsek and Duggleby, 1995)

'Tight-binding' inhibitors are further divided into rapid and slow (Morrison and Walsh,

1988). Rapid 'tight-binding' inhibitors conform to the following mechanism (mechanism

A):

kaf! A. E + 1 !; El kan

The rate of association between the enzyme and the inhibitor is given by the expression kan[E][I], where kan is a second order rate constant. Binding of the inhibitor to the target is limited by diffusion, with upper kan values in the range of 106-109M-1s-1 (Williams et al.,

1979; Morrison and Walsh, 1988). However, the true rate of El formation is dependent on the concentration of the inhibitor and is given by the pseudo first-order rate constant kan[l], which is lower than kan. Dissociation of the inhibitor from the El complex is described by the relationship kaffiEl], where ko.ff is a first order constant. For substantial binding to occur, Kaf!rnust be considerably lower than kan. Indeed, the rate of dissociation for 'tight-binding' inhibitors is in the range of 10-3 to 1O-9s- 1 (Morrisson and Walsh, 1988;

Kaplan et al., 1991; FalIer et al., 1993; Yiotakis et al., 1994; Furfine et al., 1994).

44 Slow tight-binding occurs when the initial El complex forms rapidly but undergoes a slow conformational change leading to a tighter El complex as shown below

(mechanism B):

kreverse B. E+ 1 ~ El" kan kforward

5 1 In this case, the apparent kan may be lower than 10 M-1s- . However, the actual rate of the rate limiting forward isomerisation is given by the expression kforward '[El], while the

1 2 l reverse isomerisation step is given by the expression kreverselEI*]. Kfarward (~ 1O- _1O- s- )

3 9 l and kreverse (~ 10- to 1O- s- ) are first order rate constants of the forward and reverse isomerisation steps (Morrison and Walsh, 1988).

'Tight-binding' inhibitors cannot be studied using traditional Michaelis-Menten kinetic analyses (Cha, 1975; Williams and Morrison, 1979). The assumption that the total concentration of inhibitor is equal to the concentration of the free inhibitor in the presence of enzyme does not completely hold with 'tight-binding' inhibitors (TBI) since the depletion of the initial total amount of inhibitor may be significant (Segel, 1975).

When TBI are added during the progression of the reaction, a slower rate of catalysis is reached in the order of seconds to minutes after the addition of the inhibitor. This is in contrast to rapid equilibrium inhibitors which, when added to a reaction already in progress, cause a slower rate of polymerization in milliseconds. Therefore, steady state progress curve analysis can be used to assess 'tight-binding' inhibition, while pre-steady state analysis with a rapid quench instrument is required in order to assess rapid

45 equilibrium binding (Morrison and Walsh, 1988; Cha, 1975). The progression of the reaction in the presence of a 'tight-binding' inhibitor is described by the following equation (Cha, 1975):

p = Vs • t - ( Vs - vo) . ( 1 - e -kapp' t) / kapp Equation 1.1

The apparent rate for approach to the new polymerization equilibrium (kapp ) in the presence of a TBI is described by Equation 1.2 in the case of mechanism A and by

Equation 1.3 in the case of mechanism B (Morrison and Walsh, 1988, Szedlacsek and

Duggleby, 1995):

k app = ko./J + kon • l Equation 1.2 k app = k r + kf (I / ( Ki + l » Equation 1.3

where kapp is the apparent rate for approach to a new rate of polymerization in the presence of the inhibitor and kon and ko./J are the rates of l binding to, and dissociation from, the enzyme, respectively. Moreover, kr and !if are the forward and reverse rates of enzyme isomerization on binding of the inhibitor (mechanism B), while Ki is the initial inhibitor binding constant. The overall dissociation constant in the case of mechanism B

(Kt) is defined as follows (Morrison and Walsh, 1988, Szedlacsek and Duggleby, 1995):

Equation 1.4

46 UC781 is a rapid 'tight-binding' inhibitor since its kan is in the range of 106M-I S-\ its k aff in the range of 1O-3 S-1 and its calculated Ki (~ 1 nM) is in the range of the enzyme concentration (Barnard et al., 1997).

1.8. MICROBICIDES Although the development of effective antiretroviral regimens has improved the quantity and quality of life of many illV-1 infected individuals in developed countries, it has not contributed in any way to the containment of the illV pandemie. It has also become evident that the development of a wide spectrum HIV vaccine, which might decrease the rate of infection, is unlikely in the near future due to the multiple challenges that are involved in such a task (Blocker et al., 2000; Nabel, 2001). A more realistic and immediate means for the prevention of illV infection is the development of a microbicide, especially since several promising agents of this type are already available

(Van de Wijgert et al., 2002; McCormack et al., 2002; Phillips et al., 2002). Surprisingly, even though topical microbicides have been considered in the past for the prevention of sexually transmitted diseases such as herpes simplex virus (HSV) or Neisseria gonorrhoea, this has not been the case for HIV (Weir et al., 1994). Only recently, did the development of an anti-illV microbicide become a priority (Potts, 1994; Blocker et al.,

2000).

There are several reasons why the development of a vaginal microbicide is of great importance. It appears that the rate of infection in women has been increasing (Wainberg,

1999; Blocker et al., 2000). In many cultures women are either unable to refuse sex or to insist on the use of condoms and are therefore unable to prote ct themselves. Drugs used topically to inactivate the infectious particles present in sexual fluids aimed at preventing

47 IllV infection have been tenned "virucides" or "microbicides" (Wainberg, 1999) and constitute an attractive possibility for such women.

An ideal microbicide should fulfiIl several requirements. First, it should be able to inactivate HIV at very low concentrations (high potency) in a near irreversible manner

(Rosenthal et al., 1998; Boyd and Salata, 2000). Second, it should be effective at concentrations that are not cytotoxic or irritating to the exposed genital epithelium. Third, a microbicide has to be soluble enough so that it can be absorbed topically, but unlike systemic therapies, it should not be highly bioavailable. High bioavailability would likely result in the systemic accumulation of the drug, which in tum could lead to adverse effects with continuous use. Fourth, an ideal microbicide should be able to prevent the transmission of HIV in its free, as weIl as in its cell-associated fonns. Fifth, microbicides should be inexpensive so that they can be accessible in Third World countries, but also profitable to the manufacturers so that drug production can be sustained. In order to understand the mechanism by which the various existing and newly developed microbicides act, the targets of initial HIV -1 infection have to be defined.

1.8.1. THEMECHANISMOFHIV-l SEXUAL TRANSMISSION

The sexual transmission of HIV -1 is a complex process and it appears to be dependent on the route of infection. It is weil known that infection can occur via both anal and vaginal intercourse while other practices such as oral sex are considered of low risk. Therefore, the cellular target ofHIV-l entry may vary depending on whether initial contact involves the female genitourinary epithelium, the male genital tract or the colonie/anal epithelium.

48 In aIl cases, HIV-l is able to penetrate the external 'protective' mucosal barriers and infiltrate into the draining lymphatics where its primary targets, macrophages and lymphocytes, are found.

In the female reproductive tract, CD4+ Langerhan's ceIls (LC) are present throughout

(vagina, cervix, uterus) (Hus sain et al., 1995). Langerhan's ceIls are easily infected by

HIV-l in vitro (2h viral exposure), and they in turn can infect T-ceIls (Kawamura et al.,

2000). The infiltration of infected LC into the proximal draining lymph nodes, where vulnerable macrophages and monocytes are found, constitutes one possible route of infection (Hussain et al., 1995).

With respect to the CD4- epithelial ceIls, which constitute the majority of the cells that cover the female tract, sorne groups have not observed any infection of the cervicovaginal epithelium after short exposure (2h) to free HIV-l (Green head et al., 2000). Others have reported that vaginal epithelial cells can be productively infected in cell culture by cell free virus, upon prolonged (16h) exposure (Pang et al., 2000), or by infected mononuclear cells and T-cells (Milman et al., 1994; Tan et al., 1996; Tan et al., 1998).

Infection of the endometrium and fallopian tube epithelium has also been demonstrated

(Yeaman et al., 1998).

HIV may also be able to cross the mucosa without actually productively infecting the epithelial cells. Interestingly, a number ofresearch teams have shown that HIV-l infected monocytes, present in the genital secretions, are able to come in contact with epithelial cells and induce the 'endocytosis' of HIV-I in epithelial cells (Bomsel, 1997; Hocini et

49 al., 1999; Alfsen et al., 2001). This process of'trancytosis' is rapid (20-30 min) and may involve the galactosyl ceramide receptor (Gal Cer). The internalized HIV-l can move to the basolateral surface of the epithelial cells in a polarized specifie manner from where it can theoretically access the deeper layers of the mucosa (Deschambeault et al., 1999).

Trancytosis of monocyte-associated HIV -1 has also been observed in cervix derived

HeLa cells (Morizono, 1998). In addition, trancytosis of free virus may also be possible, however, a longer exposure (3h) of the cells to HIV-l is required (Hocini et al., 2001).

Alternatively, it has been suggested that HIV-l may adhere to the surface of epithelial cells from where it is either taken up non-specifically for antigen presentation or it passes in between the somewhat 'looser' vaginal epithelium (Dezzutti et al., 2001).

Although T -cells are not abundant on the surface of the vaginal mucosa, chronic inflammation can lead to a dramatic infiltration of such cells. Inflammation of the female tract may thus play a role in the transmission of the disease (Cohn et al., 2001).

Furthermore, menstrual hormones appear to regulate the mucosal immune system.

Therefore, macrophages and CD4+ T -cells appear periodically in premenoposal women throughout the whole reproductive system, vagina, cervix, uterus and fallopian tubes

(Yeaman et al., 1998). Overall, the presence of T-cells, which are more susceptible to

HIV -1 infection than dendritic cells and epithelial cells, may facilitate the transmission of

HIV-l to women (Howell et al., 1997).

In the male genitalia CD4+ LC are present only in the foreskin and the infection of LC by

HIV-l is thus relevant mainly in uncircumcised men (Hussain et al., 1995; Szabo et al.,

50 2000). The urethral epithelium contains Fe receptors, which may play a role in mV-I infection by binding to HIV-l-antibody immune complexes found in the genital secretions ofinfected individuals (Hussain et al., 1995). The rest of the penile surface is covered by a keratinized epithelium, which is an effective barrier to infection in the absence of any physical injury.

Lastly, in the anal epithelium, specialized M cells transport HIV -1 through to their basolateral side where lymphocytes and macrophages are found (Peyer' s patches)

(Amerongen et al., 1991; Neutra, 1999). Both primary ceIl cultures and ceIllines ofCD4- colonie/intestinal epithelial cells can be productively infected by mV-l in vitro (Moyer et al., 1990; Yahi et al., 1992). CD4+ monocytes are present only below the epithelia, which lie above the lamina propria, while LC are completely absent (Hussain et al.,

1995). Therefore, infection of the anal epithelium may involve epithelial cells, M-cells or both.

Another important aspect of the initial events of infection is whether the vector of transmission is cell-associated HIV-l, free virus or both. In semen, both free mV-l and infected monocytes and macrophages have been detected (Krieger et al., 1995;

Vemanzza et al., 1994; BaIl et al., 1999; Speck et al., 1999; Tachet et al., 1999; Barroso et al., 2000; Gupta et al., 2000). Viral shedding in semen, in particular, appears to be intermittent and to be increased with advanced disease progression (Dyer et al., 1998;

Speck et al. 1999). Although there is sorne variability in the reported proportions of free

51 versus cell-associated virus, it is likely that both forms are involved in the male to female or male-to-male transmission of HIV -1 infection.

In the female genital tract, virus is shed in the cervicovaginal secretions, which may mediate the transmission ofHIV-1 to men (Mohamed et al., 1997; Critchlow et al., 1997;

Ghys et al., 1997; Mostad et al., 1998; Rasheed et al., 1998; Debiaggi et al., 2001; AI

Harthi et al., 2001; Sheck et al., 2001). Similar to male sperm, HIV-l infected cells are exfoliated in the cervicovaginal epithelium and may also be involved in the transmission of HIV-l infection from women to men (Mostad et al., 1998; Debiaggi et al., 2001).

Lastly, shedding of both free HIV and infected cells has also been described in the anal rectal mucosa (Kiviat et al., 1998).

In conclusion, microbicidal therapies should be aimed at preventing infection of

Langerhan's, epithelial and M cells, as weIl as peripherallymphocytes and macrophages.

Based on the available data, it is difficult to make any suggestions about the relative importance of each type of ceIl in the development of a microbicide. For the same reason, both free and ceIl associated HIV-l should be targeted.

1.8.2. CLASSIFICATION OF MICROBICIDES

Presently, there are three types of compounds that are being examined as potential microbicides. These include surfactants that are able to lyse the membrane of the infectious particles, and entry blockers, which bind to the surface viral antigens. Both are able to prevent the entry of HIV -1 into the target ceIl. In addition, replication inhibitors

52 prevent viral repli cation and chronic establishment of infection once HIV -1 has entered the cell.

DISRUPTING AGENTS

The surfactant N-9 was the first agent to be considered as a general STD microbicide, and more recently, as an HIV virucide (Jennings and Clegg, 1993; Feldblum and Weir, 1994;

Bourinbaiar and Lee-Huang, 1994). As a result of its established use as a spermicide, N-9 was readily considered for testing as an HIV microbicide. However, N-9 has been shown to affect the natural flora of the vaginal environment (Rosenstein et al., 1998), leading to an approximate 60 % drop in concentration of lactobacilli in women that used N-9 continuously while irregular, gram-negative bacterial colonization was also observed.

Therefore, continuous use of N-9 may lead to urinary and vaginal infections. More importantly, recent data indicate that this agent, which acts as a surfactant, causes vaginal irritation and local inflammation, which actually increases the risk of HIV and other STD infection (Kreiss et al., 1992; Zekeng et al., 1993; Stafford et al., 1998; Rosenstein et al.,

1998; Uckun and D'Cruz, 1999; Fichorova and Anderson, 2000; Milligan et al., 2000).

Similar studies of the rectal epithelium show an increase in exfoliation and damage with

N-9 use (Phillips et al., 2001; Celum, 2001). These concems are further justified by the results of several, recent phase III clinical trials. Specifically, in a Cameroon study, the

N-9 product VCF (Vagina Contraceptive Film) did not prote ct female sex workers from

HIV infection (Roddy et al., 1998) and in a more recent phase III clinical trial sponsored by UNAIDS, the use of Advantage-S, another N-9 preparation, actually increased the incidence of HIV transmission compared to the control group (Forbes et al., 2000). Use

53 of lower doses of N-9 in order to decrease its toxicity/irritant effect on the vaginal epithelium resulted in the 10ss of antiviral protection (Richardson et al., 2000).

Sodiwn dodecyl (lauryl) sulfate (SDS or SLS) is another surfactant, which is able to lyse enveloped viruses such as HIV as weIl as non-enveloped viruses such as the human papilloma virus (HP V) in vitro (Howett et al., 1999; Bestman-Smith et al., 2001). SDS reduces the infectivity of pre-treated HIV -1. Furthermore, SLS is able to inactivate celI associated HIV-1. SDS is ten-fold less toxic than N-9 in HeLa celIs (Krebs et al., 1999) and it is not irritating or toxic to the vaginal epitheliwn ofrabbits (Roy et al., 2001; Piret et al., 2002). Surfactant C31G, a citrate buffered equimolar mixture of alkyl betaine and an alkyl dimethyl amine oxide, is effective against HIV-l while it is half as toxic as N-9 in celI culture (Krebs et al., 1999). Nevertheless, the ability of SDS and C31 G to prevent

HIV infection in vivo remains to be determined.

ENTRY BLOCKERS

Cyanovirin-N (CV-N) is a small polypeptide with high inhibitory potency against HIV infection (Mori et al., 1998). It binds to the surface (SU) glycoprotein gp120 and prevents its interaction with the CD4 receptors of the cell thus completely impairing virus binding and subsequent entry (Esser et al., 1999; Dey et al., 2000; Q'Keefe et al., 2000; Mori et al., 2001; Shenoy et al., 2001; Bolmstedt et al., 2001). Distortion of the surface viral antigen gp120 upon binding to CV-N results in HIV-1 inactivation. Interestingly, CN-V is also able to prevent CD4+ independent infection (Mori et al., 2001). Although CN-V constitutes a promising HIV-specific virucide, it awaits clinical evaluation.

54 Mucibodies are peptides that are derived from the human protein mucin, and closely mimic the recognition site ofanti-gpl20 antibodies (Fontenot et al., 1998). These pre vent cellular infection by laboratory and primary macrophage-tropic HIV -1 strains. The ability ofmucibodies to prevent gp120-DC4 binding has been demonstrated in vitro. Mucibodies of different specificities against various strains of HIV and other infectious diseases may prove valuable as topical microbicides in the future.

The non-detergent microbicide PR02000 (P2K), a naphthalene sulfonate derivative, inhibits HIV-l infection by blocking binding of gp120 at nanomolar concentrations

(Rusconi et al., 1996). In the macaque animal model, P2K has been relatively effective

(50%) at preventing infection by the chimeric HIV/SIV (SHIV) virus (Weber et al.,

2001). Formulations of 0.4% P2K showed no significant inflammation/ulceration in phase 1 clinical trials (Van Damme et al., 2000) and no systemic absorption of P2K was observed. The efficacy of P2K in humans, like most other microbicides, remains to be established.

Dextrin-2-sulfate (D2S) is an anionic polymer that is also able to bind to the surface of

HIV-l and inhibit entry into the target cells. Furthermore, D2S protects peripheral blood mononuclear cells (PBMC) and macrophages from HIV-I infection (Javan et al., 1997), while it is able to inactivate SHIV in vitro (Weber et al., 2001). In animal studies, D2S was able to protect 2/4 (50%) macaques from SHIV infection with a single application.

D2S does not affect the vaginal flora and there is no evidence of induced epithelial

ss inflammation thus making this agent safe to undergo phase II clinical trials (Stafford et al., 1997; Rosenstein et al., 1998). Dextran sulfate (DS), another polysulfated carbohydrate, also has anti-IllV and anti-HSV activities in vitro. However, DS has failed to show any protective effect against HSV in the murine animal model despite its observed activity in vitro (Piret et al., 2000).

Cellulose acetate phthalate (CAP) is an "inactive" ingredient used as a camer of therapeutics in tablets and capsules (enteric coating polymer). In its microionized formulation it effectively inactivates IllV-1 and other sexually transmitted pathogens, but it does not affect Lactobacilli (Neurath et al., 1999, Neurath 2000).

Lastly, Monocaprin, the I-monoglyceride of capnc acid, has been effective in inactivating IllV-l, and other STDs, (e.g. HSV-2, Chlamydia, Neisseria) in semen upon short exposure « 5 min.) (Kristmundsdottir et al., 1999.) In addition, it has been effective in reducing the number of Ieukocytes in the semen dramatically (> 10,000 foId), thus potentially preventing cellular transmission of viral infections. In addition, monocapnn is not toxic to the vaginal epithelium of rabbits when applied for ten consecutive days (Thormar et al., 1999). In mice, intravaginal and intracutaneous infection by HSV-2, which is as sensitive to monocaprin as IllV-l, was prevented (Neyts et al., 2000). Again, the evaluation of mono caprin in being able to prevent HIV infection in humans remains to be determined.

56 REPLICATION INHIBITORS

Inhibitors of HIV-1 viral repli cation could theoretically impair the establishment of infection even past the stage of viral entry. Several HIV-1 reverse transcriptase (RT) inhibitors have been suggested for this use. Aryl phosphate derivatives of bromomethoxy zidovudine (AZT), WHI-05 and WHI-07, have been shown to have spermicidal activity

(50% sperm-immobilizing activity or SIA, 29 J.lM and 6 J.lM, respectively) in addition to their anti-HIV activity (ICso ~ 50 nM and 5 nM, respectively) (D'Cruz et al., 1998;

D'Cruz et al., 2000a; D'Cruz et aL, 2000b). This spermicidal activity is not mediated through the binding of these compounds to HIV-1 RT but rather through their toxicity to the sperm cells. Although AZT itself showed potent anti-HIV activity (6 nM), it did not inhibit sperm motility. WHI-05 and WHI-07 were not cytotoxic to the cells of the female genital epithelium at their spermicidal concentrations (D'Cruz et al., 1999). In addition, animal studies in mice and rabbits showed no adverse systemic effects and no morbidity, mortality, decreased fertility, weight loss or any kind of local inflammation or disruption of the genital epithelium even at the highest concentrations used (D'Cruz et al., 2000a;

D'Cruz et al., 2000b). Application in a form of a gel microemulsion in rnice for a period of 13 weeks resulted in a > 1OOO-fold higher level of drug concentration than required for in vitro antiviral and spermicidal activity (D'Cruz et al., 2000a; D'Cruz et al., 2000b).

Single application of WHI-05 and WHI-07 in rabbits showed 80 % effectiveness as a contraceptive in both cases.

Several non-nuc1eoside reverse transcriptase inhibitor (NNRTI) derivatives ofHIV-1 RT have also dernonstrated dual antiviral and spermicidaI activity. SpecificaIly, the thiourea

57 PETT derivative F-PBT, as weU as the HEPT derivative S-DABO were effective in inhibiting illV-1 production in PBMCs (ICso < 1 nM) while they irreversibly inactivated sperm motility (ECso - 147 J.lM) (D'Cruz et al., 1999). In contrast to N-9, these agents were spermicidal at a much lower concentration than their cytotoxic range in endocervical/ectocervical ceUs (CCso > 1 mM). Further development of the thiourea series of NNRTI has led to compounds ill-253, HI-346 and ill-445 with similar anti illV-l activity (ICso - 1-3 nM) but improved spermicidal action (ECso - 42-70J.lM).

One of the most promising microbicides of this category has been the 'tight-binding'

NNRTI, UC781 (Barnard et al. 1997). Unlike aU previously discussed microbicides,

UC781 completely inactivates isolated illV-1 upon short exposure and subsequent removal of the exogenous drug (Borkow et al., 1997). UC781-pretreated chronically infected H9 ceUs (H9+) become non-infectious, in the absence of extracellular drug.

Lastly, pre-treatment of non-infected MT2 ceUs with this NNRTI resuIts in their protection from subsequent illV-1 infection after these ceUs have been washed free of drug. In summary, UC781 prevents both free and ceU-to-ceU HIV-l transmission. The ability ofUC781 to carry its inhibitory/protective function even after it has been removed from the medium strongly suggests that this compound localizes itself in the cell for prolonged periods of time thus acting as a constant protective barrier to infection. None of these properties have been observed with any other non-tight-binding NNRTI such as

NVP orDLY.

58 Table 1.2. Representative HIV-1 microbicides

Compound Mechanism of action Special characteristics

N-9 detergent Ineffective in PIII trials, high toxicity

SDS detergent Effective against non-enveloped viruses, 10-fold less toxic than N-9 C31G detergent 2- fold less toxic than N-9

CN-V binds to illV-1 surface Prevents CD+ and CD- cell infection Mucibodies bind to gp-120 illV strain specific

P2K blocks receptor binding 50% effective (macaques, SillY), No inflammation (PI trials), No systemic absorption

D2S blocks viral entry 50% effective (macaques, SillY), Safety established (PI trials), No effect on vaginal fiora

CAP blocks viral entry Enteric coating polymer, No effect on vaginal fiora Monocaprin blocks viral entry Effective against HSV-2, Chlamydia, Neisseria Gonorrhoea, Not toxic to rabbit vaginal epithelium

Will-051WHI-07 inhibits reverse Contraceptive/spermicidal transcription (competitive)

F-PBT inhibits reverse Contraceptive/spermicidal transcription (noncompetitive)

S-DABO inhibits reverse Contraceptive/spermicidal transcription (noncompetitive)

UC781 inhibit reverse Effective upon short exposure transcription and removal of drug (tight -binding, Establishes barrier to infection of healthy noncompetitive) cells Inactivates both free and cell-associated ViruS

59 1.9. RATIONALE FOR THE PRESENT STUDIES The aim of the work that follows is to identify the distinguishing parameters that set

TBNNl apart from other rapid equilibrium NNRTI. As mentioned at the beginning ofthis introduction, the identification of UC781 as the first TBNNl (Barnard et al., 1997), as well as its exceptional pharmacological properties (high potency, delayed development of resistance, microbicidal activity (Borkow et al., 1997) etc.) triggered our interest towards identifying other TBNNI. The identification of two additional 'tight-binding' NNRTI,

CSIC (Motakis et al., unpublished data) and efavirenz (EFV) (Maga et al., 2000; Motakis et al., unpublished data), gave as the chance to actually characterize in detail the properties of TBNNl and, subsequently, examine the possible impact of 'tight-binding' inhibitors on the development of HIV -1 resistance. Furthermore, our goal was to assess whether 'tight-binding' was an important parameter in the use ofNNRTI in microbicidal applications.

60 CHAPTER 2: MECHANISM OF INHIBITION OF HIV-l REVERSE

TRANSCRIPTASE BY TIGHT -BINDING NONNUCLEOSIDE INHIBITORS

61 2.1. INTRODUCTION Most HIV -1 therapies presently available are targeted against the polymerase activities of

HIV-l RT and inc1ude both NRTI and NNRTI. The latter are allosteric inhibitors, which do not interact with the catalytic site, but bind to a distinct and proximal hydrophobie pocket. Based on crystallographic data NNR TI appear to distort the catalytic site (Esnouf et al., 1995; Rodgers et al., 1995). Data from pre-steady state kinetic analyses (Spence et al., 1995; Rittinger et al., 1995) are also in agreement with this 'active-site distortion' model (Sarafianos et al., 1998) and it is now accepted that catalysis itself, rather than the conformational change that precedes it, is slowed down dramatically in the presence of

NNRTI much as NVP or TIBO (Spence et al., 1995).

Interestingly, tight-binding NNRTIs (TBNNI) appear to differ significantly from the rest ofNNRTI. Primarily, TBNNI reach their binding equilibrium slowly (minute time range) and at concentrations that are comparable to the concentrations of the enzyme (Morrison and Walsh, 1988). This is in contrast to 'c1assical' NNRTI such as NVP where binding equilibrium is achieved rapidly (millisecond time range) and occurs only in the presence of a large excess of inhibitor over enzyme (Morrison and Walsh, 1988; Spence et al,

1995). Therefore, 'tight-binding' NNRTI demonstrate exceptional affinity to HIV-l RT and thus constitute the most pote nt type of reversible inhibitors available.

Moreover, apart from their remarkable affinity to HIV-l RT, TBNNI appear to inhibit reverse transcription in a somewhat different manner than 'c1assical' NNRTI. We have evidence that UC781, EPV and CSIC impair the ability of the RT-TIP-dNTP ternary complex to undergo the conformational changes that precede catalysis. This may offer an

62 additional explanation for the outstanding potency of TBNNI, compared to other NNRTI such as NVP; in the presence of the latter, even though catalysis is significantly slowed down, it is not completely blocked. TBNNI have additional desirable anti-HIV-l properties to offer. CeUs exposed to UC781 (Borkow et al., 1997), and as shown later in this report (see Chapter 5) EFV and CSIC, become resistant to subsequent HIV-l infection, after the drug has been removed. Similarly, pre-treatment of ceU free HIV-l with these drugs leaves the virus inactive, even after removal of the free drug as previously shown with UC781 (Borkow et al., 1997). Lastly, in addition to their microbicidal activities, UC781 and EFV appear to act synergistically with other systemic therapies such as AZT in inhibiting HIV-l activity (Borkow et al., 1999; Maga et al.,

2000). In conclusion, TBNNI have great potential as both systemic and virucidal therapies.

Barnard et al. (1997) has described the mechanism by which UC781 binds to HIV-l RT.

Here we describe how UC781, EFV and CSIC TBNNI differ from NVP in their mechanism of (1) binding to HIV-l RT and (2) inhibiting polymerization. These differences give clues for the basis of the exceptional efficacy of TBNNI. So far two

TBNNI have been identified, UC781 (Barnard et al., 1997) and EFV (Sustiva®) (Maga et al., 2000). Here we report the identification of an additional TBNNI: 5-chloro-3-

(phenylsulfonyl)indole-2-carboxamide (CSIC) also known as L-737,126 (Williams et al.,

1993). Our better understanding of TBNNI may contribute to the improvement of existing TBNNI and the development of new and more potent inhibitors of this class.

63 2.2. MATERIALANDMETHODS Reagents. Substrates C2P]d.ATP, CH]dCTP, aS-dCTP, poly(rA)-oligo(dT)12_18 and poly(rC)-oligo(dG)12-18 were from Amersham Pharmacia (Montreal, QC, Canada). DNA primers were ordered from GIBCO (Toronto, ON, Canada). T4 polynucleotide kinase was from MBI Fermentas (Flamborough, ON, Canada). UC781 was provided by A. W.

Harrison and W. G. Brouwer (UC Ltd. Research Laboratories, Guelph, ON, Canada).

NVP was from Boehringer-Ingelheim (Montreal, QC, Canada). EFV was a generous gift of Dr. M. A. Wainberg while CSIC was custom synthesized by Dalton Chemical Labs

(ON, Canada). Purified recombinant RT was prepared by a rapid method we have previously described (Fletcher et al., 1996). The concentration of the purified enzyme was determined by spectrophotometry at 280 nm using an extinction coefficient of

260,450 M-1cm-1(Furge et al., 1999).

HIV-I RT polymerization inhibition assays. Inhibition of HIV-1 RT was assessed by a fixed-time, RNA-dependent DNA polymerase activity (RDDP) assay as previously described (Fletcher et al., 1995a; Fletcher et al., 1995b). The assay involved measuring the amount of radioactive dNTP incorporated in a homopolymeric RNA template in the presence ofpurified HIV-1 RT. Briefly, RT and substrates were mixed in a 50 /-lM Tris-

HCI pH 7.8,60 /-lM KCI, 10 /-lM MgCh and 1 mM DTT buffer in a final volume of 50 /-lI.

The concentration of TIP (poly(rC)-oligo(dG)12_18) was 200 nM while the concentration of eH]dGTP was varied (0.5-20/-lM). The concentration of RT in the mixture was lOng and the concentration ofNNRTI, prepared as a stock solution in DMSO, was varied. The concentrations of DMSO in the reaction mixture did not exceed 2% and control experiments have demonstrated that this has no effect on RT activity. After 30 min

64 incubation at 37°C the reaction mixtures were quenched with 250 III of ice-cold 10% trichloroacetic acid (TCA) containing 20 mM sodium pyrophosphate (NaPPi), left on ice for at least 30 min and then filtered through glass fibre filters (FC multiScreen filtration system, type C, 1.2 micrometers) from Millipore (Bedford, MA, USA). The filters were washed sequentially, twice with 10% TCA, 20 mM NaPPi solution and twice with 100% ethanol. The level of radioactive incorporation was determined by liquid scintillation spectrometry.

Reaction Progress Curve Analysis. Reaction progress curve analysis was carried out as previously described (Barnard et al., 1997). Saturating concentrations of homopolymeric poly(rC)-oligo(dG) TIP (200 nM final concentration) and radiolabeled eH]dGTP (20 IlM final concentration) were mixed in 50 IlM Tris-HCI pH 7.8, 60 /.lM KCI and 10 /.lM

MgClz buffer to a final volume of 1 ml. The reaction was initiated by adding 250 III of

RT (2.5 ng//.ll). Two aliquots (50 Ill) were removed every min over a total period of 4 min. At 5 min, 1 III of the appropriate stock concentration of inhibitor in DMSO was rapidly added to the reaction mixture (850 /.lI). The final concentrations ofinhibitor in the mixture varied from 0 to 10 nM for UC781, 0 to 5 nM for CSIC and from 0 to 67 nM for

EFV. Reactions were carried at 37°C. Aliquots (50 /.lI) were removed at various times, quenched, filtered and counted as described above for the HIV-I RT inhibition assays.

The 50 /-lI aliquots collected before the addition of the inhibitors were also quenched filtered and counted in order to measure the amount of product produced before the addition of the inhibitor. The progress curve data were fitted to Equation 1.1. (see

chapter 1), while replots of kapp were analysed using Equation 1.2. or Equation 1.3.

65 depending on whether these replots were linear or curvilinear, respectively (Morrison and

Walsh, 1988).

Rapid-Quench Experiments. In order to measure the rates of single nucleotide incorporation the use of a heteropolimeric TIP was necessary. Only the next complementary dNTP was added to the mixture and thus extension of the TIP by one nucleotide was measured as a product. Since single dNTP incorporation occurs in the pre steady state, it is much faster than the rate of polymerisation measured under steady state conditions (see section 1.3). Therefore, a rapid-quench instrument was used to 'capture' the rates of single nucleotide incorporation (Bio-Logic quench-flow module QFM-5 and electronic controller/power supply MSP-5). The conditions used were those described previously (Spence et al., 1995; Spence et al., 1996). In aIl cases, a heteropolymeric TIP was used formed by annealing a 30 nucleotide DNA template (5' AAT CTC TAG CAG

TGG CGC CCG AAC AAG GAC 3') with a 32p labelled 18 nucleotide DNA primer (5'

GTC CCT GTT CGG GCG CCA 3'). The primer was labelled at its 5' -end with 32p by using e2p]dATP and T4 polynucleotide kinase under the conditions given by the manufacture. Annealing was obtained by heating equimolar amounts of the two oligonucleotides at 90°C and then cooling them to room temperature.

The TIP (100 nM) was preincubated with RT (100 nM) for 5 min at 37°C. The formed binary complex was then incubated with saturating concentrations ofNNRTI (> 100-fold

IC 50) or DMSO for another 5 min. Reactions were initiated by mixing equal volumes (60

,.d) ofRT-TIP (100 nM) and Mg2+-dCTP (or Mg2+-dCTPaS). The final concentration of

66 2 Mg + was 10 J.tM. Reactions were carried out for varied times and then the mixture was quenched with 0.5 M EDTA (pH 8). The products of the reaction were resolved by denaturing polyacrylamide gel electrophoresis (16%, 8 M urea) and quantified by densitometric analysis. The data from each pre-steady state burst rate measurement were analysed using the following exponential equation:

P = A • (1 - exp (kp • t) + ks • t) Equation 2.1

where P is the single-nucleotide extended product, A is the amplitude of the burst, kp is the pre-steady state rate and ks is the steady state rate of polymerization (Johnson, 1995;

Spence et al., 1995; Reardon, 1992). The pre-steady state rates (kp) obtained for each concentration of dCTP were fitted to the following hyperbolic function:

kp = (Vmax • S) 1 (S + KI) Equation 2.2

where V max is the maximal pre-steady state rate, S is the substrate concentration and KI is the substrate affinity constant (Spence et al., 1995).

Gel-Shifl A nalysis. Heteropolymeric template/primer T301P 18 and RT at vanous concentrations were incubated at room temperature (20°C) for 5 min. in the absence and in the presence of saturating concentrations of UC781, CSIC and EFV The final incubation volume was 20 J.tl and the final concentration of DMSO in aIl cases was 2 %.

The buffer used to mix the enzyme with the T301P 18 was Tris-HCl pH 7.8 (50 J.tM) and

67 KCI (60 /lM) (final concentrations). The concentration of TIP was constant (150 nM) while RT was varied from 0 to 1 /lM. Nondenaturing gel electrophoresis (6.4% polyacrylamide) was used to examine the shift in the mobility of a radiolabeled T 301P18 in the presence of increasing concentrations of IDV -1 R T. The amount of product bound to

RT was measured by densitometric analysis of the shifted bands as a percent of total radioactive T301P18. Ail data were analysed using the following quadratic equation:

[E. S] = E - 0.5{(Ki + E + S)- [(Ki + E + Si-(4. E. S)]1/2} Equation 2.3

where [E. S] is the concentration of the RT-TIP complex, E is the concentration ofRT,

Kd is the TIP dissociation constant and S the total TIP concentration (Johnson, 1995;

Reardon, 1992).

2.3. RESULTS Characterisation of TBNNJ binding. We found UC781, CSIC and EFV to be exceptionally potent inhibitors ofIDV-l polymerase activity, similar to what others have previously reported (Williams et al., 1993; Barnard et al., 1997; Maga et al., 2000).

SpecificaHy, aH three compounds inhibited DNA-dependent RNA polymerisation at concentrations that were comparable to the concentration of the enzyme in the assay (~ 2 nM). As shown in table 2.1 the IC so values of UC781, CSIC and EFV were 1.7, 2 and 7 nM, respectively, compared to 290 nM for NVP. Such low Leso values indicate that the minimal concentration of inhibitor required to completely saturate aH enzyme binding sites in the reaction mixture is approximately equal to the concentration of the enzyme in the assay. This is one of the unique characteristics of reversible, 'tight-binding' inhibitors

68 (Williams and Morrison, 1979; Morrison and Walsh, 1988). In contrast, classical non

'tight-binding' inhibitors inhibit lllV-1 RT at concentrations in excess of the enzyme concentration in the reaction mixture. In other words, a lot more drug than enzyme is required in order to push the enzyme-inhibitor equilibrium towards the bound state. For instance, the relatively potent NNRTI NVP, has an ICso of approximately 300 nM, which is 150-fold higher than the concentration of the enzyme in the reaction mixture. The effective concentrations (ECso) for inhibition of viral repli cation were also in the lower nanomolar range (1-10 nM) for UC781, EFV and CSIC, up to 40-fold more potent than

NVP (Table 2.1).

With classical NNRTI, such as NVP, Michaelis-Menten kinetics can be used to measure the actual affinity constants (Ki) for binding to lllV-1 RT (Williams and Morrison, 1979).

On the other hand, due to the fact that 'tight-binding' inhibitors are used at concentrations comparable to that of the enzyme, the Michaelis-Menten assumption that the concentrations of free and total inhibitor are equal throughout the reaction does not hold since a significant portion of the inhibitor becomes bound. Therefore, in order to assess the Ki of UC781, CSIC and EFV we used progress curve analysis, which to our knowledge is the most appropriate method for assessing the kinetics of 'tight-binding'

inhibitors (Szedlacsek et al., 1990; Barnard et al., 1997). The apparent rate (kapp) of

UC781 binding to RT increased linearly with increasing concentrations of inhibitor, as demonstrated by Barnard et al. (1997). This is indicative of a one step binding mechanism as explained in material and methods (see mechanism A, section 1.7). The rates of formation (ko,J and disappearance (kojJ) of the RT-I complex were calculated

69 3 5 1 l using equation 1.2 to be 2.1 ± 0.3 (xlO- ) S-1 and 5.1 ± 1.2 (x10 ) M- s- , respectively.

These results are in close approximation with the values, which have previously been reported with respect to UC781 (Barnard et al., 1997). Therefore UC781 binds to RT rapidly and dissociates from it slowly. The binding constant (l

The kapp for the binding of EFV and CSIC to HIV-l RT increased hyperbolically with increasing concentrations of inhibitor (Figure 2.1). This is consistent with a two-step binding model (mechanism B) as explained in section 1.7 (Morrison and Walsh, 1988;

Cha, 1975). The inhibitors bind to the enzyme to form an initial RT-inhibitor (RT-I) collision complex (Ki) and subsequently the enzyme undergoes a slow conformational change that results in a 'tighter' RT-I* complex. Therefore, under the se conditions, two kinetic inhibition constants can be calculated: Ki, which corresponds to initial binding

(Equation 1.3) and Ki*, which corresponds to overall binding including the second conformational step (Equation 1.4). Under our assay conditions, Ki and Ki* for CSIC were 8.1 ± 2.1 nM and 0.10 ± 0.02 nM, while for EFV, Ki was 77.7 ± 20.6 nM and Ki* was 8.5 ± 3.4 nM. Moreover, the unimolecular rate constants for forward and reverse isomerization (see section 1.7, Equation 1.3) between the RT-I and RT-I* complexes

3 4 3 were 3.4 ± 0.7 (xlO- ) S-I and 2.2 ± 0.2 (xlO- ) S-I for CSIC and 5.4 ± 0.4 (x 10- ) S-I and

6.4 ± 1.6 (x 10-4) S-I for EFY. This method does not allow for the calculation of the individual rates of the initial bimolecular binding as in the case ofUC781. EFV and CSIC

70 bind to HIV-l RT with an overall slower rate than UC781, but also dissociate slowly, and are therefore termed 'slow tight-binding' inhibitors (Morrison and Walsh, 1988). In order to assess how far the equilibriurn lies towards the conformationally altered RT-I* complex the ratio of the forward kisojorward and reverse k iso.reverse isomerisation rates were calculated. The kisojorwari k iso.reverse ratio was 15 in the case of CSIC and 8 in the case of

EFV. In other words the RT-I* complex is 15-fold more available (energetically favourable) than the RT -1 complex in the presence of CSIC and 8-fold more available in the case of EFV.

EjJect of TBNNI on substrate binding. The affinity constants for binding of TIP to RT were measured in the absence and the presence of saturating concentrations of TBNNI by gel-shift analysis. There were no significant differences seen in the RT-TIP mobility in the absence and presence of TBNNI. SpecificaIly, the affinity constants (.Ki) measured with respect to TIP-HIV-l RT binding were 200 nM in the absence ofNNRTI, 60 nM with UC781, 95 nM with CSIC and 120 nM in the case of EFV (data not shown).

OveraIl, there was a 2 to 3-fold decrease in .Ki with respect to aIl TBNNI. Therefore,

TBNNI, do not appear to significantly affect the affinity of the TIP for RT.

In order to measure the rate of single nucleotide incorporation in the absence of inhibitor, pre-steady state experiments with the use of a rapid quench apparatus were conducted.

The maximal rate of dCTP incorporation was 18s- 1 and the dCTP affinity constant (.Ki dCTP) was determined to be 3.4 f..I.M (Table 2.2). These values faIl within the range of previously reported measurements (Kati et al., 1992). Aiso consistent with what others

71 have demonstrated (Spence et al., 1995; Spence et al., 1996), we found that NVP slowed dCTP incorporation by 2x 103 -fold, compared to the control. Moreover, NVP increased

CTP the apparent affinity of RT-TIP for dCTP (Kl ) by 34-fold. This is in contrast to the

TBNNIs, UC781, CSIC and EFV, which only slightly altered dCTP affinity (5.6, 4.3 and

0.3 -fold, respectively), yet resulted in a dramatic inhibition of the rate of nucleotide incorporation (> 104 fold decrease in dCTP incorporation). Strikingly, UC781 and CSIC slowed down dCTP incorporation 30-fold more than NVP under drug-saturating conditions.

Evaluation of the rate of thio-dCTP incorporation. It has been suggested that for polymerases in general a 4 to 100-fold slowing of the rate of aS-dNTP incorporation, when compared to the natural dNTP substrate, is indicative of a rate limiting chemistry step (Benkovic, 1971; Herschlag et al., 1991). In the absence ofinhibitor, the rate ofaS dCTP single-nuc1eotide incorporation by HIV-l RT was the same as with the natural dCTP substrate (VdcTPN aS-dCTP - 1), as demonstrated previously by others (Reardon,

1992). The rate of incorporation of the a-thio analogue of dCTP was also measured in the presence ofUC781, EFV, CSIC and NVP. Similar to the no drug control, in the presence of saturating concentrations of TBNNI there was no decrease in the rate of polymerization with the thio analogue. As shown in Table 2.3 the elemental effects were

1.1, 1.2, 1.3 for CSIC, EFV and UC781, respectively. Surprisingly, there was no elemental effect observed with NVP.

72 Assessment of single nucleotide incorporation in the presence of homopolymeric lïP trap. The mechanism ofHIV-l RT RDDP activity follows an ordered mechanism where

TIP binds first and subsequently the dNTP binds to form a catalytically poised ternary complex (Kati et al., 1992; Reardon et al., 1993). The rate of TIP dissociation is in the

2 1 order of lxl0-4 to 5xlO- S-I while nucleotide incorporation is in the range of 2-80s-

(Reardon, 1992; Canard et al., 1998). In other words, the rate ofnucleotide incorporation

3 5 is lx10 to 8x10 - fold faster than TIP dissociation and thus TIP dissociation constitutes the rate-limiting step of the overall polymerization reaction under distributive conditions.

We assessed the extent to which the rate of nucleotide incorporation is affected by

NNRTI by using single nucleotide kinetics. In order to ensure that our experiments did not measure any effects of NNRTI on TIP dissociation, we used a homopolymeric trap, which binds to RT once the original heteroplolymeric substrate TIP dissociates and does not allow for rebinding of another TIP. Therefore, aIl polymerisation products observed are the result of aIl single polymerisation events that occurred before TIP dissociation.

Once TIP dissociation takes place, no more polymerisation can take place since RT is trapped. Theoretically, if an inhibitor could inhibit nucleotide incorporation enough so that is slower than TIP dissociation, no product would be observed.

Based on this rational, TIP was pre-incubated with RT to allow binary (RT-TIP) complex formation. Subsequently, dCTP was added together with an excess of homopolymeric poly(rA)-oligo(dT) TIP. In the absence of inhibitor 100% of the substrate was extended by one nucleotide, under these conditions, confirming that nucleotide incorporation is

73 much faster than TIP dissociation in the absence of inhibitor. In the presence of saturating concentration ofNVP more than 10% of the substrate was extended, indicating that catalysis is slowed down considerably compared to the control and that 90% of TIP dissociates before the next nucleotide can be added. Barely any product « 1%) was detectable with UC781, EFV or CSIC, indicating that catalysis was almost as slow as dissociation of TIP in their presence. Therefore, these tight-binding inhibitors appear to act as 'dead-end' inhibitors, which completely block polymerization once they are bound to HIV-I RT.

74 Table 2.1. Inhibition ofHIV-l RT by selected NNRTI

Compound ICso (nM)a (mean ± SD) ECso (nM)b (mean ± SD)

UC781 1.7± 1.2 1O± 2 CSIC 2.0 ±0.2 1.2 ±0.4 EFV 7.7± 0.9 1.7±0.2 NVP 290 ± 100 45 ± 10 a ICso values were determined with purified IDV-I RT using eH]dGTP and poly(rC) oligo(dG)I2-18 as TIP, as described under Materials and Methods. b ECso, 50% effective concentration. Inhibition of IDV-I replication in MT2 cells was determined by assessing syncytium formation.

75 Figure 2.1. Dependence of Kapp on the concentration of EFV (A), CSIC (B) or UC781

(C)

,----- 0.20 . 0.12 .-: l ! 0.24 A B C

0.15 .~ 0,09 .' ,,-.., .- 0,18 -i 1 C • E 0,06 0,10 -: -Cl.. Cl.. 0,12 ~(tJ

0.03 1 0,05 J 0,06

0,00 -+ 0.00 L_-,-, ,-,~-....-- 0.00 ,- 0 15 30 45 60 1 2 3 4 5 2 4 6 8 10 [efavirenz] (nM) [CSIC] (nM) [UC781] (nM)

The lines were calculated from the best fit of the parameters to either Equation 1.2 or 1.3 as described in Materials and Methods.

76 Table 2.2. Pre-steady state kinetic parameters ca1culated for HIV-l RT in the absence and presence of various NNR TI

Control 3.4 ± 1.0 1 18 ± 8 1 4 +CSIC 0.8 ± 0.2 4.3 3.2 ± 0.2 (x 10-4) 6 X 10 4 + UC781 0.6 ± 0.1 5.6 3.0 ± 0.1 (x 10-4) 6 X 10 3 4 +EFV 11 ± 1 0.3 1.8 ± 0.1 (x 10- ) 1 X 10 3 3 +NVP <0.1 <34 9.0 ± 1.0 (x 10- ) 2 X 10

The affinity ofHIV-l RT for dCTP was measured in the absence and in the presence of NNR TI (Kd dCTP) and in each case the maximal rate of dCTP incorporation was measured at saturating levels of substrate. The effect of NNR TI on the affinity of HIV-l RT to dCTP is reported as the ratio KdcontrollKdNNRTI , while the level of inhibition under single nucleotide incorporation conditions is reported as the ratio rateCont/ rateNNRT1. Values are the means oftwo separate experiments, each carried out in duplicate. a ratio ofthe KddCTP in the absence (control) versus in the presence ofNNRTI. b ratio of the maximum rate of nucleotide incorporation in the absence (control) and in the presence ofNNRTI.

77 Table 2.3. Rate of incorporation of dCTP and dCTP(aS) by NNRTI-inhibited RT-TIP complexes

Max rate dCTP (S-I) Max rate dCTP(aS) (S-I) Elemental effecta

Control 18 ± 8 Il ± 3 1.6 4 +CSIC 3.2 ± 0.2 (x 10- ) 2.9 ± 0.3 (x 10-4) 1.1 4 4 + UC781 3.0 ± 0.1 (x 10- ) 2.3 ± 0.1 (x 10- ) 1.3 3 3 +EFV 1.8 ± 0.1 (x 10- ) 1.5 ± 0.2 (x 10- ) 1.2 3 3 +NVP 9.0 ± 1.0 (x 10- ) 9.0 ± 0.8 (x 10- ) 1.0

Values are the means oftwo separate experiments, each carried out in duplicate. aratio of the rate of incorporation of dCTP over the rate of incorporation of dCTP( aS)

78 2.4. DISCUSSION

Despite the fact that UC781, CSIC and EFV constitute a group of chemically diverse

compounds, they are among the most potent NNRTI available to date. The

characterization of the interactions of such compounds with RT is key to understanding

their mechanism of action and developing a sense of what is required to develop NNR TI

with optimal potency (low IC 5o) and efficiency (maximallevel of inhibition). It has been

previously reported that UC781 and EFV are 'tight-binding' inhibitors of HIV-1 RT

(Barnard et al., 1997; Maga et al., 2000). We have confirmed these results and have

identified CSIC as a third TBNNI. In order to understand the relationship between the binding properties of NNRTI, their potency and their inhibitory efficiency, we have

conducted a complete and detailed kinetic comparison ofUC781, CSIC and EFV with the classical NNRTI NVP.

Unlike most classic kinetic analyses, Michaelis-Menten assumptions are not valid in the case of tight-binding inhibitors; double reciprocal and Dixon plots are not linear with

'tight-binding' inhibitors (see section 1.7). Nevertheless such studies have been used incorrectly for the estimation of the inhibition constants (KÜ of TBNNI (Maga et al.,

2000). The kinetic characterization oftight-binding inhibitors requires the use ofprogress curve analysis as previously demonstrated with UC781 (Barnard et al., 1997). In the present study, we have carried out the first direct comparison of the three identified

TBNNI, which has yielded further distinguishing characteristics among this group. In the case of UC781, the apparent rate of binding to HIV-RT (kapp) was linear with respect to inhibitor concentration; this corresponds to a mechanism where the drug associates

79 rapidly (diffusion controlled) in a single step and dissociates slowly (in the order of 10-4 s

I) (Morrison and Walsh, 1988; Barnard et al., 1997). In contrast, TIBO and NVP, rapid

4 4 equilibrium NNRTIs, bind (kon ~ 2 x 10 - Il X 10 M-1s-l) and dissociate (koff~ 2 x 10-4 -

1 4 X 10- S-I) rapidly (Spence et al., 1995). For EFV and CSIC the dependence of kapp with respect to drug concentration was hyperbolic, suggesting a two-step binding mechanism

(Morrison and Walsh, 1988; Cha, 1975). Once EFV and CSIC bind to the NNIBP, HIV-

1 RT undergoes a slow conformational change, an isomerization step which, presumably results in the rearrangement of the residues in the NNRTI binding pocket in a manner that accommodates EFV and CSIC more efficiently, thus resulting in tighter interactions. Our observations are in close agreement with a recent crystallographic report, which demonstrates that binding of EFV induces rearrangements in the NNIBP (Ren et al.,

2000). Such rearrangements have not been observed either with the UC781-RT (Ren et al., 1998) or NVP-RT (Kohlstaedt et al., 1992) crystal structures. Preliminary variable temperature NMR data suggest that UC781 is a flexible molecule, which exists both in trans and cis conformations around its thioamide bond. Therefore, the flexibility of

UC781 may make it easier for the NNIBP to accommodate its binding without necessitating dramatic side chain rearrangements (see Chapter 3). EFV and CSIC, whose structure appears to be more rigid, may therefore 'mold' the NNIBP around them, as has been previously suggested for other NNRTI (Kroeger-Smith et al., 1995). The

'reorganization' of the peripheral amino acids in the NNIBP around EFV and CSIC appears to be slow, yet is at least 8 times more energetically favourable (as inferred from the kisojorward / k iso. reverse ratio) than their arrangement upon initial collisional binding.

80 NNRTI bind to an allosteric site and the effects oftheir binding are somehow transmitted to the catalytic site, thus resulting in the inhibition of polymerization. Crystal structures do not offer conclusive explanations of how this is possible, since several different structural changes have been observed in and around the catalytic site, giving rise to three models (Sarafianos, 1998). However, such data have been substantially complimented by pre-steady state analyses which have shown that NVP inhibits reverse transcription by slowing down the chemistry step ofpolymerization (Spence et al., 1995).

Normally, in the absence of inhibitor, the RT-TIP complex binds dNTP, giving rise to

RT -TlP-dNTP, the first ternary complex. The latter undergoes a slow (rate Iimiting) conformational change, and this second ternary complex (RT-TIP-dNTP*) is poised to undergo virtually spontaneous catalysis (see section 1.3). In other words, RT-TIP-dNTP* is a short-lived transition intermediate in which the dNTP is bound tighter than in the RT

TIP-dNTP complex. Consequently, the dNTP affinity constant (:Ki dNTP), quantifiable by pre-steady state analysis, is a measure of the rate of formation of the initial ternary complex (RT-TIP-dNTP) and is in the range of 5-10 /.lM (Reardon, 1992~ Spence et al.,

1995). In the presence ofNVP, a dramatic (~ 35-fold) increase in affinity to dNTP has been interpreted as the consequence of the chemistry step becoming slower than the dNTP conformational change. In other words, when catalysis is slowed down by an

NNRTI the affinity of dNTP measured is with respect to the RT-TIP-dNTP* complex since under such conditions the latter has a much longer half-life. Although we have confirmed the se results with respect to NVP, as discussed below, we chose to use elemental effect analysis to further ascertain the validity of this interpretation. With

81 dNTP respect to CSIC, UC781 and EFV there was no significant change in KI . This observation is consistent with two independent interpretations. First, NNR TI do not slow down the chemistry step and the dNTP conformational step remains the slower of the two steps (see section 1.3). Second, chemistry may be significantly slowed down, yet the dNTP conformational step is slowed down even more by TBNNI. In order to acquire further cIues conceming the slowest step during nucleotide incorporation in the presence ofNNRTI we used elemental effect analysis as with NVP.

Elemental effect analysis has often been used in the study of various polymerases to distinguish between the rate limiting status of the chemistry and the conformationaI steps.

An elemental effect greater than 4 is indicative of a rate limiting chemistry step

(Benkovic, 1971; Herschlag et al., 1991; Reardon, 1992) as is the case with E.co/i polymerase (Klenow) and T7 DNA polymerase (Kuchta et al., 1987; Patel et al., 1991).

In the case of HIV-RT an elemental effect of approximately 1 has been previously reported, consistent with dNTP conformation being the slowest step (Reardon, 1992). We have confirmed this result and in addition we have found no elemental effect in the presence of NVP (Table 2.3.). The only other report which examined the mechanism of

NVP inhibition using pre-steady state kinetics (Spence et al., 1996) did not take the elementaI effect into consideration in interpreting the mechanism of NVP inhibition.

Therefore, based on the data on dNTP binding alone they speculated that catalysis itself becomes rate-limiting in the presence of NVP. Our resuIts suggest that even though chemistry may be significantly affected, the conformational change that precedes catalysis may actually remain slower in the presence of NVP. There was no elementaI

82 effect observed with any of the TBNNls (UC781, EFV, CSIC) examined either, which was consistent with the notion that TBNNI do not make the chemistry step slower than the dNTP conformational step.

In order to assess to what extent nucleotide incorporation (dNTP conformational and catalytic steps) was slowed down when compared to TIP dissociation we used steady state kinetics in the presence of an RT trap. The slowest step in the overall process of

IDV-I RT polymerization (lOOO-fold slower than the dNTP conformational change which precedes chemistry) is the dissociation ofTIP. Consequently, RT-TIP can undergo dNTP polymerization (conformational change and catalysis) before TIP dissociates from the enzyme. We examined whether TIP dissociation remains the rate-limiting step in the presence of NNRTI. In that case, complete polymerisation (100% product production) would be expected before TIP dissociation. In order to prevent rebinding of T/P and subsequent polymerisation, a homopolymeric trap, which binds RT tightly upon TIP dissociation, was used. This R T -trap, formed only once the TIP would dissociate, was unavailable for any further polymerization past that stage. Under these conditions, NVP, slowed down polymerization by 8,000-fold. Nevertheless, we detected significant residual activity, with approximately 10 % of the substrate being extended. In order for this polymerization to be observed, TIP dissociation had to remain the slowest step.

Therefore, despite the fact that classical NNRTI such as NVP appear to slow down both the chemistry and/or conformational steps of dNTP incorporation, TIP dissociation remains the slowest step of the overall polymerization process. In the case of TBNNI, there is no product detected in the presence of trap « 1%). Therefore, nucleotide

83 incorporation (conformation and/or chemistry) is significantly slowed down when

TBNNI are bound to RT, preventing any significant substrate extension before TIP dissociates. As a result, we conclude that TIP dissociation ceases to be rate limiting with

TBNNI.

One of the main differences between classical NNRTI and TBNNJ is in the extent at which they inhibit nucleotide incorporation once they are bound to the enzyme

( efficiency). Under drug saturating conditions, using single-nucleotide kinetics we measured a 20,000 to 40,000-fold decrease in the rate of polymerization with tight binding NNRTI, compared to a 2,000-fold decrease observed with NVP. In other words,

NVP is lOto 30 times less effective than the TBNNI in inhibiting polymerization.

Similarly, in the presence of trap, 10% of product was detected with NVP, while barely any polymerization « 1%) was detectable with UC781, EFV or CSle. Furthermore, under steady state processive conditions no polymerization product was detected for up to a period of one hour in the presence of saturating TBNNI concentrations, in contrast to

NVP (data not shown). TBNNI demonstrate maximal inhibitory efficiency while non tight-binding NNRTI such as NVP do not completely block polymerization. Therefore,

TBNNI can be considered true 'dead-end' inhibitors ofHIV-1 RT polymerization.

In summary, the effectiveness with which TBNNI inhibit HIV-I RT polymerization appears to be the result of two distinct components. First, TBNNI bind to HIV-l RT tightly, which results in the improvement of potency observed with UC781, CSIC and

EFV. Second, TBNNI are 'dead-end' inhibitor of DNA synthesis, showing the highest

84 possible inhibitory efficiency (> 99%). With non-tight-binding NNRTI, although the conformational and/or chemistry step is slowed down significantly, TIP dissociation remains rate limiting. On the contrary, TBNNI slow down nucleotide incorporation

( chemistry and/or dNTP conformational step) to the extent that it cannot occur before T IP dissociates. In light of the fact that NNR TI are increasingly being used in anti-IllV-l therapy, we propose that in the future development of other effective NNRTI these two critical parameters, 'tight-binding' and 'dead-end' inhibition, are taken into consideration.

85 CHAPTER 3: DEVELOPMENT OF RESISTANCE TO TIGHT-BINDING NNRTI

86 3.1. INTRODUCTION The development of effective anti-HIV agents has been seriously hampered by the rapid appearance of viral drug-resistance. HIV -1 R T is a very 'plastic' enzyme that is able to tolerate amino acid substitutions almost everywhere in its structure (Tantillo et al., 1994).

Moreover, HIV-l RT has low fidelity (Preston et al., 1988) and lacks a proofreading mechanism (Roberts et al., 1988). Consequently, under the continuous presence of any

NNR TI, even when used in highly active antiretroviral therapy (HAAR T) drug combinations, HIV -1 resistant variants appear rapidly both in cell culture and in patients

(Larder et al., 1999).

The development of compounds to which the appearance of resistance is delayed (or ideally does not occur at aIl) constitutes an immense challenge. NNRTI that form a greater number of stronger interactions, primarily with highly conserved residues in the

NNIBP, could theoretically be resilient to HIV-l resistance (Pelemans et al., 2000).

TBNNI such as UC781 appear to interact in an exceptionally tight manner with the

NNIBP (Barnard et al., 1997). Here we report that a single substitution has little effect on the inhibitory potency of TBNNI. Such NNRTI constitute promising candidates for the future development of improved antiretroviral therapy. As described previously, three

'tight-binding' inhibitors of HIV-l RT have been identified to date: UC781, EFV and

CSIC (Barnard et al., 1997; Maga et al., 2000). In this chapter we show data that confirms that TBNNI show excellent inhibitory potencies in cell culture (Table 2.1.). We also show that development ofresistance to UC781 is slow when compared to non-tight- binding NNRTI such as UC84 and NVP. Our data suggest that this delay is due to the fact that at least two substitutions are required in order to achieve high-Ievel resistance to

87 UC781, CSIC and EFV as previously suggested (Ren et al., 2000). This is in contrast to what we observed with other NNRTI such as NVP and DLV where inhibitory activity is significantly impaired in the presence of a single NNIBP substitution (Schinazi et al.,

1997). The data presented below, which cornes from cell culture, recombinant RT and molecular modeling experiments, has helped us identify several important interactions between TBNNI and the residues in the NNIBP described below. This knowledge may prove valuable in the design of novel TBNNI with increased resilience to muItidrug resistance.

3.2. MATE RIALS AND METHODS The laboratory viral strain HIV -1 HIB was obtained from NIH AIDS Research and

Reference Reagent Program, Division of AIDS, NIAID (Washington, DC, USA) and courtesy of Dr. R. C. Gallo. The CD4 expressing MT2 lymphocyte cell tine was purchased from American Type Culture Collection (Rockville, MD, USA). The carboxanilide NNRTIs, as mentioned before in this thesis, were provided by Dr. W.G.

Brouwer and Uniroyal Chemical Ltd. Research Laboratories (Guelph, ON, Canada).

Media for aIl cell culture experiments (RPMI 1640) and fetal bovine serum (FBS) were from Canadian Life Technologies, GIBCO (Toronto, ON, Canada). Primers were also from GIBCO. The homopolymeric template/primer (TIP) poly(rC)-0Iigo(dG)12_18 and

CH]dGTP, as weIl as the T7 Sequencing Kit and the bacterial plasmid used for the cloning ofHIV-l RT, pKK223-3, were aIl obtained from Amersham Pharmacia Biotech

(Montreal, QC, Canada). Taq polymerase and the lnsT/Aclone PCR Cloning Kit were available at MBl Fermentas (Flamborough, ON, Canada). Lastly, both DNA purification

88 kits, QIAamp DNA Mini Kit and QIAGEN Plasmid Purification Kit, were from

QIAGEN (Mississauga, ON, Canada)

Cell culture and virus replication. AlI celI culture experiments were carried out under conditions identical to these described in the material and methods of Chapter 2. Briefly,

CD4-expressing MT2 lymphocytes were cultured in RPMI containing FBS. Viral stocks were prepared by collecting the supematant from infected MT2 cells. The viral infectivity of each stock was assessed (TCID50) and the appropriate multiplicity of infection (M.O.I.

~ 0.01) was used in each experiment. AU drug stock solutions were prepared in dimethyl sulfoxide (DMSO), the final concentration of which in all solutions did not exceed 1%.

Inhibition of infection was examined by incubating MT2 cells with different concentrations of drug. The 50% effective drug concentration (EC50) was assessed when the no-drug control showed 100% cytopathic effect (CPE), 4 to 5 days post-infection.

Infection of MT2 lymphocytes was assessed by observation of syncytia formation, p24 elisa and R T activity assays in duplicate samplesas previously described.

Development of resistance to NNRTI in cell culture. MT2 ceUs (3 x 105 cells/l ml of media) were incubated with sub-IC5o or IC 50 concentrations of NNRTI and with the 5 appropriate infectious dose of IllV-lIIIB (5x10 TCIDso) for a period of 3 h at 37°C. The cells were then washed in order to remove any residual virus and were recultured in the presence of the same concentration of drug as before. The development of infection was observed on a daily basis and the media was changed every 3 to 4 days. In cases were there was no, or low, infection observed the drug concentration used in the fresh media

89 remained unchanged. When 70% syncytia was observed, 250 /-11 of the clarified virus containing supernatant was used to infect a new uninfected MT2 population (750 /-11, 3 x

105 cells). The final drug concentration in each one of these cycles was increased until there was no infection observed during a long incubation period or until the drug concentrations were beginning to affect cell growth. Samples of the viral populations from each cycle were collected as cell-free supernatant and stored at -80°C for further characterization.

Identification ofmutations involved in NNRTI resistance. The viral sample of interest was used to infect 5 x 106 MT2 cells in 10 ml of media. When the cells were 50% infected they where spun down to remove the virus-containing supernatant and the QIAamp DNA purification kit was utilized to extract the genomic DNA of these cells. The R T sequence of the integrated provirus was amplified by PCR using Taq polymerase and the following forward and reverse primers: 5' -CTG AA T TCA TGC CCA TT A GCC CTA TTG AG-3' and 5' -CTA AGC TTA CTA TAG TAT TTT CCT GAT TCC AG-3'. The PCR product was cloned into a vector using the InsT/Aclone PCR Cloning Kit and the newly ligated plasmid was then used to transform JM109 E.coli bacteria. The presence of the insert was verified by restriction digestion of purified plasmids from ten colonies. Six of the positive clones were sequenced with respect to resistance for each NNR TI. Several primers corresponding to various regions of the RT sequence were used to read the whole RT gene.

90 Site-directed mutagenesis. AIl recombinant R T mutants were constructed using the Muta gene M13 In vitro Mutagenesis Kit from BIO-RAD (Richmond, CA, USA). The wt RT gene was cloned into an M13 vector and the appropriate oligonucleotides were used to introduce single and double nucleotide alterations in the RT gene. The presence ofthese mutations in M13 were verified by sequencing as described above and a positive clone was used to subclone the mutant RT gene into the bacterial plasmid pKK223-3. This plasmid was then used to transform JM109 bacteria and the heterodimeric p66/p51 RT protein was expressed and purified as previously described (Fletcher et al., 1996). The inhibitory potency (IC50) for aIl mutant enzymes was assessed by using homopolymeric

TIP poly(rC)-oligo(dG)12_18 and eH]dGTP in a procedure also described in detail in

Chapter 2.

Mo/ecu/ar modeling of TBNNI in RT. The coordinates of the X-ray crystal structure of

RT-(cis-UC781) (Ren et al., 1998; protein data bank entry Irt4) were used as the basis for the construction of the model of the RT -(trans-UC781) complex. In order to obtain the trans form of the inhibitor, the C-N bond of the thioamide of UC781 was rotated by

176.8°. The UC781 was docked into the RT structure and the energy of the overall structure was minimized using Sybyl 6.5 (Tripos Inc., St. Louis, MO, USA). The

Gastreiger-Huckel method was used to calculate alI charges while the Tripos force field was used to minimize the structure until the energy between iterations differed by less than 0.01 kcal/ mol! Â. The structures ofCSIC and EFV were also docked in the NNIBP of RT based on the RT-(cis-UC78I) crystal structure and were minimized as described above for the trans conformer ofUC781.

91 3.3. RESULTS Selection of TBNNI-resistant viruses. The two carboxanilides UC84 and UC38, although chemically and structurally similar to UC781, have 30 and 6-fold lower antiviral potencies than the latter, respectively (Borkow et al., 1997). The development of resistance to UC84 and UC38 (Figure 3.1), similar to non-tight-binding NNRTI NVP

[Borkow et al., unpublished data] was relatively rapid, within 40 days. On the other hand, resistance to UC781 (Figure 3.1) and CSIC (data not shown) developed slower, that is, in

55 days. In aIl cases, the resistant viruses had IC50s that were > 250 the IC50s of the wt virus. Sequencing of UC84- and UC38-resistant HIV-l revealed a single RT mutation present in each case, V106A and LlOOI, respectively. Mutagenic analysis of UC781- resistant virus revealed the presence of various sets of mutations in the HIV -1 R T gene.

These included KI03T/V106A (Figure 3.1), V179D/Y181C, VI79D!E138K, and

V179D!E138K1YI81C. The V1081 mutation was also observed in combination with the latter three groups of R T mutations. With CSIC aIl clones screened carried the

L 100I/K 103N set of mutations.

Determination ofTBNNI viral cross-resistance. UC84-resistant virus was also resistant to

UC38 and vice versa, UC38-resistant HIV-l was resistant to UC84. However, UC781 was effective against both UC84- and UC38-resistant viruses, while the UC781-resistant virus was completely resilient to both UC38 and UC84 inhibition (data not shown).

Interestingly, CSIC and EFV were moderately effective « 200-fold resistance) against

UC781-resistant virus compared to wt type HIV-l (Table 3.2). FinaIly, CSIC-resistant

92 virus was highly resistant against aIl, tight-binding (UC781, EFV) and non-tight-binding

(UC38, UC84), NNRTI « 500-fold).

Determination of the level of NNRTI-resistance conferred by single and double recombinant HIV-l RI' mutants. The K103T and V106A mutations, which were found to appear with resistance to UC781, were introduced in recombinant HIV-l RT both alone and in combination (K103TNI06A). UC781 and structurally similar, but not tight binding, UC38 were tested against all three recombinant enzymes. As shown in Table 3.1 the potency of UC781 was only slightly altered in the enzyme with single substitutions

(4.5- and 7-fold resistance with K103T or V106A mutants, respectively). In contrast, the double mutant (K103TN106A) induced a high level of resistance to UC781 (207-fold), while UC38 was ineffective against any of the mutant enzymes.

Moreover, the inhibitory potency of UC38 was highly compromised by the presence of other single or double substitutions in the NNlBP that are se en with resistance to various

NNRTI (30 to 160-fold). This was not the case with UC781 where inhibitory effectiveness was comparable to that seen with wt HIV-I RT against aIl single and double mutants with the exception of KI03TN106A (Table 3.1). Specifically, UC781 was active against single mutants Y18IC and Y188C, which are involved in cross resistance to most classical NNRTI. Furthermore, the P225HN106A substitution containing virus involved in resistance to the quinoxaIinethione S-2720, was inhibited by

UC781 (Pelemans et al., 1997).

93 Importantly, CSIC and EFV effectively inhibited the entire spectrum of mutants shown in

Table 3.1, including the double mutant K103N106A that is resistant to UC781 (data not shown).

Modeling ofNNRTI in HIV-l RT. Although a crystal structure ofUC781 complexed with

RT has been pub li shed (Ren et al.; 1998), our modeling studies have led to data that are in closer agreement with the biochemical and drug-resistance profiles available. In the crystal structure UC781 is bound to the NNIBP in its cis conformation. However, our docking experiments to the same RT molecule have revealed that the trans form of this inhibitor about its thioamide bond can also be accommodated in the NNR TI binding pocket.

Nevertheless, there are sorne common interactions observed in both the cis (Fig. 3.3) and the trans (Fig. 3.4) structures. For instance the strong hydrogen bond between the thioamide portion of UC781 and the backbone of residue K 10 1 is always observed. The majority of aIl other interactions are hydrophobic in nature. The furan ring of UC781 interacts with residue V179 and its phenyl ring interacts with residue F227 and the backbone of L234. Lastly, the phenyl ring of UC781 seems to interact with the highly conserved amino acid Y318 (Pelemans et al., 2000) in both conformers.

However, there are sorne important interactions that are unique to the trans-UC781 model

(Fig. 3.4). Specifically, only in the trans model does the furan ring interact with the side chains of residues E138 and K103. Moreover, side chain P225 interacts with the phenyl

94 moiety of trans-UC781 and residues Y181, W229 and L234 make strong interactions with the pentenyl side chain of only trans-UC781. Residues W229 and L234 are highly conserved amino acids and have never appeared mutated in any resistant viral strains

(Esnouf et al., 1997; Pelemans et al., 2000).

Based on the same UC78I crystal structure, we modeled CSIC in the NNIBP of HIV-I

RT (Fig. 3.5). Unlike UC781, CSIC is a relatively rigid structure with no significant rotation around its structure and thus, has only one spatial conformer. Our modeling shows CSIC to interact with residues L100 and K103. Specifically, L100 is in close contact with the indole/carboxamide moiety of CSIC, while K103 interacts with its sulfonyl portion. Further, the sulfonyl side chain of the CSIC molecule interacts with the backbone of residue KIOl. The backbones of amino acids E138, Y181 and I180 are involved in the binding of the phenyl moiety ofCSIC.

The modeling of EFV in the NNIBP of the RT crystal structure showed several similar interactions to these se en with CSIC (Fig. 3.6). The phenyl moiety of EFV interacts with the backbone of residues E138, I180 and Y181 (weakly), while the cyclohexenyl group shows extensive interactions with the side chain ofL100. The cyclopropyl group ofEFV makes contacts with the side chains of VI06 and F227 while the trifluoro portion interacts mainly with the side chain of K 103 and partly with the side chain of V 179 and the backbone of K 10 1.

95 Table 3.1. Inhibitory potency of UC781 and UC38 against various NNRTI recombinant RT mutants

HIV-l RT ICso (nM)

UC38 Fold resistance UC781 Fold resistance

WT 30 ± 10 1.7±1.2 K103T 4600 ± 640 150 12.2 ± 1.2 7 VI06A 3300 ± 850 110 7.6 ±4.2 4.5 K103TNI06A 5000 ± 1200 160 352.5 ± 43.0 207 E138K 1000 ± 200 30 12.5 ± 2 7 Y181C 2600 ± 500 87 11.6 ± 3.4 7 Y188H 1700 ± 140 57 6.2 ± 1.0 4 P225H 810 ± 260 27 2.7 ± 0.4 1.6 VI06NP225H 5000 ± 800 160 39.1 ± 16.5 23

The concentrations at which the RNA-dependent DNA polymerase activity of the wt and various recombinant RT mutants is inhibited by 50% (IC so) were determined for UC38, a non-tight-binding carboxanilide, and UC781, a TBNNI. Although there is a > 30-fold increase in UC38 ICso in aIl cases, only the KI03TNI06A shows high-Ievel resistance to UC781. AIl values are averages and standard deviations of the mean were calculated from three or more independent measurements each carried out in duplicate.

96 Figure 3.1. Development ofresistance to UC84, UC38 and UC781

250

of?,--- 200· x ""-" 150 ..

100 -J

50 -

o o 10 20 30 40 50 Time (days)

Resistance to the classical UC carboxanilides, UC84 C-) and UC 38 C.) develops rapidly, while resistance to TBNNI UC781 ce) is significantly delayed.

97 Table 3.2. Cross-resistance profiles of UC781, CSIC and EFV with UC781 and CSIC resistant viruses

NNRTI wt HIV-l CSIC-resistant HIV-l UC78l-resistant HIV-l

ECso ECso resistance ECso resistance (nM) (nM) (fold) (nM) (fold)

UC781 10 ± 2 >5000 >500 >4400 >440 CSIC 1.2±0.4 >2000 >1700 183± 96 Il EFV 1.7 ± 0.2 >5000 >2900 345± 95 203

UC781-viruses were highly resistant to UC781 but not to CSIC and EPY. On the contrary, CSIC-resistant virus was resistant to aU three TBNNI.

98 Figure 3.3. Interactions of cÎs-UC781 with the residues of the NNRTI binding pocket

179

Model of the RT-(cis-UC781) complex based on the available crystal structure (top). A two-dimensional (2-D) representation of the interactions involved in cis-UC781 binding is also shown below (bottom). Residues shown are aIl in a 4 Â radius from the inhibitor molecule and interactions (broken tines) are shown only for distances that are less than 3.7 Â.

99 Figure 3.4. Interactions oftrans-UC781 with the residues of the NNRTI binding pocket

iIm[jf.L88

Model of the RT-(trans-UC781) complex based on the available RT-UC781 crystal structure (top) (Ren et al., 1998). A two-dimensional (2-D) representation of the interactions involved in trans-UC781 binding is also shown below (bottom). Residues shown are aH in a 4 Â radius from the inhibitor molecule and interactions (broken hnes) are shown only for distances that are less than 3.7 Â.

100 Figure 3.5. Interactions ofCSIC with the residues of the NNRTI binding pocket

Model of the RT-CSIC complex based on the available RT-UC781 crystal structure (top). A two-dimensional (2-D) representation of the interactions involved in CSIC binding is also shown below (bottom). Residues shown are aIl in a 4 Â radius from the inhibitor molecule and interactions (broken lines) are shown only for distances that are less than 3.7 Â.

101 Figure 3.6. Interactions ofEFV with the residues of the NNRTI binding pocket

100

Model of the RT-EFV complex based on the available RT-UC781 crystal structure (top). A two-dimensional (2-D) representation of the interactions involved in EFV binding is also shown below (bottom). Residues shown are aIl in a 4 A radius from the inhibitor molecule and interactions (broken lines) are shown only for distances that are less than 3.7 A.

102 3.4. DISCUSSION The rapid development of resistance to nonnucleoside monotherapy has restricted their use in multidrug remedies. Even though NNR TI are increasingly being used in highly active antiretroviral therapy (HAART) for the treatment of HIV-l infected patients, the development of resistance through the long-term use of these combination therapies has been inevitable (Geretti et al., 2001; Violin et al., 2002; Seifert 2002). Our understanding of the mechanism by which CUITent therapies fail will propel the rational design of new

NNRTI, which may overcome such limitations.

Resistance to NVP and DL V develops due to the appearance of a single substitution

(Schinazi et al., 1997). Corn mon substitutions include Y181C, Y188H and K103N.

Furthermore, we, like other groups, have found that experimental NNRTI such as UC84 and UC34 also require just a single substitution such as V106A, E138K caalzarini et al.,

1995b) and LlOOI for high-Ievel resistance. On the other hand, newer NNRTI such as

EFV require multiple substitutions for high-Ievel resistance (Young et al., 1995) and have been termed "second generation NNRTI" (De Clercq, 1999). Moreover, UC781, an experimental second generation NNRTI, has been previously shown to be effective against single NNR TI mutants (Balzarini et al., 1996b, Balzarini et al., 1996c). Our results from recombinant mV-l RT experiments suggest that aH three TBNNI (EFV,

UC781 and CSIC) are indeed effective against single NNffiP mutants, which are seen with resistance to a wide range of experimental and clinical NNRTI. Our recombinant RT data, in combination with our sequencing analysis, strongly support the concept that at least a double mutant is necessary to significantly reduce the inhibitory activity of UC781 in vitro. Multiple substitutions have been observed in CSIC-resistant virus, while similar

103 results have been reported previously for EFV (Young et al., 1995). The requirement for two or more mutations translates to a delay (> 15 days) in the development of resistance to UC781, compared to UC84, UC38 or NVP. The identification of other 'tight-binding' inhibitors will likely result in therapeutic alternatives to which the development of resistance is slower than observed with CUITent NNR TI containing remedies.

We have identified a senes of different mutational combinations in highly UC781- resistant viruses: K103TNI06A, E138KN179D and E138K.1YI81C as well as combinations of the latter two mutations with V108I. Moreover, Buckheit and his group

(1997b) have previously reported mutation sets KlO1IV108I1Y181C while Balzarini et al.

(1998) have found V106A1F227L and KlOOIIV106A1F227L to be present in UC781- resistant strains. Therefore, more than one set of at least two substitutions in the NNIBP appears to confer high-level resistance to UC781. We suspect that this is a consequence of the flexibility of the UC781 molecule. NMR studies suggest that both the trans and cis forms around the thioamide C-N bond of UC781, exist [Borkow, G. et al., unpublished data]. Although, the published crystal structure shows UC781 bound in its cisoid conformation (Ren et al., 1998), the interactions deduced from this structure do not adequately explain the biochemical and resistance data available. Our molecular modeling studies indicate that the NNIBP can accommodate the transoid conformer and that a number of additional important interactions are observed in the trans-UC78 1 model when compared to the cis-form. Only in the trans model are significant interactions with residues K103 and E138 observed. The latter residues would be expected to play a key role in the binding of UC781 since they are mutated in UC781 resistant viral enzyme.

104 Moreover, recombinant single KI03T and E138K RT mutants showed the highest level of resistance observed with a single substitution (7-fold). Therefore, we suggest that both conformers of UC781 bind in the enzyme thus resulting in the complex resistance profiles reported above.

There are several factors that may contribute to the delayed development of resistance se en with second generation NNRTI. UC781 and EFV interact with all three mechanistic forms of the enzyme (RT, RT-TIP and RT-TIP-dNTP; see section 1.3) (Barnard et al.,

1997; Maga et al., 2000), unlike other c1assical NNRTIs such as UC84, which binds to free RT and the binary complex, and UC38, which interacts with only the ternary RT form (Fletcher et al., 1995a). Although crystallographic data suggest that UC84 and

UC38 bind to RT in their cisoid forms (Ren et al., 1998), NMR studies indicate that the most energetically favourable configuration is cis for UC38 but that with UC84 the trans configuration is the most likely structure (Dmitrienko et al., unpublished data). The fact that UC84 and UC38 interact with different RT mechanistic forms implies that the conformation of the carboxanilides may be important in determining to which mechanistic formes) they bind. The fact that UC781 interacts with all three mechanistic forms (see section 1.3) suggests that this results from its ability to bind as both a cis and trans isomer. Overall, binding to multiple enzyme mechanistic forms may contribute to the tight-binding nature of EFV and UC781 and the sustained activity observed in the presence of NNRTI substitutions. Similar experiments with CSIC will be useful in establishing this hypothesis.

105 Furthennore, there appears to be a fine balance between the size of the NNR TI molecule and its ability to retain inhibitory activity in the presence of a substitution in the NNIBP.

UC84 and UC38 are smaller than UC781, which allows for more rotation and fewer interactions in the pocket. This 'loose fit' ofUC84 and UC38 probably contributes to the dramatic loss of binding with single substitutions. Based on the published crystal structure, the anilide rings of UC38 and UC84 are closer to residues Y181 and Y188, which could explain the reduced sensitivity of the Y181C and Y188L mutants to these

UC NNRTI compared to UC781 (Ren et al., 1998). Moreover, the furanyl ring ofUC781 is more flexible than the oxathiin moiety ofUC84, which would allow for the rearranged binding of UC781 in the presence of single substitutions. On the other hand, it has been suggested that the reason that EFV is effective against K103N is its smaller size compared to NVP (Ren et al., 2000). EFV is able to move deeper into the pocket of the mutant K103N and bind differently yet still maintain a tight association in comparison to the wt type enzyme (Ren et al., 2000; Lindberg et al., 2001). It is important to note that

EFV is a relatively rigid molecule in contrast to UC781. Therefore, in order for an

NNR TI to be able to bind to a modified NNR TI pocket it has to be able to accommodate itself in the pocket, either by altering its own structure or through its small size. Large molecules such as NVP, which interact strongly with residues Y181 and Y188, are therefore, very susceptible to minor facile changes in the pocket (Ren et al., 2000; Ren et al., 2001). Altematively, UC781 possesses flexibility, which enables it to fit into the

'mold' of the mutated NNRTI pocket. UC84 and UC38, although smaller than UC781, are less flexible, while they make significant interactions with residues Y181 and Y188.

Such interactions appear to be to the detriment of these NNRTI's inhibitory potency in

106 the presence of single substitutions (Ren et al., 2001). CSIC, like EFV, is a small, rigid structure with the same set of mutants observed with resistance (K103N, LlOOI). There have been no CSIC-HIV-l RT crystal structures published, but based on our modeling studies, CSIC is effective against single NNRTI-resistant mutants because ofits ability to mold the pocket around its structure, as seen with EFV (Ren et al., 2000; Linberg et al.,

2002). The ability of CSIC, like EFV, to rearrange the pocket around its structure is consistent with our observation that a slow conformational change occurs upon binding of the TBNNI to HIV-l RT, which leads to a tighter E-I complex (see Chapter 2).

Our modeling of EFV in the NNRTI pocket shows several similarities and differences with the recently published crystal structure ofEFV with HIV-l RT (Ren. et al., 2000;

Linberg et al., 2002). In both cases, interactions with the backbone of residue K 10 1 and side chains ofresidues K103 and L100 were observed. However, in the EFV-RT crystal structure aIl these interactions are seen with the benzoxazin portion of EFV, while in the model discussed here the trifluoromethyl group makes significant contributions to this binding. In the model aIl these contacts are within a 3.7 A radius.

In addition, it has been suggested that drug interactions with highly conserved amino acids may also contribute to the delay in the development of resistance. UC781 interacts hydrophobically with highly conserved residue W229 as se en in other modeling studies

(Esnouf et al., 1997; Pelemans et al., 2000). Moreover, both UC781 and EFV appear to interact with the highly conserved amino acid Y318 (Pelemans et al., 2000). Therefore,

107 focusing on interactions with highly conserved amino acids such as W229 and Y318 constitutes a rational goal for the development ofnon-resistant NNRTI interactions.

Interestingly, UC781-resistant mutants do not dramatically affect the activity of the two other TBNNI, CSIC and EFV. As suggested above, CSIC and EFV are able to bind deeper in the mutated NNIBP pocket forming new interactions. Nevertheless, it is remarkable that CSIC and EFV retain moderate inhibitory potency even in the presence of multiple substitutions such as V179DIE138K1YI81C, which would normally abolish the activity of most available NNRTI. In contrast, UC781 is inactive against the CSIC resistant virus indicating that the combination of substitutions LlOOI and K103N substantially interferes with its binding. Resistance to UC781 caused by the se latter substitutions may be due to either the direct loss of important interactions or due to the distortion in the NNIBP. Expectedly, CSIC-resistant virus is cross-resistant to EFV, since they share the same resistance profiles. It is noteworthy that in patients a different set of mutations has been observed with EFV, specifically, K103N, V1081 and P225H (Miller et al., 1998). The reason behind this variation remains unc1ear.

The differences in the resistance and cross-resistance profiles, taken together with the differences in the mode of binding, distinguish between two different types of TBNNI: one that includes flexible, rapid binding TBNNI, such as UC781, and another that encompasses more rigid, smaller molecules, that mold the NNIBP around them through a conformational change, such as CSIC and EFV. AIl TBNNl show a delay in the development of resistance, a property that is specific to select 'second generation'

108 NNRTI (De Clercq, 1999). In summary, the development ofnewNNRTI should focus on compounds that bind to aIl mechanistic forms of HIV -1 R T and whose binding does not depend as much on easily mutable residues such as Y181 and Y188. Instead binding to more conserved residues such as W229 and Y318 would be expected to result in NNRTI with greater resilience to resistance. Lastly, screening ofnew NNRTI should test for their ability to either mold their structure within the NNIBP or, altematively, mold the structures surrounding them in order to optimize their binding. The tlexibility by which

TBNNI interact with the NNIBP appears to be an important reason behind their prolonged efficacy. Whether slow TBNNI show greater resilience to the development of resistance than rapid TBNNI, or vice versa, remains to be determined.

109 CHAPTER 4: MECHANISM OF HIV-l RESISTANCE TO TIGHT-BINDING

NNRTI: STUDIES WITH UC781

110 4.1. INTRODUCTION Even though only three NNRTI are currently approved as therapeutics for the clinical treatment of HIV-l infection (NVP, EFV, DEL), a much larger number of nonnucleosides have been identified in the laboratory (see section 1.4.2). Among the most extensively characterized NNRTI are the UC series of carboxanilide derivatives

(Bader et al., 1991; Balzarini et al., 1995b; Fletcher et al., 1995a; Fletcher et al., 1995b;

Balzarini et al., 1996; Barnard et al., 1997; Ren et al., 1998; Hahn et al., 2000). The carboxanilide NNRTI possess a number of interesting R T inhibitory and HIV -1 antiviral properties. For example, certain UC NNRTI bind selectively to different RT mechanistic forms (Fletcher et al., 1995a), and combinations ofthese provide synergistic inhibition of

HIV-l RT polymerization (Fletcher et al., 1995b). Continued development of the UC carboxanilide series has resulted in UC781, an exceptionally potent inhibitor and a 'tight- binding' inhibitor ofHIV-l RT (Barnard et al., 1997), a property that is, to date, unique among the NNRTI. In addition, crystal structures of free RT with several UC NNRTI, including UC781, have been published (Ren et al., 1998).

Despite the successful identification of several potent NNRTI, the rapid development of viral drug-resistance discussed in Chapter 3 is a serious concern. Resistance to NNRTI correlates with substitutions in the NNIBP. Single point substitutions in NNIBP residues such as K103N, Y181C, etc. result in high-Ievel resistance to most NNRTI (Tantillo et al., 1994), including many of the ue carboxanilides (Balzarini et al., 1996b; Buckheit, Jr. et al., 1995). However, UC781 is active against NVP-resistant HIV-l (Balzarini et aL,

1996b). The development of HIV -1 resistance to UC781 in vitro is significantly delayed when cornpared to NVP and other non-tight-binding UC carboxanilide NNRTI (Chapter

III 3). We have chosen one out of the several UC781-resistant genotypes we have identified, the double mutant K103T N106A, in order to identify the phenotypic changes that are associated with resistance to this TBNNI.

We compared the recombinant wt RT enzyme with the single K103T or V106A RT mutants, and with RT containing the K103T/VI06A double substitution. The results described below show that the introduction of one and then two substitutions into the RT

NNIBP results in a sequential elimination of specific inhibitory properties of UC781, thereby leading to high-Ievel resistance to this compound.

4.2. MATERIALS AND METBODS The carboxanilide NNRTI UC38 and UC781 were provided by Dr. W. G. Brouwer

(Uniroyal Chemical Ltd. Research Laboratories, Guelph, ON, Canada). Ultrapure dNTP, eH]dGTP and homopolymeric template/primer (TIP) poly(rC)-oIigo(dG)12_18 were from

Amersham Pharmacia Biotech (Montreal, QC, Canada).

Site-specific mutagenesis, expression and purification of wt and mutant HIV -1 R T were carried out as previously described (Fletcher et al., 1996). Briefly, the K103T, V106A and K103TNI06A substitutions were introduced into the HIV-l RT gene using the

Muta-Gene M13 In Vitro Mutagenesis System (BIO-RAD, Richmond, CA) with appropriate oligonucleotide primers. Mutant clones were verified by DNA sequencing.

The mutated RT genes were then subcloned into expression vector pKK223-3

(Amersham-Pharmacia Biotech) and used to transform E.co/i JM109. Heterodimeric p66/p51 recombinant HIV -1 R T proteins were purified as previously described (Fletcher

112 et al., 1996). The RNA-dependent DNA polymerase (RDDP) activity ofpurified RT was assayed using the homopolymeric TIP poly(rC)-0Iigo(dG)12-18 and eH]dGTP, as described (Fletcher et al., 1995a; Fletcher et al., 1995b; Barnard et al., 1997). No significant differences in either kcat or Km for either dNTP or TIP were noted among the recombinant RT enzymes used in this study in the absence ofinhibitor.

Characterization of NNRTI inhibition of HIV-l RT. RT RDDP activity was measured using the same reaction assay conditions as described above, with the addition of varying concentrations of NNRTI. The latter were prepared as stock solutions in dimethyl sulfoxide (DMSO), and added to the reaction mixtures prior to the addition of RT in a volume such that the final concentration of DMSO did not exceed 1%. Assays to characterize the kinetics of UC781 inhibition of K103TN106A RT with respect to TIP were carried out using 24 f..lM dGTP and variable poly(rC)-oligo(dG)12-18 (ranging from

0.5 to 10 x Km, Km ~ Il nM). Similarly, assays to characterize the kinetics of UC781 inhibition ofK103TN106A RT with respect to dNTP substrate were carried out using 33 nM poly(rC)-oligo(dG)12-18 TIP and variable concentrations of dGTP (ranging from 0.5 to

10 x Km, Km- 8 f..lM).

Assay of HIV-l RT polymerisation under saturating drug concentrations. RT RDDP assays used saturating levels of the dNTP substrate eH] dGTP (50 f..lM dGTP) and the

TIP poly(rC)-0Iigo(dG)12_18 (500 nM). The RT concentration was fixed at 20 nM of p66/p51 heterodimer. The NNRTI concentrations for 'complete' inhibition ofRT activity were determined empirically in control experiments by titrating RT with increasing

113 concentrations of inhibitor until no further change in RT activity was note d, or until no

RT activity remained. In aIl cases, the 'plateau' value for maximal inhibition was in the range of 250 x IC so. To ensure complete saturation, we used inhibitor concentrations equal to 1500 x IC so . This corresponded to 50 /-lM for UC38 and 3 /-lM for UC781. RT was pre-incubated with TIP and NNRTI inhibitor for 10 min before initiation of DNA synthesis by the addition of dGTP. Aliquots (10 /-lI) were collected at intervals of one minute (in the absence ofNNRTI) or 10 minutes (in the presence ofNNRTI). In aIl cases, product formation was linear over the time periods used, and the rates of polymerisation were calculated from these data.

Analysis oftight-binding inhibition. 'Tight-binding' inhibition was analysed as previously described (Barnard et al., 1997). Briefly, the kinetics of RT-catalyzed DNA polymerisation product formation was determined at a number of UC781 concentrations.

Product formation was analysed using Equation 1.1. The dependence of kapp on UC781 concentration was fitted to the linear Equation 1.2. This corresponds to a one step mechanism for the binding of inhibitor to the enzyme (Morrison and Walsh, 1988; Cha,

1975) that we have previously shown applies to UC781 inhibition ofHIV-l RT (Barnard et al., 1997).

Characterization of UC781 binding ta different RT mechanistic forms by fluorescence spectroscopy. The change in steady state intrinsic prote in fluorescence of different RT mechanistic forms due to inhibitor binding was measured using a PTI C61 QuantaMaster

T-format spectrofluorometer, with excitation at 280 nm and emission scanned between

114 300 and 400 nm. All measurements were carried out at 20°e. Samples contained 100 nM

RT in 50 mM Tris-HC1 (pH 7.8, 20°C) containing 60 f.lM KCl and 10 f.lM MgCh. In measurements involving interaction ofNNRTI with the RT-TIP binary complex, RT was pre-incubated with 500 nM of a heteropolymeric 30-nt template annealed with a 19-nt

DNA primer possessing 2',3' -dideoxycytidine monophosphate as the 3' -terminal nucleotide, prior to titration with NNRTI. In measurements involving interaction of

NNRTI with the RT-TIP-dNTP temary complex, RT was pre-incubated with 500 nM ofa heteropolyrneric 30-nt template annealed with a 19-nt DNA primer possessing 2' ,3' dideoxycytidine monophosphate as the 3' -terminal nucleotide, together with 50 f.lM of dGTP, the nucleotide complementary to the first available template base. The presence of ddCMP at the 3' -terminus of the primer prevented polymerisation from taking place.

Each of the different RT samples was then titrated by addition of small aliquots of

UC781.

In each case, 0.5 f.ll aliquots ofUC781 stock solutions in DMSO were added to the RT mechanistic form, and the change in fluorescence was measured. The final concentration of DMSO in the titrated samples never exceeded 3%, and data were corrected for any quenching of RT fluorescence due to DMSo. No significant inner filter effects were noted at the concentrations of UC781 used. The fluorescence data were fitted to the following equation (Eftink, 1997; Fersht, 1985):

Equation 4.1

115 L1F is the change in fluorescence, L1Fmax is the maximum change in intrinsic fluorescence,

Ki is the inhibitor dissociation constant and [L] is the concentration of drug. UC781 behaves as a 'tight-binding' inhibitor for each of wt, KI 03T and V106A RT and Ki was app calculated as Ki=KiaPP/[E] where Ki is the value obtained from Equation 4.l. and E is the RT concentration.

4.3. RESULTS Inhibition of wt and mutant recombinant HIV-l RT by UC781. As described in the previous chapter, while the K103T or V106A single mutants showed high-Ievel resistance to UC38, they showed only minimal resistance to inhibition by UC781 (Table

3.1). The Y181C and Y188H mutations have been implicated in high-Ievel resistance to other NNRTI such as NVP (Spence et al., 1996) and RT possessing these substitutions also showed high-Ievel resistance to UC38, whereas none of these single substitutions elicited significant resistance to UC781 (Table 3.1.).

In contrast, significant resistance (> 200-fold) to UC781 was only provided by the presence of two substitutions in the NNIBP. Our studies on the in vitro development of

UC781-resistant HIV-l showed that high-Ievel viral resistance required at least two substitutions in the RT nonnucleoside binding pocket (NNIBP), namely K103T and

V106A. The K103T/V106A double mutant RT showed a 200-fold resistance to UC781 in vitro (Table 3.1.). The V106AIP225H mutants, which give high-Ievel resistance to the quinoxaline S-2720 (Pelemans et al., 1997), provided only 20-fold resistance to UC781.

116 Tight-binding inhibition by UC781. 'Tight-binding' inhibition was assessed by analysis of R T RDDP reaction progress curves, as previously described (Barnard et al., 1997).

Despite the low-Ievel resistance to UC781 produced by the K103T and V106A single substitutions, UC781 retained the tight-binding mode of inhibition of these mutant enzymes. The rates of association of UC781 to wt, K103T and V106A RT were rapid

5 5 3 3 (5.1 x 10 -5.8 X 10 M-1s-I) while dissociation was slow (1.4 x 10- - 2.1 X 10- S-I)

(Table 4.1). A linear dependence ofkapp on UC781 concentration was observed with both the K103T and V106A (data not shown) single mutant enzymes, indicating a one-step mechanism for UC781 binding to RT (Morrison and Walsh, 1988), as previously shown with wt RT (Barnard et al., 1997).

In contrast, UC781 did not act as a 'tight-binding' inhibitor of the K103TN106A double mutant RT, but rather followed rapid-equilibrium inhibition kinetics (data not shown).

UC781 inhibition of the KI03T/V106A double mutant RT was linear non-competitive with respect to both dNTP (Ki = 294 ± 40 nM) and TIP (Ki = 600 ± 14 nM).

Fluorescence spectroscopie measurements of the binding of UC781 to wt and mutant RT.

The kinetic mechanism ofHIV-l RT catalyzed DNA synthesis is such that there are three mechanistic forms of the enzyme in the steps leading to catalysis (section 1.3), namely free RT, the RT-TIP binary complex, and the RT-TIP-dNTP ternary complex (Majumdar et al., 1988; Kedar et al., 1990; Kati et al., 1992; Reardon, 1992). Studies of protein fluorescence quenching have proven useful in analysis of the binding of substrates and inhibitors to HIV-l RT (Rittinger et al., 1995; Thrall et al., 1996). Previous studies have

117 demonstrated that UC781 interacts tightly with each of the three mechanistic forms ofwt

RT (Barnard et al., 1997; see section 1.3). Similar experiments were carried out in order to examine the binding ofUC781 to the K103T and V106A single mutants enzymes, and the K103TN106A double mutant RT. UC781 was able to interact with aH three mechanistic forms (see section 1.3) of each of the mutant enzymes (Table 4.2). No significant changes were observed with respect to binding of UC781 to the KI 03T and

V106A single mutants enzymes relative to wt RT. In contrast, the K103T/V106A double mutant showed significantly reduced affinity for UC781 (> 300-fold). The affinity for

UC781 of each of the mechanistic forms of the K103TNI06A RT was decreased to a similar extent relative to wt RT.

RT-catalyzed DNA synthesis in the presence of saturating concentrations of UC781.

UC781 is one of the most potent inhibitors of wt RT RDDP activity when compared to most other NNRTI (see Table 1.1) (Barnard et al., 1997). Previous studies have shown that NNRTI such as TIBO and NVP are unable to completely inhibit the RDDP activity even when RT is fully saturated with inhibitor (Spence et al., 1995). In our studies, in order to ensure complete saturation of RT by inhibitor, RT RDDP activity was titrated with increasing concentrations of NNRTI until no further change in enzyme activity was noted. Where possible, this limiting concentration was then increased by 100% in subsequent reaction assays, to ensure complete saturation by inhibitor. In general, concentrations equivalent to 1,500 times the in vitro ICso were used.

118 The rate of RDDP activity of wt RT in the presence of saturating levels of UC38 was about 1.5% of the uninhibited enzyme, whereas virtually no RDDP activity was shown by wt RI in the presence of saturating UC781 (Table 4.3). In contrast, the K103T and

V106A single mutants enzymes showed readily discernable rates of RDDP activity in the presence of saturating UC781; the rate of RDDP activity catalyzed by these mutants in the presence of UC781 was up to 3% of that seen in the absence of inhibitor. This residual activity corresponds to a 40-fold decrease in the efficiency of the inhibitor, that is, in its ability to block polymerization while bound. The KI03T/VI06A double mutant showed substantial RDDP activity in the presence ofUC781 (more than 10% of the rate of the uninhibited enzyme). However, it should be noted that the UC781 concentration employed in these experiments was only about 300-fold greater than the in vitro IC so, due to limitations ofUC781 solubility in aqueous media.

119 Table 4.1. Rate constants for binding and dissociation ofUC781 with wt and mutant RT determined from kinetic analysis of 'tight-binding' inhibition

5 3 HIV-l RT k on (x 10 , M-1s-l) koff(X 10- , s-I) KcJ(nM)

WT 5.1 ± 1.2 2.1 ±0.3 2.4 ± 0.9 K103T 5.5 ±0.5 1.7 ± 0.4 3.3 ±0.9 V106A 5.8 ± 0.6 1.4 ± 0.2 2.5 ± 0.6 K103TN106A N.D. N.D. N.D.

The calculation of the binding parameters could not be determined (N.D.) for the KI03AN106A. The values are means ± S.D. calculated from three or more independent measurements.

120 Table 4.2. Dissociation constants for binding of UC781 to different RT mechanistic forms determined from steady state fluorescence measurements

K.t(nM) HIV-l RT FreeRT RT-TIP RT-TIP-dNTP (binary complex) (temary complex)

WT 0.8 ± 0.03 0.2 ± 0.06 0.3 ± 0.03 K103T 1.0±0.2 1.4 ± 0.2 1.9 ± 0.3 V106A 1.1 ± 0.001 0.3 ± 0.1 0.4 ± 0.02 K103TN106A 300.0 ± 0.09 471.0± 0.6 363.0 ± 0.3

Values are the means ± S.D. from three independent experiments.

121 Table 4.3. RT RDDP activity of wt and mutant RT in the presence of saturating concentrations ofUC781 as compared to UC38

HIV-l RT kcat (S-l) kcat (S-l) a residual activity (- inhibitor) (+ inhibitor) (%)

UC781

wt 0.82 ± 0.14 0.0007 ± 0.0002 0.1 KI03T 0.85±0.14 0.028 ± 0.0008 3.3 V106A 0.83 ± 0.13 0.018 ± 0.003 2.2 K103TN106A 0.83 ± 0.10 0.140 ± 0.012 16.9

UC38 wt 0.85 ± 0.12 0.013 ± 0.003 1.5

RT assays were carried out as described in Materials and Methods. Values are the means ± S.D. from three independent experiments, each carried out in duplicate. a % residual activity of R T -catalyzed DNA synthesis in the presence of saturating concentrations of inhibitor over RT polymerization activity in the absence of inhibitor. The inhibitor concentrations used with wt RT were 3 IlM for UC781 and 50 IlM for UC38, which are 1,500 x the in vitro IC so values for these inhibitors. Similarly, 10 IlM UC781 was used in assays with K103T and V106A RT, and 100 IlM UC781 was used in assays with the K103T/V106A RT. 100 IlM is about the maximum attainable concentration of UC781 in aqueous media.

122 4.4. DISCUSSION

NNRTI bind to the nonnucleoside inhibitor binding pocket (NNIBP), a well-defined region of HIV-l RT close to, but distinct from, the active site (Tantillo et al., 1994).

Single substitutions in the NNIBP, such as V106A, Y181C and Y188H, confer high-Ievel resistance to structurally dissimilar NNRTI such as NVP, TIBO, BHAP and TSAO, among others (Tantillo et al., 1994; Balzarini et al., 1994; Maga et al., 1997; Soriano et al; 2002). We have also found that single substitutions in the NNIBP provide high-Ievel resistance to UC NNRTI, such as UC38 and UC84. However, single substitutions in the

NNIBP do not provide significant resistance to UC781 (Table 3.1).

Several groups, including ours, have found that high-level HlV-l resistance to UC781 requires two or more substitutions in the NNIBP of RT (Buckheit, Jr. et al., 1997b;

Balzarini et al., 1998). Interestingly, each group has identified different mutation patterns.

However, none of these studies identified the phenotypic basis underlying the need for multiple mutations in UC781 resistance. UC781 is among the most potent NNRTI antivirals (Barnard et al., 1997; Balzarini et al., 1994; Balzarini et al., 1996c). There are a number of characteristics of UC781 inhibition that likely contribute to this potency. The compound is unique among the characterized NNRTI in that it acts as a 'tight-binding' inhibitor ofHIV-l RT and, unlike other UC NNRTI such as UC38 and UC84, UC781 is able to interact with aIl mechanistic forms of RT (Barnard et al., 1997). In addition,

UC781 is essentially a 'dead-end' inhibitor of wt RT RDDP activity (Table 4.3). We hypothesized that resistance to UC781 would most likely involve modulation of one or more of these inhibitory characteristics.

123 Both UC38 and UC84 are less potent antivirals than UC781. Furthermore, unlike UC781,

UC38 interacts only with the RT-TIP-dNTP temary complex, whereas UC84 binds primarily to free RT and the RT-TIP binary complex (Fletcher et al., 1995a). UC781 is still able to bind to aIl three mechanistic forms of the K103TN106A double mutant, albeit with much reduced affinity (Table 4.2), indicating that resistance is not due to the loss of interaction with any particular R T form.

Previous studies conceming the mechanism of resistance to NVP arising from a single substitution in the NNIBP indicated that the decreased potency of the NNRTI against the mutant RT is attributable to alterations in the rates of inhibitor association (kan) and/or dissociation (kaff) from the enzyme (Spence et al., 1996; Maga et al., 1997). However, we found no significant changes in the rates of binding or dissociation of UC781 from the

K103T and V106A single mutant enzymes relative to wt RT (Table 4.1, Table 4.3). Thus,

UC781 continued to act as a 'tight-binding' inhibitor of the single mutant RT species (Kd

= 2.4 - 3.3 nM).

However, UC781 lost the ability to act as a 'dead-end' inhibitor of the RDDP activity catalyzed by the K103T and V106A enzymes. Residual RDDP activity of these mutants in the presence of saturating levels of UC781 was about 3% of the rate seen in the absence of UC781 (efficiency ~ 97%), compared to < 0.1 % for wt RT (efficiency

-99.9%) in the presence ofUC781 (Table 4.3). This change reflects a > 30-fold decrease in inhibitory efficiency. This decrease in efficiency of UC781 inhibition appears to be in conflict with the IC50 values reported in Table 4.3. The drop in potency ofUC781 in the

124 presence of any single substitution is less than 7-fold as compared to the wt RT. How can this slight drop in potency be in agreement with a greater than 30-fold drop in efficiency?

Simply, a drop in efficiency from 99.9% (wt RT) to 97% (K103T or V106A), that is a drop of ~ 3%, would not be expected to result in a significant decrease in ICso. In other words, even a 30-fold decrease in the exceptional efficiency of UC781 (seen in the presence of a single substitution) is not sufficient to significantly alter the overall inhibitory potency of this TBNNI. Introduction of a second substitution in the NNIBP eliminates the tight-binding nature of UC781 inhibition, by dramatically increasing the rate of dissociation of the inhibitor (Kd = 300-600 nM) from the mutant enzyme as compared to wt or single mutant enzymes (Table 4.3.). This resistance phenotype is similar to the mechanism of resistance to NVP resulting from the Y 181 C mutant, which results in a 55-fold increase in the rate ofNVP dissociation from the mutant compared to wt enzyme (Spence et al., 1996).

As discussed in the previous chapter, high-Ievel resistance to UC781 requires multiple substitutions in the NNIBP in order to sequentially eliminate critical inhibitory parameters. The first substitution eliminates the ability of UC781 to act as a 'dead-end' inhibitor of RT-catalyzed DNA synthesis, without altering the tight-binding nature of the

RT-UC781 interaction. The introduction of a second substitution eliminates tight binding, presumably by increasing the rate of dissociation of the enzyme-inhibitor complex. Each of the RT mechanistic forms appears to be equally affected in this respect.

Our studies suggest that the order of introduction of substitutions into the NNIBP is

125 probably not important, but rather that it is the number of substitutions that is critical in

UC781 resistance. Different mutation patterns have been identified in the R T gene from

UC781-resistant IDV-l. For example, Buckheit and colleagues reported the presence of

KlOIEN108I1Y181C (Buckheit, Jr. et al., 1997b), whereas Balzarini's group identified the V106A1F227L and LlOOIlV106A1F227L groups (Balzarini et al., 1998). We found the K103TN106A mutation set in IDV-l that provides more than 200-fold resistance to

UC781 in vitro. We therefore also suggest that the actual identity of the NNIBP mutations is probably less important in UC781 resistance than is the nurnber of mutations.

126 CHAPTER 5: MICROBICIDAL (VIRUCIDAL) ACTIVITY OF TIGHT-BINDING

NNRTI

The material presented in this chapter was published in June 2002.

(Motakis,D. and Pamiak,M.A. (2002) A tight-binding mode of inhibition is essential for anti-human immunodeficiency virus type 1 virucidal activity of nonnucleoside reverse transcriptase inhibitors. Antimicrob.Agents Chemother., 46: 1851-1856.)

127 5.1. INTRODUCTION In the last decade, there has been rapid development of therapies designed to treat HIV-1 after infection has occurred. In contrast, most attempts to prevent the spread of HIV infection have been disappointing, partly, due to the fact that a vaccine is not yet available. However, the development of topical microbicides may constitute an alternative to vaccines in reducing the rate of transmission of HIV to non-infected individuals (Potts, 1994; Wainberg, 1999; Van de Wijgert et al., 2002).

In order for anti-HIV vaginal or rectal agents to be effective, they should completely attenuate HIV and prevent cell-to-cell transmission; they should preferably block several of the pre-integrational stages of viral replication and act rapidly. Furthermore, an ideal microbicide should not be cytotoxic to the vaginal epithelium and not be absorbed systemically, while it should be inexpensive and easy to produce in a topical formulation.

Potential compounds include detergents that disrupt the viral architecture such as SDS

(Howett et al., 1999; Bestman-Smith et al., 2001), blockers of viral binding such as dextran sulfate (Baba et al., 1988; Mitsuya et al., 1988; Neurath et al., 1996) and NNRTI such as UC781 (Borkow et al., 1997).

Previous studies have demonstrated that UC781, a 'tight-binding' inhibitor ofHIV-l RT, has potent virucidal activity against HIV-l (Borkow et al., 1997) and acts as a 'chemical barrier' in MT2 lymphocytoid cells to HIV-l infection, a property that identifies UC781 as a potential microbicide. Importantly, what di stinguishes UC781 from other microbicides is its ability to be 'absorbed' both by the virus and the cells allowing virucidallprotective activities to persist even after aIl extracellular drug has been

128 completely removed. These microbicidal properties of UC781 have not been observed with other non-tight-binding NNRTI such as NVP (Borkow et al., 1997).

Recently, two additional NNRTI, CSIC and EFV (Maga et al., 2000) have been identified as 'tight-binding' inhibitors (see Chapter 2). Their availability has enabled us to examine the hypothesis that 'tight-binding' is a critical parameter ofNNRTI microbicidal activity.

In order to study the use of NNRTI as microbicidal agents we used a group of selected

NNRTI: UC781, EFV, CSIC, UC84, NVP and DLY. As UC84 is structurally similar to

UC781, this NNRTI was chosen as an essential control in order to examine whether virucidal activity can be solely attributed to the chemicallstructural characteristics of the

UC inhibitors. Moreover, NVP and DL V, two NNRTI currently in clinical use, are quite potent inhibitors of RT (Table 5.1, Table 1.1). Therefore, these NNRTI were used in the experiments described in this chapter to assess the role of antiviral potency alone in cellular protection and viral inactivation. Lastly, NVP, DLV and UC84 are rapid equilibrium inhibitors while UC781, CSIC and EFV, as established earlier, are 'tight binding' inhibitors of HIV-l RT (see Chapter 2). This enabled an assessment of the role of rapid equilibrium versus "tight-binding" inhibitors in defining microbicidal potency.

Here we report that UC781, CSIC and EFV, establish a long-term safety barrier in lymphocyte-like MT2 cells from subsequent HIV-I infection. This protective effect is not observed with rapid equilibrium inhibitors (REl) UC84, NVP and DL V (DL V).

Furthermore, aIl TBNNI are able to completely attenuate both free and cell associated

HIV-l, while exposure of chronically infected H9 ceUs to TBNNI renders them non-

129 infectious. The fact that the aforementioned virucidal properties of TBNNI are not compromised by the removal of aIl exogenous inhibitor prior to infection may have important implications in the efficacy of these compounds in vivo. OveraIl, our data suggest that the applicability ofNNRTI as potent microbicides is restricted to TBNNI.

5.2. MATERIAL AND METRODS The HIV -1 HIB laboratory strain of HIV -1 was obtained from the NIH AIDS Research and

Reference Reagent Program, Division of AIDS, National Institute of Allergy and

Infectious Diseases. The CD4+ MT2 cell line was obtained from the American Type

Culture Collection. The carboxanilide NNRTI UC781 and UC84 were provided by W. G.

Brouwer from Uni royal Chemicals Ltd., Research Laboratories (Guelph, ON, Canada).

NVP was a gift from Boehringer-Ingelheim (Laval, QC, Canada). EFV and DLV were a kind gift from Dr. M. A. Wainberg, and CSIC was synthesized by Dalton Chemical Labs

(ON, Canada). HIV-1 chronically infected H9 cells (H9+ cells) were developed in our labo RPMI-1640 cell culture medium and heat-inactivated fetal bovine serum (FBS) were obtained from Canadian Life Technologies (GIBCO, Toronto, ON, Canada). Purified recombinant RT was prepared by a rapid method previously described (Fletcher et al.,

1996). HIV-1 RT RNA-dependent DNA polymerase activity (RODP) was measured as previously described (Fletcher et al., 1995a~ Fletcher et al., 1995b). HIV-1 p24 antigen elisa reagents were from SAIC-Frederic (Frederic, MO, USA).

Cel! culture and virus replication. MT2 and H9+ lymphocytoid cells were cultured in

RPMI 1640 with 10% FBS. Stock solutions of NNRTI were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO was always 1% or less since

130 control experiments showed that there was no effeet on viral infeetivity or ceIl viability at these concentrations. The level of infection of MT2 lymphocytes was determined by microscopic observation of syncytia formation (Yao et al., 1992b; Rooke et al., 1991).

AlI data points were averages of at least two independent determinations. Cytotoxicity was determined by ceIl growth kinetics and trypan blue exclusion (Hirabayashi et al.,

1989).

4 HIV-l virus pre incubation with NNRTI. Isolated HIV-l uIB (TCIDso = 5.5 x 10 ) was incubated with various concentrations of tight-binding NNRTI in a final volume of 200

JlI of media without FBS for 2 h at 37°C. Subsequently, the virus was diluted by adding drug-free media to a final volume of 15 ml and was then concentrated down to 0.5 ml using centriprep-100 centrifugaI concentrators. This step was repeated three times in total. Control experiments confirmed that aIl extravirion drug was effectively removed by this method. The drug-pre-treated virus (0.5 ml) was used to infect MT2 lymphocytes (3 x lOs cells) for 2 h at 37°C, the cells were then isolated by centrifugation, washed and fresh media (without any NNRTI) was added. The development of infection was assessed by microscopie assessment of syncytium formation and measurements of the p24 antigen levels.

Pre incubation of chronicaUy infected H9+ ceUs with NNRTl. Chronically infected H9+

4 cells (5 x 10 ) were incubated with various NNR TI concentrations in RPMI 1640-10%

FBS for 18 h at 37°C. In each case, the drug was removed from the cells by gentIe centrifugation at 300 x g for 10 min. To ensure elimination of all exogenous drug these

131 ceUs were subsequently washed twice with a volume of 15 ml of RPMI-1640 with no

FBS. These H9+ ce11s were then recultured for a 12 h period at 37°C in order to produce new virus. The culture media was subsequently centrifuged at 1,000 x g for 10 min in order to remove a11 ce11s and the virus-containing supematants were coUected. Viral production was not affected by any of the NNRTI tested as detennined by p24 antigen elisa. Equal amounts of virus (50 !-lI; 0.25 ng of p24) were used to infect 5 x 104 MT2 lymphocytoid ceUs. Infection was measured after 4 days by microscopie evaluation of the cytopathic effect (CPE). In experiments assessing the effect of NNRTI on ceU-to-celI

HIV-l transmission, H9 lymphocytes (6 x 103 ceUs) were pre-treated with various

NNRTI concentrations in RPMI 1640-10% FBS for 18 h at 37°C. The drug was then removed by centrifuging the ceUs (300 x g for 10 min). To ensure elimination of aIl exogenous drug these ceUs were subsequently washed twice with 15 ml of media and isolated each time by low speed centrifugation. FinaUy the drug pre-treated H9+ ceUs were resuspended in fresh media with FBS and 103 of these H9+ceUs were co-cultured with 3 x lOS MT2 lymphocytoid ceUs. Therefore, the majority of the cells observed microscopica11y were MT2 ceUs. The level of infection of the MT2 cells was quantified by assessment of the cytopathic effect (syncytia fonnation) after 4 days.

Pre-treatment of MT-2 ce Us with NNRTI prior to HIV-l infection. Uninfected MT2 lymphocyte-like ceUs (3 x lOs ceUs) were incubated with different concentrations of

NNRTI for 18 h at 37°C and the drug was subsequently removed by centrifugation. The ceUs were washed extensively as described above and then infected with equal amounts

3 of HIV-l (TCIDso = 1.5 x 10 ). Virus infectivity was assessed by microscopic

132 observation of syncytia fonnation on a daily basis for up to 30 days. In experiments that examined the effect of viral load on NNRTI protection, MT2 cells (5 x 104 cells) were pre-treated with varying concentrations of NNR TI as described above. These cells were then infected with different concentrations of virus with a final muItiplicity of infection

3 (M.O.!.) ranging from 0 to 0.18 (TCID50 =7.1 x 10 ). The leve1 of MT2 infection was measured again by microscopie assessment of the cytopathic effect 4 days post-infection.

5.3. RESULTS Inactivation ofisolated HIV-l by TBNNl. We examined the ability ofNNRTI to attenuate the infectivity of isolated HIV-l virus upon short exposure. Incubation of isolated virus with UC781, EFV or CSIC for 2 h at 37°C attenuated HIV-l even though the drug had been completely removed prior to assessing infectivity. Preincubation with concentrations higher than 2.5 /lM aboli shed HIV-1 infectivity in aIl cases (Figure 5.1).

CSIC showed a slightly more potent virucidal profile in this respect. UC84, DL V and

NVP did not compromise the ability of HIV -1 to infect MT2 lymphocyte-like cells even at the highest concentrations of drug preincubation (10 /lM).

Inhibition of ceU-to-ceU HIV-l transmission. We assessed the effect of NNRTI on the transmission of infection from infected to non-infected lymphocyte-like cells in two independent ways. First, we assessed the infectivity of the virus produced from H9+ cells, which were treated with NNRTI for 18 h at 37°C. HIV -1 produced by these cells was isolated 24 h after the complete removal of the drug from the cellular environment. This virus was used to infect MT-2 cells. At 30 /lM ofUC781, CSIC or EFV no infection was detectable in 4 days (Figure 5.2).

133 Second, we examined the ability of tight-binding NNRTI to prevent infection of MT2 cells from pre-treated chronically infected H9 cells. We pre-treated H9+ cells as described above and after removing aIl extracellular drug these cells were cocultured with uninfected MT2 ceIls. There was a marked decrease in the ability of H9+ cells to infect

MT2 cells upon UC781, CSIC and EFV pre-treatment. CSIC was the most effective

NNRTI in this respect (Figure 5.3).

Assessment of the cellular protection from HIV-I infection offered by TBNNI. To deterrnine if TBNNI could prote ct cells from HIV infection, MT -2 ceIls were pre-treated with NNRTI and then the ability of HIV to infect the cells was assessed. As shown in

Figure 5.4, pre-treatment with DLV offered no protection against infection compared to the no drug control and complete infection was observed in 5 days. Similar results were observed with high concentrations ofUC84 and NVP (data not shown). In contrast, pre treatment with UC781, EFV or CSIC showed a concentration dependent delay in the appearance of cytopathic effect (Figure 5.4). CSIC was the most effective in establishing a barrier to HIV -1 infection; no cytopathic effect was observed with 10 J..lM of CSIC for up to 30 days (data not shown).

We also examined whether the protective effect provided by UC781, EFV and CSIC could be overcome by increasing the viral load (Figure 5.5). Different amounts of virus were used in order to challenge drug-treated MT2 cells after aIl exogenous drug was removed. Significant protection was observed throughout the range of viral loads tested

(M.O.I.: 0.0007-0.18). Interestingly, CSIC showed the broadest spectrum of protection as

134 can be seen from the size of the 'floor' area in Figure 5.5. (C), corresponding to 0% infection.

135 Table 5.1. Surnrnary of sorne properties of the NNRTI used in this study

C Drug ICso (nMt ECso (nM)b Type of inhibition

Clinical NVP 290± 100 45 ± 10 REl DLV 24±4 16 ± 5 REl EFV 7.7± 0.9 1.7±0.2 TBI Experimental UC84 117 ± 4 240 ± 70d REl UC781 1.7 ± 1.2 10 ± 0.2 TBI CSIC 2.0 ± 0.2 1.2 ± 0.4 TBI

a determined by measurernent of recombinant wild type p511p66 R T RDDP activity using [3H]-dGTP and poly(rC)-oligo(dG)12-18 as described in Materials and Methods. b Inhibition ofHIV-l infection ofMT2 cells.

C Inhibition ofHIV-l reverse transcriptase in vitro: REl, rapid equilibrium inhibitor; TBI, 'tight-binding' inhibitor. dBorkow et al., 1997

l36 Figure 5.1. Inactivation of isolated HIV -1 virus particles following exposure to different concentrations ofNNRTI

ü tê 60 ID

20 -

o 2 4 6 8 10 Viral preincubation drug concentration (pM)

HIV-luIB was incubated with UC781 (e), CSIC ( ... ), EFV (-), DLV (0), NEV (0), and UC84 (.6.) for a period of 2 h at 37°C. The exogenous drug was then removed and the virus was washed extensively, as described in Materials and Methods. The exogenous drug-free virus was then used to infect MT2 lymphocytes and the extent of HIV-l induced cytopathic effects were assessed four days post-infection. Data points are the means ± S.D. ofthree separate determinations.

137 Figure 5.2. Inactivation of HIV-l produced by TBNNI-pre-treated chronically infected H9 cells

120 5 ---g 100 1". 4 t5 & 80 Q) -- .~ 3 E .c:...... 0> (Il -C ...... 0. 60 0 ..q- N >. 2 0.. ü 40 :::R0 1 20

0 o 0 5 10 15 20 25 30 35 40

Time (da ys)

H9+ cells were incubated with UC781 (e), CSIC (Â) and EFV (-) for 18h at 37°C, and then the cells were washed free of exogenous drug. The H9+ cells were then cultured for 24h in the absence of drug, and the virus produced during that period was separated from the cells by centrifugation, as described in Materials and Methods. Equal amounts of virus were added to MT2 cells and the extent of infection of these cells was determined after 4 days by microscopie evaluation of syncytium formation (left axis, full symbols). TBNNI pre-treatment did not affect viral production by H9+ cells as assessed by measurement of p24 levels (right axis, empty symbols: UC781 (0), EFV (0), CSIC (6». The data presented are the means of more than two independent determinations.

138 Figure 5.3. Effeet ofNNRTI treatment ofH9+ cells on cell-to-cell transmission offfiV-1 in the absence of extracellular drug

----._~_.~._".~" .- _. f 100 -

80 -, ...u ~ - ~ 20

0 0 10 20 30 40 Pretreatment druQ concentration ülM)

H9 chronically infected cells (H9+) were incubated with the indicated concentrations of UC781 (e), CSIC (Â) and EFV (-) for 18h at 37°C, then the cells were separated from extracellular drug and virus as described in Materials and Methods. The isolated H9+ cells were then co-cultured with uninfected MT2 cells (1:300 ratio ofH9+ to MT2 cells), and the extent of infection of the latter was determined 4 days later by microscopie assessment of HlV-1 induced syncytium formation.

139 Figure 5.4. Effect of "transient" exposure of uninfected MT2 cells to vanous concentrations ofNNRTI on subsequent viral infection.

A B

100 ' 100 , 80' 80 UJ : UJ ~ 60~,, ~ 60 ?f!. 40' o o 20 5 $' 5 10 ~ Q;-. 10 f :::::-. 15 § 'cf 15 (\'b Cl 20 ~rr:r 20 ~ o ~ o

c o

100 " 100

UJ 80 u.I 80 ~ 60 rs 60 ?f!. 40 o ?f!. 40 20\ 5 ;::-.. 20 o 10:! 0 10 $' N- ~ 15 § 15 (y § 0- . 20 ':!,...frJ 20 ~ o ~ o

Uninfected MT2 cells were incubated with the indicated concentrations of NNRTI for 18h at 37°C, then washed to rem ove exogenous drug. The cells were then exposed to 3 identical inocula of HIV-l (M.O.I. = 7.5xlO- ). HIV-l induced cytopathic effects were assessed daily by microscopie examination of syncytium formation as described in Materials and Methods. DLV (A), UC781 (B), EFV (C), CSIC (D).

140 Figure 5.5. Effect of viral load on infection of MT2 ceUs pre-treated with various concentrations oftight-binding NNRTI

100'" u.l D 75; 0.1 <,>"'- 50\ 25' o 5 10 15 20 25 [U C(81)( liA !-hvl)

100' ~ 75 ü 0.1 §!. 50 ", 25:, 0'

15 20 25 [Ëfavirenz] ( Illv!) c

100 '5: 75', <.) ~ 50' 25 o

Uninfected MT2 cells were incubated with the indicated concentrations of NNR TI for 18h at 37°c, then washed to remove aU exogenous drug. The cells were th en inoculated with the indicated amounts of HIV-l (M.O.!. ranging between 0.0007 to 0.18). HIV-l induced cytopathic effects were assessed 4 day post-infection by microscopie examination of syncytium formation as described in materials and methods. (A), UC781; (B), EFV; (C), CSIC.

141 5.4. DISCUSSION UC781 was the first NNR TI that was shown to completely attenuate both free and cell associated HIV -1 (Borkow et al., 1997). In the same publication, UC781 was shown to

'shield' MT2 lymphocyte-like cells from infection upon pre-treatment and subsequent removal of the drug. Notably, this is the first, and until recently the only, tight-binding

NNRTI with 'microbicidal' properties identified. 'Tight-binding' inhibitors (TBI) dissociate from the enzyme slowly, once bound, while rapid equilibrium inhibitors such as NVP bind and dissociate from HIV-1 RT rapidly (see Chapter 1). Consequently,

'tight-binding' inhibitors are effective at very low nanomolar concentrations, which are in the range of the enzyme concentration inhibited, in contrast to non-tight-binding inhibitors. Since NVP, a rapid equilibrium NNRTI, did not show any of the virucidal or protective effects ofUC781, it suggested that the microbicidal activity ofUC781 was due to its tight-binding properties. However, further evaluation of this hypothesis has been difficult until the recent identification of two other tight-binding NNRTI, CSIC and EFV

(Maga et aL, 2000).

Pre-treatment of HIV -1 for a short period of time with EFV and CSIC attenuated the infectivity of isolated HIV -1, similar to UC781 (Borkow et al., 1997). UC781, CSIC and

EFV must be able to diffuse through the lipid membrane coat and capsid of HIV-1 and subsequently bind and inhibit HIV-1 RI. No analogous virucidal effects were observed with NVP, DLV or UC84 even though these NNRTI are relatively potent RT inhibitors

(Table 5.1). Similarly, aIl three TBNNI inactivated the virus produced by chronically infected cells. These data validate our hypothesis that 'tight-binding' inhibition is a prerequisite of virucidal activity with NNRTI.

142 Furthennore, MT2 lymphocyte-like celIs, pre-treated with EFV and CSIC, became refractory to HIV-l infection, as previously observed with UC781 (Borkowet al., 1997).

AlI three TBNNI showed a prolonged protective effect after single drug exposure whereas DLV and UC84 did not. Surprisingly, even though aIl NNRTI including DLV and UC84 are lipid soluble compounds and should be able to penetrate into cells,

'shielding' from HIV-l infection was observed only with tight-binding NNRTI.

Therefore, the ability of TBNNI to block infection is likely due to an effective cellular reservoir of drug that is available upon viral exposure. It has been suggested that TBNNI localize at the plasma membrane of celIs and are subsequently taken up by HIV -1 upon entry (Borkow et al., 1997). The ability of TBNNI to establish a cellular reservoir is consistent with the observation that drug pre-treated H9+ cells produce inactive HIV-I, even after the drug has been effectively removed. The identification of the cellular compartment that sequesters TBNNI awaits further investigation.

The fact that very small amounts (comparable, but not lower than the enzyme concentration) of TBNNI are required before HIV-l RT is saturated with drug also appears to be a key factor in these protective properties. The ability of TBNNI to stay bound to HIV-l RT for prolonged periods oftime (in the order of minutes to hours) when compared to other NNR TI should theoretically contribute to the protective effect of

TBBNI (Barnard et al., 1997; Borkow et al., 1997). Blocking of reverse transcription, both intracellular and endogenous (as previously shown with UC781) (Borkow et al.,

1997), and subsequent degradation of the viral ribonucleoprotein complex likely results

143 in the inactivation of the virus prior to integration, thus rescuing cells from chronic infection.

In the assessment of microbicidal candidates it is important to take into consideration their ability to remain locally distributed, as opposed to diffusing systemically.

Obviously, systemic therapies used clinically such as DL V, EFV and NVP have high oral bioavailability. Similarly, UC781 has been shown to have moderate bioavailability in animaIs (Buckheit et al., 1997). Therefore, the long-term use of UC781 or EFV as a preventative, topical microbicide may lead to an undesirable circulating level of these inhibitors in the blood. For instance the systemic use of EFV has a nwnber of side effects

(retinal toxicity, gynecomastia, vasculitis, hepatotoxicity etc.), which may not be acceptable in the case of a preventative ointment (Lewis et al., 2002; Boffito et al., 2002;

Fumaz et al., 2002; Domingo et al., 2002; De Santis et al., 2002; Tseng et al., 2002;

Fundaro et al., 2002; Sulkowski et al., 2002). On the contrary, the fact that the bioavailability of CSIC may be lowered by its low solubility (see section 1.4.3) might constitute an advantage in the application of this TBNNI as a microbicide in vivo; it is likely that CSIC will exert its local protective effects against HIV -1 infection and will be devoid of any hazardous systemic accumulation. In addition, although aIl TBNNI are equally effective in attenuating isolated and ceIl-associated HIV-l, CSIC is superior to

UC781 and EFV in protecting MT2 cells from infection (Figure 5.4). Possibly, CSIC can attain greater concentrations in the cell than the other two TBNNI, a hypothesis that should be further assessed in Phase l clinical trials.

144 Tight-binding NNRTI fulfill a number of the criteria for an ideal microbicide. They are very potent and are able to completely inactivate HIV -1 and prevent cell-to-cell transmission by protecting healthy cells and rendering infected cells non-infectious.

TBNNI do not require activation in order to be effective; they act directly and rapidly.

TBNNI have been shown to be effective against a variety ofHIV-l strains (Young et al.,

1995; Buckheit et al., 1997b) while they are effective at low concentrations, have low cytotoxicity (Table 1.1), and are unlikely to affect the vaginal epithelium. CSIC appears to have an additional advantage over other NNRTI; its low bioavailability will likely prevent the systemic diffusion of this NNRTI, thus limiting possible side effects as mentioned above. Nevertheless, the efficacy of TBNNI against epithelial, Langerhan's,

M cells as well as macrophages, which are the likely initial targets of HIV -1 infection

(see section 1.8.1), should be assessed in the future.

These data indicate that TBNNI are excellent virucidal candidates for the prevention of

HIV-l infection. We suggest that tight-binding to HIV-IRT should constitute the primary selection criterion in the further selection of NNR TI microbicides. There have been several different approaches to the development of microbicides, such as the use of membrane surfactants and viral binding inhibitors (see section 1.8.2). TBNNI in formulations with such types of viral microbicides could maximize the potential of these agents in attenuating HIV -1 before permanent infection is established.

145 CHAPTER 6: GENERAL DISCUSSION

146 There have been three main drawbacks in the effort to contain the spread of HIY. First, it has been difficult to prevent the appearance of resistance to the available drug therapies.

Second, most of the existing theurapeutic regimens have been out of reach for underprivileged countries due to their high cost. Third, the inadequate use of the condom and the lack of an effective vaccine have also contributed to the dissemination of HIV infection. It is evident that there is a need for more effective yet less costly therapies, which are resilient to the development of viral resistance. This would benefit those who already suffer from the devastating complications of this disease. On the other hand, even though the efforts to make available a vaccine should be reinforced, other avenues of prevention should also be sought. The development of an agent that could contain the spread ofHIV infection to uninfected individuals is long overdue (Potts, 1994; Wainberg,

1999).

What is the role of NNRTI in the effort to improve the prognosis of HIV infection?

Currently, three NNRTI are available clinically (NVP or Viramune®, DL V or

Rescriptor® and EFV) while several others are undergoing clinical investigation such as tivirapine, loviride, MKC-442 and HBY 097 (De Clercq, 1999). DL V, NVP and EFV in combination with two other NRTI such as AZT + ddI, AZT + 3TC or with an NRTI and a

PI have been shown to be the most effective in suppressing the viral plasma load and restoring lymphocyte counts in patients (De Clercq, 1999; Barreiro et al., 2002; Ghani et al. 2002; Mathews et al. 2002; Plana et al., 2002). Such therapies are more effective than drug combinations involving NRTI and PI alone. Although administration of such triple therapies early in infection is successful in delaying the appearance of resistance, multidrug resistant strains have been reported to appear with such therapies (Geretti et al.,

147 2001). Currently, overcoming multidrug resistance is one of the greatest challenges in anti-illV drug development. It has been suggested that the periodic alteration of the regimens administered to the patient or the use oflarge 'knock out' doses ofinhibitor will help prevent the appearance of resistant HIV strains. These recommendations may prove useful in improving the resilience of existing NNR TI to III V-1 resistance. On the other hand, exceptionally potent NNRTI may be able to effectively inhibit HIV-l at relatively low drug concentrations despite the presence of resistance mutations (De Clercq, 1999).

Compounds with low nanomolar or even picomolar inhibitory concentrations, against wild type IllV, such as the TBNNI, remain active against mutated HIV at nanomaolar or lower micromolar concentrations.

The development of NNRTI in the last decade was largely initiated by the screening of numerous compounds with anti-illV activity in cell culture. This approach has been of tremendous help in the initial identification of potential drug targets and structures.

Subsequently, biochemical and crystallographic data, in combination with drug modeling studies, have been crucial in optimizing the effectiveness of many experimental compounds. Both screening and rational drug design have yielded numerous potential compounds that may prove use fui in the future as clinical therapies. However, the facile development of resistance to aIl therapeutic NNRTI demands more innovative drug design, guided by the knowledge gained from the study of the mechanism of drug resistance.

148 Crystal structures have helped deduce the role of individual residues in drug binding.

Although NNRTI bind to the same pocket, different patterns of interactions have been identified giving rise to different resistant profiles. Nevertheless, mûst of our understanding of the interactions involved in drug binding, inhibition and resistance are derived from structures that precede the ternary complex of HIV -1 R T and are therefore not representative of the catalytically poised enzyme. Molecular mûdeling has been useful in compiling the available crystallographic data in one structure and creating a

"dynamic" NNIBP. In one such model, the NNIBP is depicted as a "plastic" pocket; the potential space occupied by various NNRTI differs, while flexible residues rearrange themselves in order to accommodate the drug (Mao et al., 2000). Such an approach has led to new potent NNRTI (ICso~ 7 -70 nM), which are effective against K103N/Y181C

HIV-l at acceptable concentrations (ICso < 3 /lM). Hopefully in the future drug modeling willlead to compounds, which are resilient to resistance.

Kinetic and mutational studies have also aided the screenmg of NNRTI. The characterization and classification of NNRTI, based on their inhibitory mechanism can guide the identification of even more potent NNRTI. Tight-binding inhibitors offer several pharmacologie advantages: low toxicity, the possibility of resilience to resistance and protection of the target tissues from infection. The characterization of tight-binding

NNR Tl sets the stage for the identification of other potent NNR Tl based on their kinetic profile rather thanjust their structure. CSIC, UC781 and EFV, unlike mûst other NNRTI, stay bound on HIV -1 R T for prolonged periods of time and prevent the conformational

149 change of polymerisation from occurring. Similar studies should identify other existing or newly developed NNRTI with similar pharmacologic properties.

As mentioned earlier (section 1.4.2), recent data (Shulman et al., 2001; Haubrich et al.,

2002; Whitcomb et al., 2002) have demonstrated that NNRTI may not only be useful in combination with NRTI, but they may also be useful as salvage therapy since they appear to be up to 60% more effective against NRTI-resistant HIV-l strains than against the wild type virus. Most likely, this is due to the rearrangement of the residues in the NNIBP secondary to the neighbouring amino acid substitutions introduced in the polymerase catalytic site with NRTI-resistance. Overall, in these reports EFV has shown the greatest effectiveness. Therefore, in the future, it would be interesting to test how 'tight-binding'

NNRTI compare with rapid equilibrium NNRTI with respect to inhibition of NRTI resistant HIV -1. It is quite possible that due to the fact that TBNNI make stronger interactions with the NNIBP, UC781, CSIC as well as other 'tight-binding' NNRTI will be found to be consistently more effective in this respect. If the latter is actually shown to be true, TBNNI use may become useful salvage therapy in the delay of development of antiviral drug resistance.

Unfortunately, the majority of the population affected by HIV does not have access to the costly therapeutics, which are currently available. Microbicides may help contain the spread of infection in the absence of a vaccine (Van de Wijgert et al., 2002). Although several promising leads are already available, their efficacy in vivo is questionable, mainly due to the difficulties associated with ensuring adequate exposure of the not easily

150 accessible anatomy of the vagina and the rectum. Various means such as foams and gels have been suggested, which may play a role in increasing efficacy. Combinations of multiple inhibitors that block different steps in the binding and replication of HIV -1 may also impact on efficacy. In any case, obtaining a microbicide that is 100% effective is unlikely. Variability in parameters such as the type of exposure, anal vs. vaginal sex, viral load in secretions, compliance and proper usage will definitely impact on microbicidal effectiveness. Nevertheless, the use of microbicides will have an immense benefit in the control of the HIV pandemie despite these obstacles. For instance, a formulation with an efficacy of 50% would be expected to reduce the worldwide HIV burden by more than 20 million, a significant accomplishment in light of this health catastrophe. Tight-binding

NNRTI are among the most promising microbicides available and should be considered as potential theurapeutic agents in this area.

The overall cost of microbicide usage should also be 10w. Such ointments will be used only as required, while facile self-administration would further reduce educational and health care costs. The choice of cheap, yet effective small molecules such as NNR TI may make microbicides more accessible. The development of resistance with the topical use ofNNRTI is not a problem since there is no continuous exposure of the virus to the drug.

Prevention of infection by already resistant strains may be attained by the use of TBNNI or combinations of these compounds with other microbicidal agents.

Although other R T inhibitors have been reported to have microbicidal properties, TBNNI have been shown to be effective after a short exposure to the compound and its

151 subsequent removal (Borkow et al., 1997; Motakis et al. 2002). These data support the

premise that NNRTI based microbicides will likely establish 'intraepithelial' reservoirs

able to offer more effective protection than other microbicides, which are not absorbed

intracellular at sufficiently high concentrations. We suspect that the ability of TBNNI to remain in the cell and in the virus for prolonged periods of time will resuIt in remarkable protection in vivo. It is unlikely that NNRTI alone will be completely effective in preventing HIV-I entry and infection. However, combination of TBNNI with other agents that are available today, and that oppose HIV-I at the pre-integrational stages of infection, should prove efficacious in this respect. TBNNI await clinical evaluation in order to establish their role in the future prevention ofHIV-1 infection.

152 CONTRIBUTIONS TO ORIGINAL KNOWLEDGE

The following section is a requirement by the Faculty of Graduate Studies and Research

at McGill University (Montreal, Canada) as part of a Ph.D. thesis.

153 The focus of the work presented in chapters 2 to 5 has helped us understand the mechanism of binding, inhibition, and microbicidal activity of TBNNI. Moreover, we now have a clearer understanding of the mechanism ofHIV-l resistance to such uniquely potent NNR TI. Below is a summary of the novel knowledge contributed by this thesis.

Chapter 2. We have found that NNRTI, CSIC and EFV, like UC781 (Barnard et al.,

1997), interact with R T and completely inhibit polymerization at concentrations that approximate the concentration of the enzyme. This is a characteristic of 'tight-binding' inhibitors (Morrison, 1969). Although UC781 binds to R T rapidly (Barnard et al., 1997), via a single-step mechanism, it appears that RT undergoes a conformational change upon binding to CSIC or EFV. This conformational change is not seen with UC781, presumably due to the greater flexibility of this molecule. Dissociation from RT is slow, occurring on a minute to hour time scale, in aIl three cases. Moreover, TBNNI appear to share a distinct mechanism of inhibition as compared to other NNRTI. We are the first to report that this group ofNNRTI inhibit by slowing down the conformational step. This is different from what others have found with non-tight-binding NNRTI for which the rate limiting step is the chemistry step (Spence et al., 1995).

Chapter 3. We have also found that the development of resistance to TBNNI is delayed compared to non-tight-binding NNRTI such as UC84, UC38 and NVP. We are the first to establish in vitro that this delay is due to the requirement for multiple RT mutations for high-Ievel resistance. In this work we report that mutational pairs KI03T/VI06A,

E138QNI79D and E138QIY181C, in combination with the mutation V108I, are among

154 the multiple resistant genotypes of UC781. Also, we have found that mutant

LI00IIKI03N confers resistance to CSIe. The same mutations are found in EFV-resistant virus, which is consistent with what others have previously reported with EFV (Young et al., 1995). Our molecular modeling is in close agreement with aIl data above since residues LlOO and KlO3 show close contacts with different moieties of both EFV and

CSIC. In the case of UC781 we believe that the identification of several different sets of substitutions that may confer resistance to this TBNNI may partially reflect the fact that this inhibitor interacts in more than one way with the NNRTI binding pocket. Our modeling studies show that UC781 can bind both in its cisoid as weIl as its transoid form.

On the contrary, CSIC and EFV are rigid structures, which appear to bind to RT in a single manner.

Chapter 4. Our group has been the first to look at the mechanism of resistance to a tight binding NNRTI. We have found that a single substitution is unable to confer high-Ievel resistance in vitro. Mutants KlO3T and VlO6A did not affect the binding ofUC781 to the

NNR TI binding pocket. However, both single mutants are able to polymerize at a low level while the drug is bound. On the other hand, the presence of both substitutions abolishes the binding of the drug to the enzyme and 'dead-end' inhibition. The low affinity with which UC781 binds to the KI03TNlO6A double RT mutant is in agreement with the level of resistance seen both in vitro and in vivo.

Chapter 5. AlI three TBNNI, UC781, CSIC and EFV, were able to inactivate HIV-I upon short exposure of the free virus to, and subsequent removal of, the drug. Similarly,

155 aIl TBNNI-pre-treated chronically infected H9 ceIls produced non-infectious virus. The latter pre-treated cens were unable to infect other uninfected lymphocytes. Therefore,

TBNNI pre-treatment resulted in the inactivation of both free and cel! associated virus and thus the inhibition of infection. Moreover, pre-treatment of non-infected MT2 cells and subsequent removal of an exogenous drug resulted in extensive protection of these cells from subsequent mv-1 infection. The protective effect of these NNR TI was dependent on the viral challenge and the drug concentration used. None of the above attenuating and protective effects were se en with any of the classical NNRTI such as

NVP, DLVand UC84.

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