Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors

Send Orders for Reprints to [email protected] Journal Name, Year, Volume 1

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors

Catherine F. Latham1*, Jennifer La1,2, Ricky N. Tinetti1, David K. Chalmers2, Gilda Tachedjian1,3,4,5

1Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, VIC, 3004, Australia; 2Medicinal Chemistry, Monash Institute for Pharmaceutical Sciences, Monash University, 381 Royal Pde, Parkville, VIC, 3052 Australia; 3Department of Microbiology, Monash University, Clayton, VIC Australia; 4Department of Infectious Diseases, Monash University, Melbourne, VIC Australia; 5Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, VIC, Australia.

Abstract: Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment- based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

Keywords: antiretrovirals, HIV-1, fragment-based drug discovery, reverse transcriptase, integrase, fragment screening.

1. HUMAN IMMUNODEFICIENCY VIRUS (HIV) Treatment with cART can control viral loads and prevent REMAINS A GLOBAL HEALTH ISSUE development of AIDS, although it cannot eradicate the virus completely so HIV positive individuals must maintain Human immunodeficiency virus (HIV) was identified as the continuous drug therapy throughout their lives. Viral causative agent of acquired immunodeficiency syndrome suppression by cART has been highly successful for (AIDS) over 30 years ago [1-4]. HIV is a retrovirus that decreasing morbidity and mortality associated with HIV infects CD4+ immune cells and is primarily sexually infection [6-11], providing patients with a good quality of transmitted, although infections also occur by transfer of life in the absence of a permanent cure. infected blood such as needlestick injuries or needle-sharing among injecting drug users. If an HIV infection is left The effectiveness of cART is often limited by the untreated, the eventual depletion of the CD4+ T lymphocytes emergence of drug-resistance mutations and the availability leads to severe and fatal immunodeficiency. HIV remains a of resources for monitoring virological failure due to these major burden on health resources worldwide as mutations (reviewed in [12]). Like many other viral and approximately 35 million people are infected [5] and there is bacterial pathogens, HIV can rapidly develop resistance currently neither a cure for HIV infection nor an effective mutations under the selective pressure of drug treatment and vaccine to block HIV transmission. There are, however, over those that favour survival of the virus persist in the genome. 25 antiretroviral drugs (ARVs) used to treat HIV. ARVs are Mutations constantly emerge in the HIV genome due to the usually administered as a cocktail of two or more drugs, a high replication rate, the high error rate and recombination regime that was initially termed highly active antiretroviral mediated by reverse transcriptase [13], an HIV enzyme that therapy (HAART), but now referred to as combined generates a double-stranded DNA copy of the viral RNA antiretroviral therapy (cART). genome. Without monitoring of patients on cART, their viral

loads can increase and drug resistance mutations can emerge. These mutations can circumvent their current combination of *Address correspondence to this author at the Centre for Biomedical ARVs and therefore severely limit the number of drugs Research, Burnet Institute, 85 Commercial Rd, Melbourne, VIC, 3004, available for future treatment and/or result in transmission of Australia; Tel/Fax: +61-9282-2130, +61-9282-2100; E-mail: drug-resistant HIV strains [14]. Recent surveillance has [email protected] shown that transmission of drug-resistant strains is on the

XXX-XXX/14 $58.00+.00 © 2014 Bentham Science Publishers 2 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al. rise, as high as 6.6% in resource-limited countries [15]. the NRTI class [35]. NNRTIs have diverse chemical ARV-based formulations are currently being developed for structures but can still induce common cross-resistance pre-exposure prophylaxis (PrEP) as either topical or oral mutations in HIV-1 RT, reducing effectiveness of all or most formulations as a means to address the millions of new HIV of the NNRTIs available in the clinic [36]. NNRTI- infections occurring each year [5]. PrEP products have resistance mutations reduce inhibitor efficacy predominantly shown efficacy in early clinical trials [16-18] leading to a by sterically blocking inhibitor binding to the NNIBP of burst in PrEP development based on ARVs aimed for use in HIV-1 RT, such as Y181C/I/V, L100I and K101P [37], or high-risk populations. However, application of some ARVs blocking inhibitor entry to the pocket, like K103N [38]. The for HIV prevention that are already used for treatment pose a integrase strand transfer inhibitors (INSTIs) are one of the risk for the emergence and transmission of drug-resistant newest classes of HIV-1 ARVs in clinical use with the HIV strains, particularly since some of these products are approval of raltegravir by the United States Food and Drug designed for continuous and long-term use. Administration in 2007, elvitegravir in 2012 and dolutegravir in 2013. While these three drugs have only been available Since current PrEP uses the same pool of ARVs that is for a short period, drug-resistance surveillance has revealed used for the treatment of HIV infections, new drugs are that resistance to raltegravir is emerging in patient urgently needed. Ongoing research in both industry-based populations (e.g. IN mutants N155H, Y143R and particularly and academic communities is focussed on discovery and Q148H/G140S) [39]. While elvitegravir can overcome the development of novel anti-HIV drugs, that are not affected Y143R resistance mutation [40], several other cross- by clinically-relevant drug resistance mutations, to provide resistance mutations have been reported for elvitegravir [41]. more options for both the treatment of HIV and preventing Currently there are no reports of drug resistance mutations its transmission. This review discusses a new strategy for the selected by dolutegravir when used in first line therapy [42]. development of novel ARV lead compounds known as There is, however, some evidence that drug resistance fragment-based drug discovery (FBDD) with particular focus mutations selected by elvitegravir or raltegravir could reduce on two proven HIV-1 drug targets; reverse transcriptase (RT) the effectiveness of dolutegravir [41], although the clinical and integrase (IN). impact of these mutations remains to be seen. 2. CURRENT ANTIRETROVIRALS ARE NOT The continual development of drug resistant HIV SUFFICIENT TO MAINTAIN LONG-TERM strains, influence of associated side effects of some ARVs EFFICACY AGAINST HIV-1 [43] and problems with adherence and failure of cART in The 26 ARVs currently used in the clinic target the treatment-naïve and treatment-experienced patients drives a three HIV-1 enzymes: RT, IN, protease (PR), and the viral continuous need for new classes of antiretrovirals. To entry machinery comprising HIV-1 envelope or its cellular address these problems, there are currently several ARV coreceptor, CCR5. Over half of these clinically-available drugs in clinical development (Table 1). Some are new drugs drugs inhibit the polymerase function of HIV-1 RT and are that fall into known classes and others are new classes of divided into two classes, the nucleoside/nucleotide RT ARV drugs, targeting other proteins in the viral life-cycle. inhibitors (NRTIs or NtRTIs) and non-nucleoside RT While the identification of new HIV drug targets is a inhibitors (NNRTIs). NRTIs are active once they have been promising approach for generating diverse drug classes, phosphorylated by host cell kinases [19] and designed to many programs remain focussed on the proven targets RT terminate extension of the nucleic acid chain during DNA and IN. Here, we highlight recent studies for defining novel polymerisation [20]. NNRTIs are allosteric inhibitors of drug scaffolds using a newer approach called FBDD for HIV-1 RT that bind to a pocket adjacent to the polymerase development of novel antiretrovirals against HIV-1 RT and active site that does not form until bound by inhibitor [21]. IN. When NNRTIs bind to this NNRTI binding pocket (NNIBP), 3. USING FRAGMENT BASED DRUG DISCOVERY they cause a large conformational change in RT that affects polymerase activity [22-23], RNase H activity [24] and 3.1. What are “fragments” and why use FBDD? enhances RT dimerisation [25-26]. The remaining drugs in Current industrial drug discovery programs are initiated clinical use target the strand-transfer reaction mediated by using high-throughput screening (HTS) of large chemical IN, viral polypeptide cleavage by HIV-1 PR, and viral entry libraries consisting of 105-106 compounds in a search for by directly blocking viral fusion by HIV-1 envelope, or by binding to the host CCR5 receptor that engages the HIV-1 detectable activity in a biological assay. The compound “hits” identified in such a screen would typically have virion during entry to the host cell. activities in the low micro- to nanomolar range deriving Despite an apparent abundance of ARVs, the utility of from a level of intrinsic chemical specificity for their target. many drugs is limited by the development of viral mutations Conversely, FBDD, also referred to as fragment based ligand that confer resistance to multiple drugs within the same design or fragment based lead generation, is a relatively new class, drastically limiting ARVs available for ongoing approach designed to identify minimally functionalised treatment. Mutations in HIV-1 RT that evade inhibition by compounds as chemical building blocks for strategic NRTIs usually sterically interfere with drug binding (e.g optimisation into larger potent drug molecules [44]. The core M184V, Y115F, Q151M, L74V and K65R) [27-32]. principle of FBDD is that small compounds (fragments) are Thymidine analog mutations (TAMs) (M41L, D67N, K70R, capable of making high quality interactions with the target L210W, T215Y/F, and K219Q/E/N) in HIV-1 RT develop in protein and, even though their affinity for the protein is low, the presence of NRTIs that are thymidine analogs, they can be elaborated into larger, high affinity inhibitors zidovudine and stavudine, and can reverse the primer with good biopharmaceutical properties [45]. FBDD is also termination step [33-34]. Mutations in HIV-1 RT such as distinct in its focus on biophysical methods to assess binding TAMs are known to decrease susceptibility to most drugs in affinity rather than exclusively focussed on biological Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 3 activity assays to prioritise chemical compounds. FBDD index (BEI) [60] which is appropriate if only potency data is screening requires far fewer compounds to yield hits than available. Other optimisation parameters can also be conventional library screening as the fragments are smaller included in the calculation such as the partition coefficient, (MW <300) and are able to cover a greater amount of logP (ratio of concentration of the compound in octanol to its chemical space and diversity as they represent less complex concentration in water), an indication of compound starting scaffolds [46]. With a predicted 1063 possible hydrophobicity, to generate ligand lipophilicity index molecules with 30 heavy atoms or less [47], simplifying the (LLEAT)[61], or even polar surface area for the surface starting scaffolds to fragment sized compounds (<300 MW) efficiency index (SEI) [62]. This list is by no means means fewer molecules need to be screened to achieve exhaustive. While LE and other indices relating binding similar coverage of chemical diversity as a collection of affinity to molecular size/shape have been adopted widely hundreds of thousands of larger, more functionalised for FBDD and HTS programs, their value for decision compounds (>300 MW). The FBDD ethos is that fragments making in drug discovery has recently been debated in the are far better suited as starting scaffolds for innovative literature [63-65]. molecular design and chemical elaboration in order to One of the key concepts underlying FBDD is that the produce better quality leads than traditional drug design careful selection of low MW and low lipophilicity (logP) strategies [44, 48]. compounds during library design, screening and hit Shuker et al [49] and Hajduk et al [50] reported the first validation can provide a greater chance of producing such practical fragment-based screening approaches in the mid favourable properties in the late stages of generating drug 1990s called ‘SAR by NMR’ - structure-activity relationship candidates [48]. Lipinski [66] famously proposed the ‘rule of (SAR) by nuclear magnetic resonance (NMR). Since then, five’, defining favourable physical properties for drug-like FBDD has been used to generate drug candidates for many compounds based on the properties of oral drugs that had drug targets and has been validated by the FDA’s approval reached the market. The success of FBDD in preserving of vemurafenib (Zelboraf, PLX4032) in 2011, Plexxikon’s favourable properties is difficult to evaluate given the drug for treatment of metastatic melanoma [51-53]. Several diversity in strategy and priorities in any given drug other compounds generated using FBDD are currently under discovery program; however, two separate analyses of evaluation in clinical trials including three in Phase III available FBDD data, Astex [48] and Astra-Zeneca [67] studies for various indications including Alzheimer’s disease sought to evaluate whether fragments generate leads with (MK-8931, BACE1 inhibitor) [54-55], breast cancer more favourable properties than larger, more conventional (LEE011, CDK4 inhibitor) [56] and lymphocytic leukemia HTS library hits. Both studies are in agreement that fragment (ABT-199, Bcl-2 inhibitor) as well as almost 30 more hits and their corresponding lead compounds are candidates in Phase I and II trials (www.clinicaltrials.gov). significantly smaller and less lipophilic when compared to a set of published HTS drugs. 3.2. Fragment-based approaches are driven by binding efficiency 3.3. The FBDD pipeline and methodology The small size of fragment hits means they have low FBDD approaches can be initiated on a much smaller affinity for their target (usually high µM to mM). However, scale than conventional drug screening which otherwise low affinity does not necessarily mean that the functional limits drug screening programs to the better resourced groups within the molecule are not individually making laboratories with access to large compound collections strong interactions with the target protein, just that there are typically found in the pharmaceutical industry. few such interactions. Consequently, FBDD programs have Consequently, FBDD has been very popular for initiating adopted efficiency metrics that establish binding quality, by drug discovery programs in academic settings that are combining measures of compound affinity (or potency) with intellectually invested in identifying and characterising other optimisable parameters such as molecular size or innovative drug targets and are already suitably equipped lipophilicity. One of the most widely used metrics is ligand with sensitive instrumentation for detecting weakly binding efficiency (LE) which the free energy of binding (ΔGbind = - molecules. Smaller libraries of fragment-sized compounds RT ln Kd) and the number of non-hydrogen or heavy atoms are accessible to both academic and industry-based to assess the contribution of each atom in the compound to laboratories and can be purchased from commercial sources, the overall binding interaction with its protein target [57-58]: or compiled in-house based on library design principles derived from over a decade of FBDD programs (reviewed in LE = ΔG / HAC = - RT ln (K ) / HAC bind d [68-69]). where LE is ligand efficiency, R is the ideal gas constant, T The FBDD toolkit often consists of multiple is temperature in Kelvin, K is the dissociation constant for d complementary biophysical and structural biology methods the binding interaction between the compound and the target used in combination to screen a fragment library against a protein and HAC is the “heavy atom count” or number of desired target [46]. NMR [70] and surface plasmon non-hydrogen atoms in the compound. While fragments have resonance (SPR) [71-72] are popular first-pass screening low affinity, they have only a small number of non-hydrogen methods as they are rapid and very sensitive. Ligand- atoms indicating that most of the molecule is contributing to observed NMR methods (such as saturation transfer binding. Constraints on the ultimate size of the target difference NMR or STD-NMR) are advantageous as the size compound (e.g. MW 500) and potency (e.g. 10 nM) dictate a of the protein target is not a limiting factor. With hardware minimum LE, which is often considered to be 0.3 and software now available for high throughput crystal kcal/mol/HAC although target LE values may vary [59]. screening, data measurement and processing, X-ray Other efficiency metrics can be used to evaluate ligand crystallography can be extremely informative in the quality; IC divided by MW provides the binding efficiency 50 screening phase for suitable targets [54, 73-75] as it can 4 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al. detect binding as well as elucidate the structural details of Other classes of RT inhibitors have been reported that the fragment binding site in one step. Both NMR and are under development but have not yet reached the clinic crystallography can resolve signals from cocktails of (Table 2). Nucleotide-competing RT inhibitors (NcRTIs) are compounds so libraries can be screened in mixtures of 5-10 distinct from NRTIs as they are not nucleoside analogs and compounds to decrease sample number and screening time do not cause chain termination. No inhibitors of HIV-1 RT [76]. Different methods used for fragment screening have RNase H activity are in clinical use although there have been also been reported to produce distinct sets of hits when using numerous studies in the area (Table 2). Inhibitors of HIV-1 the same fragment library against the same protein target RT dimerisation, T/P binding and inhibitors that bind to [77]. Using clever experimental design and high-throughput multiple sites are also subject of recent investigations (Table advancements, other biophysical methods have more 2). Maximising the number of lead ARV candidates entering recently been optimised for use in fragment screening and clinical evaluation is essential to increase the possibility that validation such as mass spectrometry [78-79], isothermal drugs in novel classes will be available for clinical use given titration calorimetry [80-82] and weak affinity the high failure rate in progressing a drug lead through the chromatography [83-86]. preclinical and clinical pathway to approval. A greater number of drug classes available for use in cART could Once hits have been confirmed to inhibit or bind to the drastically reduce the ability of HIV to develop viable drug- target, a medicinal chemistry program can be established to resistant strains, and thus increase the efficacy of cART elaborate the fragment scaffold into a suitable lead candidate. during long-term use. During this process, ligand affinities are measured using biophysical methods and/or bioassays and are almost always 4.2. HIV-1 RT as a target for FBDD strategies guided by X-ray structures of the fragment hits bound to the HIV-1 RT is a highly flexible enzyme [94]. This protein target which help to characterise binding sites and flexibility makes it an ideal target for allosteric inhibition, guide the medicinal chemistry. In most cases, fragment particularly since it is known that inhibitors bound to one site elaboration proceeds by fragment ‘growing’ but in some cases, multiple scaffolds can be ‘merged’ or linked to rapidly can affect the enzyme at a distant location. For example, NNRTIs can affect HIV-1 RT RNase H activity and enhance generate high affinity compounds. dimerisation as well as primarily blocking polymerase 4. FBDD AND HIV-1 REVERSE TRANSCRIPTASE activity (Section 2). A wealth of structural information is available for this target, with over 200 crystal structures of 4.1. HIV-1 RT structure and known drug binding sites HIV-1 RT in the Protein Data Bank (PDB) HIV-1 RT is an essential viral enzyme that converts the (http://www.rcsb.org/), in apo form or bound to various single-stranded viral RNA genome to a double-stranded inhibitors and/or substrate variants. One advantage of an DNA copy to be integrated into the host genomic DNA. RT FBDD approach is that fragments are small enough to act as acts early in the viral life cycle, soon after entry to the host probes to identify allosteric binding sites of a cell mediated by the HIV-1 envelope complex. It is a multi- conformationally flexible target. Depending on the screening functional enzyme with both RNA-dependent and DNA- strategy used, fragment screens have the potential to either dependent DNA polymerase activities as well as RNase H find novel chemical scaffolds that target clinically relevant activity, which is responsible for digesting RNA in the RNA- binding sites, or to probe for new allosteric binding sites to DNA duplex. All three functions are necessary to generate a exploit for drug design. Both approaches have been used in double-stranded DNA copy of the viral RNA genome [21, three FBDD programs against HIV-1 RT. 87]. HIV-RT is a heterodimer comprised of the p66 and p51 4.3. FBDD screening against HIV-1 RT to find novel subunits, where p66 is catalytic subunit and p51 is structural. NNRTI scaffolds It has been described as a hand-shaped molecule where p66 forms the polymerase domain from fingers, thumb and palm Geitmann and colleagues at Beactica AB (Sweden) [95] subdomains, as well as a connection subdomain that joins the used an FBDD approach to identify novel scaffolds as a RNase H and polymerase domains [88-89]. The nucleic acid basis for developing new NNRTIs with activity against well- substrate, or template/primer (T/P), binds in a groove that known drug-resistant variants of HIV-1. Their approach was runs through both the polymerase and RNase H active sites. to find scaffolds that likely bind to the NNIBP of wild-type Only two classes of HIV RT inhibitor are available for HIV-1 RT using a combination of a binding assay and an clinical use, the NRTI and NNRTIs, which have been activity assay. A library of ~1000 fragment sized compounds described in section 2 above. (MW <300 Da) was first screened for binding to HIV-1 RT using a SPR assay [96] yielding 96 fragments (~9% library). There are four NNRTIs in clinical use, two first These hits were then simultaneously screened for inhibition generation NNRTIs, nevirapine and efavirenz, and two next- of RT polymerase activity in an enzymatic assay as well as generation NNRTIs, rilpivirine and etravirine [90]. The for their ability to compete with the NNRTI, nevirapine, for NNRTIs show more variety in their chemical structures binding to the NNIBP. Only 8 compounds competed with compared to the NRTIs, although all four inhibit enzyme nevirapine and reproducibly demonstrated an IC value less function by binding the allosteric NNIBP. While cross- 50 than 1 mM. The final screen was resistance profiling to find resistant HIV-1 mutants have been reported for the compounds that maintained activity against the drug- clinically-available NNRTIs extensive structural analyses resistant RT variants (K103N, Y181C, L100I). In the final suggest that properties such as strategic inhibitor flexibility analysis, one of the 8 fragments, compound 1 (Table 3), had could help maintain a high genetic barrier to clinically- reproducible inhibitory activity in the enzymatic assay relevant resistance mutations that arise in the NNIBP [91- against all four variants of HIV-1 RT (IC < 25 µM). 93]. 50 Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 5

FBDD offers several advantages for a drug discovery domain of HIV-1 RT (428, RNase H primer grip adjacent, program aimed at finding novel NNRTI scaffolds. NNRTIs 507). bind to HIV-1 RT by an “induced fit” mechanism, where the Three of the seven novel binding sites were binding pocket, NNIBP, is evident only when an inhibitor is demonstrated to be inhibitory when the fragments bound in bound [21]. Due to their small molecular size compared to compounds used for conventional HTS, fragments could be these pockets were tested in a dual activity assay that detects inhibition of either RNase H or DNA polymerase activity better able to probe the NNIBP for new binding modes with simultaneously. The fragments bound in these sites have the ability to evade resistance mutations as they arise. varying potency in the mid-micromolar range (Table 3). Conventional screening strategies are usually based on broad 5 6 Based on the structural analysis, these three binding sites activity assays and use very large libraries (>10 -10 ) of have differing potential as druggable sites. The “NNRTI highly functionalised compounds that would be much less likely to have yielded similar numbers of hits. One novel adjacent” binding site is located near the NNRTI binding site identified by the binding of 2 ( . This site is located scaffold from a library of ~1000 compounds represents a Table 3) at the dimerization interface between the RT subunits. very respectable hit rate of 0.1%. The potency threshold for a Compound 2 made contacts with side chains from both p66 desirable compound is also likely to be much more stringent and p51 (Figure 1A). Although this pocket is discrete and for larger compounds in HTS, limiting the likelihood of contains conserved residues less likely to tolerate mutation identifying compounds as hits, and for progressing to competition or resistance screens so early in the process. under selective drug pressure, the authors note that any inhibitors designed to bind to the NNRTI adjacent pocket Because Geitman et al [95] could incorporate selective would need to address less conserved residues forming key screening strategies like the competition assay and resistance inhibitor interactions such as T165 and T139, and NNRTI- profiling so soon after the initial library screen, they were resistant mutations such as Y181C and E138K. The second able to identify a novel NNRTI scaffold with desired novel inhibitory site described by Bauman and colleagues inhibitory properties from a small number of compounds in only three screening steps. [98] was the Incoming Nucleotide Binding Site, located adjacent to both the polymerase active site and the nucleotide The path required to chemically evolve a low binding pockets of RT. Movement of residue Q151 on potency/low affinity fragment scaffold with desirable binding of 3 (Table 3) opens up the pocket, where compound properties into a high potency NNRTI drug lead is less clear. 3 makes contacts with a catalytic aspartate (D185) as well as No further reports describing optimisation of 1 (Table 3) into other highly conserved residues that comprise the pocket a novel NNRTI lead or candidate have been published, (Figure 1B). The authors highlight this pocket as one of the although the authors of the SPR fragment screening study two most appealing for drug design initiatives against RT as [95] published a second study in the same issue of J Med an inhibitor bound here could directly interfere with both Chem [97] analysing the effectiveness of fragment-based nucleotide binding and polymerase catalytic activity. The strategies to find NNRTI fragment scaffolds binding to HIV- third novel inhibitory binding site that was identified as very 1 RT. The authors focused their analysis on understanding appealing for future drug design is the “Knuckles” site, how best to optimise fragments into high potency leads by referring to its location between the p66 “fingers” and characterising fragment-sized compounds derived directly “palm” domains of the hand-shaped RT molecule. This site from substructures of known NNRTIs with nanomolar has been characterised by structures of RT bound to three binding affinity constants (KD). Surprisingly, they found that separate compounds, 4, 5 and 6 (Table 3) that have defined many of the NNRTI substructures had very weak binding the flexibility in this pocket and suggest several affinities and low LE values compared to the parent NNRTI. opportunities for elaborating fragments. Although only Furthermore, analysis of binding energies of the NNIBP led compound 5 was found to be inhibitory [98](Table 3), the to this group reporting that there might not be a fragment trifluoromethoxy and phenol groups of compounds 4 and 5 binding site or “hotspot” in the NNIBP. These findings make key interactions in the Knuckles pocket, exposing highlight challenges faced in elaborating smaller, weaker S117 and M164. Bauman et al suggest expansion at residue binding fragments into higher potency compounds against a I167, as movement of K166 allows binding of larger conformationally flexible target such as HIV-1 RT. compounds such as 6 (Figure 1C). All three of these sites demonstrate potential for further elaboration of fragments or 4.4. FBDD screening against HIV-1 RT to identify novel de novo structure-based drug design towards developing new allosteric binding sites for drug design classes of HIV-1 RT inhibitors. The Arnold group at Rutgers University took an Crystallography has proven to be a powerful and highly alternate approach and used fragment screening to search for informative tool to screen fragment libraries, as it not only novel allosteric binding sites rather than mining a known determines binding to the target, but also the location of the inhibitory pocket [98]. A library of 742 fragment-sized binding site and direction for chemical elaboration of molecules was screened by X-ray crystallography, fragments. It does, however, require significant target- discovering 34 fragments that bind to RT at 16 different specific optimisation including the ability to generate very binding sites. Seven of these sites have been reported in high resolution structures of fragment complexes. The detail [98]. By blocking the NNIBP during the screening Arnold group had to engineer a construct of HIV-1 RT, process using rilpivirine, their screen was able to select for RT52A, to produce crystals that diffracted well enough to fragments that bound to previously unknown allosteric resolve fragments bound to RT [99]. While this fragment pockets. The seven identified binding sites are located in the screening strategy was successful in identifying three polymerase domain (incoming nucleotide binding site, inhibitory sites, the remaining four of the seven sites knuckles, NNRTI adjacent and 399) and the RNase H described were not found to be inhibitory. While lack of inhibition discourages pursuit for drug development, these 6 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al. pockets could require larger inhibitors to exert any inhibitory improved potency from only a small structure-activity study effect [98]. For example, structural analysis of 7 (Table 3) suggests that these scaffolds are promising candidates for bound to the RNase H Primer Grip Adjacent site suggests it further optimization in an ongoing drug development could be expanded into the RNase H active site guided by program. This approach shows that mechanistic the crystal structure to generate an inhibitor. Since characterization could be an effective tool in an FBDD publication of this crystallographic fragment screen in 2013 program, particularly given the uncertainty of obtaining [98], there have been no further reports of inhibitors cocrystal structures of the compound hits attracting the exploiting these novel sites. greatest interest. 4.5. FBDD screening against HIV-1 RT to discover novel 5. FBDD AND HIV-1 INTEGRASE allosteric inhibitors 5.1. HIV-1 IN structure and known drug binding sites In the most recently reported FBDD approach targeting HIV-1 RT, the Tachedjian group in Melbourne, Australia HIV-1 IN catalyses the insertion of the double-stranded [100] used STD-NMR to screen a library of 630 fragments DNA copy of the viral genome, generated by RT, into the against HIV-1 RT to search for allosteric inhibitors at sites host chromosomal DNA [105]. The viral genome then other than the NNIBP. STD-NMR was followed by a persists in the host genome whereby the host secondary screen using a HIV-1 RT polymerase activity transcription/translation machinery propagates both viral assay, where eight fragment hits were identified with proteins and RNA to generate new viral particles. IN- micromolar potencies against the DNA polymerase function mediated integration is a two-step process consisting of of wild-type (WT) HIV-1 RT. Only three of the hits, 8-10 firstly, excision of a dinucleotide from the viral DNA called (Table 3) maintained a similar potency against WT RT and the “3’-processing step”, followed by “strand transfer three clinically relevant NNRTI-resistant RT mutants, reaction” integrating viral DNA into the host genome. The K103N, Y181C and G190A. In contrast to Bauman et al functional IN unit is a tetramer consisting of two active sites, [98], this group [100] then used biochemical assays to gather where each IN monomer has three domains with distinct evidence about the mechanisms of action and possible functions – an N-terminal domain (NTD) HHCC-type zinc binding sites on the enzyme for the top three fragment hits. finger for protein multimerisation, a central core domain Two of three fragments, 9 and 10, were shown to compete (CCD) for DNA substrate recognition and containing the with the HIV-1 RT substrates, nucleotide and T/P, catalytic “DDE motif”, and a C-terminal domain (CTD) for respectively, suggesting that these two compounds could be non-specific DNA binding, stabilising the IN-DNA complex inhibiting the enzyme allosterically with fragment 9 likely during translocation. acting at or near the polymerase active site, such as the The three IN inhibitors currently in clinical use are INSTIs NcRTI binding site [101] or another allosteric site. Fragment that bind to the catalytic IN active site. Other classes of IN 10 was also shown to inhibit RNase H activity, although it is inhibitors have been reported, primarily allosteric inhibitors unclear whether inhibition of both polymerase and RNase H targeting various sites on the IN CCD domain (Table 2). The activities are the result of binding at one or more sites on RT. most prominent class of allosteric IN inhibitors are the While both these inhibitory mechanisms have been described ALLINIs (allosteric IN inhibitors) that target a site on HIV-1 in the literature for structurally unrelated inhibitors (Table IN at the dimer interface that interacts with a host cell factor, 2), their binding sites could be distinct. Fragment 10 was lens epithelial-derived growth factor (LEDGF), which is even shown to have activity against HIV-1 replication in cell essential for integration [106-109]. LEDGF tethers the IN- culture (EC50 = 20 µM), likely at the reverse transcription DNA complex to the chromosome, and is also packaged into step as determined in a time of addition assay. These virions [110]. The ALLINIs, first coined as LEDGINs properties are remarkable for such a small molecule with few (LEDGF-IN inhibitors) by the Debyser group [111], bind at distinctive chemical features, particularly since other known the CCD-CCD interface of two IN subunits and compete inhibitors of T/P (i.e. SY-3E4 [102], KM-1 [103] and with LEDGF for binding to the same pocket. These APEX57219 [104], Table 3) are large and undesirable as inhibitors have been the subject of extensive characterisation drug development candidates. A small structure-activity with some compounds in advanced preclinical development relationship analysis revealed three related compounds with (reviewed in [112-114], Table 2). While there is not yet a 3-5 fold higher potency than 8-10, and one of these related clinical study evaluating the genetic barrier of ALLINIs to compounds, 11 (Table 3), was shown to maintain T/P the emergence of HIV resistance mutations, in vitro selection competition and activity against HIV-1 in cell culture, has suggested that single mutations could confer drug similar to its parent compound, 8. resistance to this class although there is limited cross- This study represents a novel approach to FBDD for resistance with the existing INSTIs [111, 115]. Although this this HIV-1 target. While the focus for FBDD is often on class of inhibitors were designed to inhibit IN function to biophysical assays and characterization, it can be difficult to block HIV replication, ALLINIs also stabilize interacting IN judge the appropriate timing to assess the inhibitory potential subunits, and promote aberrant, higher order IN of allosteric inhibitors or binding sites. In this study [100], multimerization leading to impaired maturation of virus activity assays were used early in the screening process particles [115-116]. The effect on multimerization of IN is while taking steps to minimise false positives due to now being exploited directly as a strategy for drug compound aggregation at high concentrations. Mechanistic development (Table 2)[117]. Other classes of inhibitors have assays were also used to characterise and prioritise hits from been reported in a handful of studies including peptide-based the fragment screen for further development. The fact the inhibitors of the LEDGF interaction and multimerisation, as Tachedjian group [100] were able to identify additional well as one inhibitor in a new class called allosteric, non- compounds related to the top three fragment hits that have catalytic site inhibitors (NCINIs)(Table 2). Very few studies have been published on NCINIs, most likely due to the Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 7 proprietary nature of the lead compound [118-119], which structures of 15 of these fragment hits with HIV-1 INCORE123 has recently entered Phase I clinical evaluation (Table 1). (IN-CCD with four solubilising mutations C56S, W131D, W139D and F185H) were determined, revealing several 5.2. HIV-1 IN as a target for FBDD strategies fragments bound to a novel binding site, termed the fragment HIV-1 IN is an essential viral enzyme that has been a binding pocket (FBP), which is located at the dimer successful drug target for HIV-1 treatment, with three ARVs interface, adjacent to the LEDGF binding site. Two in clinical use – raltegravir, elvitegravir and dolutegravir. IN fragments bound in overlapping sites in the FBP, KM00835 inhibitors are well tolerated in patients and the two newer IN (12) and SB00942 (13) (Table 4). Based on the position of inhibitors, elvitegravir and dolutegravir, have a high genetic the overlapping chemical groups, the thiophene of 12 and the barrier to resistance, which makes IN an attractive target for aromatic ring of 13, the two compounds were merged to development of new ARVs. Nevertheless, HIV strains that generate a series of four new compounds. Crystal structures are cross-resistant to IN inhibitors have already begun to of the new series bound to HIV-1 INCORE123 revealed that emerge (section 2 above) as these inhibitors are all members some features of the original compounds were maintained of the same class, the IN strand transfer inhibitors (INSTIs). and bound with similar weak affinity (KD ~ 3-6 mM). There is now increasing motivation to develop novel classes Introduction of a bromine to one of the compounds in the of IN inhibitors for HIV-1 treatment and prevention. merged series, 14 (Table 4), displaced a key water-mediated hydrogen bond with the S195 side chain seen in the Like HIV-1 RT, HIV-1 IN is a multimeric flexible structure, INCORE123-12 (Figure 2A, left), and formed a direct enzyme with the potential for novel allosteric sites perhaps interaction with S195 (Figure 2A, right). This new with an “induced fit” mechanism that could be identified interaction was exclusive to one compound, 14, and resulted using fragment-sized probes in an FBDD screening program. in a 17- to 35-fold improvement in the binding affinity [128] IN is, however, a large tetrameric protein that has posed a over the parent compounds (Table 4). The fragments and greater structural challenge than RT. Currently no crystal merged compound series were not tested in any integrase structures are available for the full-length HIV-1 IN, activity assays for their inhibitory potency and the functional although structures of the three isolated domains are relevance of the FBP remains unknown. available [120-123] and the crystal structure of a related retroviral integrase complex from prototype foamy virus The second group screened the Maybridge Ro3 [124] has been used to generate full-length homology models fragment library against HIV-1 IN using a combination of [125-126] that can be used for rational drug design against SPR and STD-NMR [130]. The library was purchased from HIV-1 IN. Most drug discovery programs are using relevant Maybridge at slightly different time from Wielens et al [128] portions of IN depending on the site to be targeted, as well as meaning that the two libraries had ~90% compounds in substantial engineering to introduce mutations to improve common. Crystal structures were determined for six of protein solubility for screening assays and structural studies sixteen fragment hits from the screening phase, in complex [127-130] (see section 4.2 & 4.3). with another optimised IN-CCD construct, INcore3H [134]. One of these hits, a N-benzyl indolinone, 15 (Table 4) was Similar to the approaches used for HIV-1 RT, FBDD studies bound in a novel pocket on HIV-1 IN defined by Q62, H183 targeting HIV-1 IN have exploited both the LEDGF/IN and S147, located near the mobile loop at the dimer interface interaction as well as searching for novel allosteric binding (Figure 2B). Compound 15 inhibited HIV-1 IN with sites using a variety of fragment-based methodologies. In micromolar potency (IC50 = 259 µM) in a strand transfer addition to a few computationally-based screens of virtual assay. A similar binding site had been previously observed in libraries [131-133], there have been two fragment-based a crystal structure of a related integrase from avian sarcoma screening studies targeting HIV-1 IN [128, 130] leading to virus (ASV) complexed with inhibitor Y3 [135]. Using X- several novel inhibitor scaffolds targeted to the LEDGF site ray crystallography, molecular modelling and the strand and two new allosteric sites on HIV-1 IN, the “FBP” and transfer assays, SAR studies of 19 compounds in three “Y3 site”. These two screening programs are described related chemical series, N-benzyl indoline-2,3-diones, N- below. benzyl indolin-2-ones, N-benzyl indolinones revealed 5.3. STD-NMR and SPR screening of a fragment library several avenues for further development of potent inhibitors against HIV-1 IN at this novel site. One compound, 16 (Table 4), was 45-fold more potent than the parent compound, 15. Molecular Two groups in Melbourne, Australia screened the same docking of compound 16 bound to HIV-1 IN-CCD suggests Maybridge fragment library against HIV-1 IN using two that the increase in potency potentially arises from different approaches [77]. A group at Monash University strengthening interactions with residues Q62 and H183 that used STD-NMR as the primary screen [128] while a second define the binding site. However, molecular dynamics group at CSIRO used SPR [130] to screen the library. Both simulations suggest that the conformation of HIV-1 IN that groups used X-ray crystallography to characterise fragment forms these key interactions with 16 is not the most hits using a similar IN-CCD construct. Despite using almost energetically favourable and that this inhibitor could be the same set of compounds to screen against the same target, binding in multiple conformations [136]. these two different approaches produced distinct sets of hit compounds. Two structurally similar fragments, 17 and 18 (Table 4), identified by Rhodes et al [130] were bound to the In the first approach, NMR screening of the Maybridge LEDGF site on HIV-1 IN, and their SAR was explored in a Ro3 fragment library of 500 compounds yielded 62 follow-up study [136]. Analogues of 17 and 18 were confirmed fragment hits [128]. Compounds were first investigated using X-ray crystallography, a strand-transfer screened as cocktails of ~5 compounds by STD-NMR then 15 assay, a cell-based assay and the Alphascreen assay for the followed by N-HSQC of individual hits. Cocrystal IN-LEDGF binding site [137] designed to specifically assess 8 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al. compounds that compete for LEDGF binding to HIV-1 IN. cytopathic retroviruses (HTLV-III) from patients with aids The most promising compound from this study, 19 was and pre-aids. Science. 1984, 224 (4648), 497-500. active in both biochemical assays in the micromolar range as 3. Schupbach, J.; Popovic, M.; Gilden, R. V.; Gonda, well as against HIV-1 replication in cell culture (EC50 = 29 M. A.; Sarngadharan, M. G.; Gallo, R. C., Serological µM). analysis of a subgroup of human t-lymphotropic retroviruses The crystal structures of the IN-fragment complexes (HTLV-III) associated with aids. Science. 1984, 224 (4648), determined by Wielens et al [77, 128] have been used in 503-505. subsequent virtual fragment screens against HIV-1 IN [133, 4. Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.; 138] although no further development of the inhibitor Nugeyre, M. T.; Chamaret, S.; Gruest, J.; Dauguet, C.; scaffolds or exploitation of the novel binding sites from Axler-Blin, C.; Vezinet-Brun, F.; Rouzioux, C.; Rozenbaum, either study has been published to date. Since Peat et al W.; Montagnier, L., Isolation of a t-lymphotropic retrovirus [136] published the study describing the novel LEDGF from a patient at risk for acquired immune deficiency inhibitors, the additional abilities of LEDGF inhibitors to syndrome (aids). Science. 1983, 220 (4599), 868-871. promote aberrant HIV-1 IN multimerisation and HIV virion 5. UNAIDS Unaids report on the global aids epidemic maturation have been characterised (see section 5.1.), 2013; 2013. suggesting that development of this class of inhibitors is not 6. Zhang, F.; Dou, Z.; Ma, Y.; Zhao, Y.; Liu, Z.; as straight forward as first anticipated. However, recent work Bulterys, M.; Chen, R. Y., Five-year outcomes of the china [113, 116-117, 139] has provided a greater understanding of national free antiretroviral treatment program. Ann Intern how these effects could be exploited in the development of Med. 2009, 151 (4), 241-251, W-252. novel classes of ARVs targeting HIV-1 IN. 7. Kumarasamy, N.; Solomon, S.; Chaguturu, S. K.; CONCLUSION Cecelia, A. J.; Vallabhaneni, S.; Flanigan, T. P.; Mayer, K. H., The changing natural history of HIV disease: Before and There is a constant need for new ARVs to use for HIV- after the introduction of generic antiretroviral therapy in 1 treatment and prevention strategies to combat the southern india. Clin Infect Dis. 2005, 41 (10), 1525-1528. emergence of drug resistance. While ongoing research has 8. Palella, F. J., Jr.; Delaney, K. M.; Moorman, A. C.; identified new inhibitory binding sites on proven targets such Loveless, M. O.; Fuhrer, J.; Satten, G. A.; Aschman, D. J.; as HIV-1 RT and HIV-1 IN, many of the ARVs currently in clinical trials for HIV-1 fall into the same classes as existing Holmberg, S. D., Declining morbidity and mortality among drugs in clinical use. While only a relatively new technique, patients with advanced human immunodeficiency virus FBDD has been successfully applied to identify high quality infection. HIV outpatient study investigators. N Engl J Med. drug scaffolds and new inhibitory binding sites against HIV- 1998, 338 (13), 853-860. 1 RT and IN as discussed above, as well as several other 9. Vittinghoff, E.; Scheer, S.; O'Malley, P.; Colfax, HIV-1 drug targets of HIV-1 including PR, TAT and G.; Holmberg, S. D.; Buchbinder, S. P., Combination envelope proteins, gp120 and gp41. Whether fragments have antiretroviral therapy and recent declines in aids incidence a high genetic barrier to the development of drug resistance and mortality. J Infect Dis. 1999, 179 (3), 717-720. and if they are able to inhibit HIV that are resistant to 10. Hogg, R. S.; Heath, K. V.; Yip, B.; Craib, K. J.; existing drugs remains to be determined. Overall, FBDD- O'Shaughnessy, M. V.; Schechter, M. T.; Montaner, J. S., based programs are making a valuable contribution to the Improved survival among HIV-infected individuals ongoing development of efficacious agents to be used in following initiation of antiretroviral therapy. JAMA. 1998, therapeutics and PrEP for HIV-1 worldwide. 279 (6), 450-454. CONFLICT OF INTEREST 11. Detels, R.; Munoz, A.; McFarlane, G.; Kingsley, L. A.; Margolick, J. B.; Giorgi, J.; Schrager, L. K.; Phair, J. P., The authors declare no conflict of interest. This work was Effectiveness of potent antiretroviral therapy on time to aids supported by the National Health and Medical Research and death in men with known HIV infection duration. Council of Australia (NHMRC) (Project Grant 1064900) and Multicenter aids cohort study investigators. JAMA. 1998, the Percy Baxter Charitable Trust, Perpetual Trustees, 280 (17), 1497-1503. awarded to C.F.L. and G.T. G. T. was supported by the 12. Kumarasamy, N.; Krishnan, S., Beyond first-line NHMRC Senior Research Fellowship 543105. HIV treatment regimens: The current state of antiretroviral ACKNOWLEDGEMENTS regimens, viral load monitoring, and resistance testing in resource-limited settings. Curr Opin HIV AIDS. 2013, 8 (6), The authors gratefully acknowledge the contribution to 586-590. this work of the Victorian Operational Infrastructure Support 13. Coffin, J. M., HIV population dynamics in vivo: Program received by the Burnet Institute. Implications for genetic variation, pathogenesis, and therapy. REFERENCES Science. 1995, 267 (5197), 483-489. 14. Pham, Q. D.; Wilson, D. P.; Law, M. G.; Kelleher, 1. Gallo, R. C.; Salahuddin, S. Z.; Popovic, M.; A. D.; Zhang, L., Global burden of transmitted HIV drug Shearer, G. M.; Kaplan, M.; Haynes, B. F.; Palker, T. J.; resistance and HIV-exposure categories: A systematic Redfield, R.; Oleske, J.; Safai, B.; et al., Frequent detection review and meta-analysis. AIDS. 2014, 28 (18), 2751-2762. and isolation of cytopathic retroviruses (HTLV-III) from 15. Stadeli, K. M.; Richman, D. D., Rates of emergence patients with aids and at risk for aids. Science. 1984, 224 of HIV drug resistance in resource-limited settings: A (4648), 500-503. systematic review. Antivir Ther. 2013, 18 (1), 115-123. 2. Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C., Detection, isolation, and continuous production of Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 9

16. Grant, R. M.; Lama, J. R.; Anderson, P. L.; K.; Hattox, S.; Adams, J.; et al., Inhibition of HIV-1 McMahan, V.; Liu, A. Y.; Vargas, L.; Goicochea, P.; replication by a nonnucleoside reverse transcriptase Casapia, M.; Guanira-Carranza, J. V.; Ramirez-Cardich, M. inhibitor. Science. 1990, 250 (4986), 1411-1413. E.; Montoya-Herrera, O.; Fernandez, T.; Veloso, V. G.; 24. Radzio, J.; Sluis-Cremer, N., Efavirenz accelerates Buchbinder, S. P.; Chariyalertsak, S.; Schechter, M.; Bekker, HIV-1 reverse transcriptase ribonuclease H cleavage, leading L. G.; Mayer, K. H.; Kallas, E. G.; Amico, K. R.; Mulligan, to diminished zidovudine excision. Mol Pharmacol. 2008, 73 K.; Bushman, L. R.; Hance, R. J.; Ganoza, C.; Defechereux, (2), 601-606. P.; Postle, B.; Wang, F.; McConnell, J. J.; Zheng, J. H.; Lee, 25. Tachedjian, G.; Orlova, M.; Sarafianos, S. G.; J.; Rooney, J. F.; Jaffe, H. S.; Martinez, A. I.; Burns, D. N.; Arnold, E.; Goff, S. P., Nonnucleoside reverse transcriptase Glidden, D. V.; iPrEx Study, T., Preexposure inhibitors are chemical enhancers of dimerization of the HIV chemoprophylaxis for HIV prevention in men who have sex type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 2001, with men. N Engl J Med. 2010, 363 (27), 2587-2599. 98 (13), 7188-7193. 17. Thigpen, M. C.; Kebaabetswe, P. M.; Paxton, L. A.; 26. Srivastava, S.; Sluis-Cremer, N.; Tachedjian, G., Smith, D. K.; Rose, C. E.; Segolodi, T. M.; Henderson, F. L.; Dimerization of human immunodeficiency virus type 1 Pathak, S. R.; Soud, F. A.; Chillag, K. L.; Mutanhaurwa, R.; reverse transcriptase as an antiviral target. Curr Pharm Des. Chirwa, L. I.; Kasonde, M.; Abebe, D.; Buliva, E.; Gvetadze, 2006, 12 (15), 1879-1894. R. J.; Johnson, S.; Sukalac, T.; Thomas, V. T.; Hart, C.; 27. Sarafianos, S. G.; Das, K.; Ding, J.; Boyer, P. L.; Johnson, J. A.; Malotte, C. K.; Hendrix, C. W.; Brooks, J. T.; Hughes, S. H.; Arnold, E., Touching the heart of HIV-1 drug Group, T. D. F. S., Antiretroviral preexposure prophylaxis resistance: The fingers close down on the dntp at the for heterosexual HIV transmission in botswana. N Engl J polymerase active site. Chem Biol. 1999, 6 (5), R137-146. Med. 2012, 367 (5), 423-434. 28. Sluis-Cremer, N.; Arion, D.; Kaushik, N.; Lim, H.; 18. Baeten, J. M.; Donnell, D.; Ndase, P.; Mugo, N. R.; Parniak, M. A., Mutational analysis of lys65 of HIV-1 Campbell, J. D.; Wangisi, J.; Tappero, J. W.; Bukusi, E. A.; reverse transcriptase. Biochem J. 2000, 348 Pt 1, 77-82. Cohen, C. R.; Katabira, E.; Ronald, A.; Tumwesigye, E.; 29. Martin, J. L.; Wilson, J. E.; Haynes, R. L.; Furman, Were, E.; Fife, K. H.; Kiarie, J.; Farquhar, C.; John-Stewart, P. A., Mechanism of resistance of human immunodeficiency G.; Kakia, A.; Odoyo, J.; Mucunguzi, A.; Nakku-Joloba, E.; virus type 1 to 2',3'-dideoxyinosine. Proc Natl Acad Sci U S Twesigye, R.; Ngure, K.; Apaka, C.; Tamooh, H.; Gabona, A. 1993, 90 (13), 6135-6139. F.; Mujugira, A.; Panteleeff, D.; Thomas, K. K.; Kidoguchi, 30. Deval, J.; Selmi, B.; Boretto, J.; Egloff, M. P.; L.; Krows, M.; Revall, J.; Morrison, S.; Haugen, H.; Guerreiro, C.; Sarfati, S.; Canard, B., The molecular Emmanuel-Ogier, M.; Ondrejcek, L.; Coombs, R. W.; mechanism of multidrug resistance by the q151m human Frenkel, L.; Hendrix, C.; Bumpus, N. N.; Bangsberg, D.; immunodeficiency virus type 1 reverse transcriptase and its Haberer, J. E.; Stevens, W. S.; Lingappa, J. R.; Celum, C.; suppression using alpha-boranophosphate nucleotide Partners Pr, E. P. S. T., Antiretroviral prophylaxis for HIV analogues. J Biol Chem. 2002, 277 (44), 42097-42104. prevention in heterosexual men and women. N Engl J Med. 31. Ray, A. S.; Basavapathruni, A.; Anderson, K. S., 2012, 367 (5), 399-410. Mechanistic studies to understand the progressive 19. Furman, P. A.; Fyfe, J. A.; St Clair, M. H.; development of resistance in human immunodeficiency virus Weinhold, K.; Rideout, J. L.; Freeman, G. A.; Lehrman, S. type 1 reverse transcriptase to abacavir. J Biol Chem. 2002, N.; Bolognesi, D. P.; Broder, S.; Mitsuya, H.; et al., 277 (43), 40479-40490. Phosphorylation of 3'-azido-3'-deoxythymidine and selective 32. Boyer, P. L.; Sarafianos, S. G.; Arnold, E.; Hughes, interaction of the 5'-triphosphate with human S. H., Analysis of mutations at positions 115 and 116 in the immunodeficiency virus reverse transcriptase. Proc Natl dntp binding site of HIV-1 reverse transcriptase. Proc Natl Acad Sci U S A. 1986, 83 (21), 8333-8337. Acad Sci U S A. 2000, 97 (7), 3056-3061. 20. Mitsuya, H.; Jarrett, R. F.; Matsukura, M.; Di 33. Meyer, P. R.; Matsuura, S. E.; So, A. G.; Scott, W. Marzo Veronese, F.; DeVico, A. L.; Sarngadharan, M. G.; A., Unblocking of chain-terminated primer by HIV-1 reverse Johns, D. G.; Reitz, M. S.; Broder, S., Long-term inhibition transcriptase through a nucleotide-dependent mechanism. of human T-lymphotropic virus type III/lymphadenopathy- Proc Natl Acad Sci U S A. 1998, 95 (23), 13471-13476. associated virus (human immunodeficiency virus) DNA 34. Arion, D.; Kaushik, N.; McCormick, S.; Borkow, synthesis and rna expression in t cells protected by 2',3'- G.; Parniak, M. A., Phenotypic mechanism of HIV-1 dideoxynucleosides in vitro. Proc Natl Acad Sci U S A. resistance to 3'-azido-3'-deoxythymidine (AZT): Increased 1987, 84 (7), 2033-2037. polymerization processivity and enhanced sensitivity to 21. Sarafianos, S. G.; Marchand, B.; Das, K.; Himmel, pyrophosphate of the mutant viral reverse transcriptase. D. M.; Parniak, M. A.; Hughes, S. H.; Arnold, E., Structure Biochemistry. 1998, 37 (45), 15908-15917. and function of HIV-1 reverse transcriptase: Molecular 35. Whitcomb, J. M.; Parkin, N. T.; Chappey, C.; mechanisms of polymerization and inhibition. J Mol Biol. Hellmann, N. S.; Petropoulos, C. J., Broad nucleoside 2009, 385 (3), 693-713. reverse-transcriptase inhibitor cross-resistance in human 22. Das, K.; Martinez, S. E.; Bauman, J. D.; Arnold, E., immunodeficiency virus type 1 clinical isolates. J Infect Dis. HIV-1 reverse transcriptase complex with DNA and 2003, 188 (7), 992-1000. nevirapine reveals non-nucleoside inhibition mechanism. Nat 36. Clavel, F.; Hance, A. J., HIV drug resistance. N Struct Mol Biol. 2012, 19 (2), 253-259. Engl J Med. 2004, 350 (10), 1023-1035. 23. Merluzzi, V. J.; Hargrave, K. D.; Labadia, M.; 37. Ren, J.; Nichols, C.; Bird, L.; Chamberlain, P.; Grozinger, K.; Skoog, M.; Wu, J. C.; Shih, C. K.; Eckner, Weaver, K.; Short, S.; Stuart, D. I.; Stammers, D. K., 10 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

Structural mechanisms of drug resistance for mutations at D'Andrea, K.; Koehler, A.; Stumm, M.; Lin, P. S.; Lee, R. J.; codons 181 and 188 in HIV-1 reverse transcriptase and the Grippo, J.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G. improved resilience of second generation non-nucleoside A.; Sosman, J. A.; Chapman, P. B.; Flaherty, K. T.; Xu, X.; inhibitors. J Mol Biol. 2001, 312 (4), 795-805. Nathanson, K. L.; Nolop, K., Clinical efficacy of a raf 38. Hsiou, Y.; Ding, J.; Das, K.; Clark, A. D., Jr.; inhibitor needs broad target blockade in braf-mutant Boyer, P. L.; Lewi, P.; Janssen, P. A.; Kleim, J. P.; Rosner, melanoma. Nature. 2010, 467 (7315), 596-599. M.; Hughes, S. H.; Arnold, E., The lys103asn mutation of 52. Lee, J. T.; Li, L.; Brafford, P. A.; van den Eijnden, HIV-1 rt: A novel mechanism of drug resistance. J Mol Biol. M.; Halloran, M. B.; Sproesser, K.; Haass, N. K.; Smalley, 2001, 309 (2), 437-445. K. S.; Tsai, J.; Bollag, G.; Herlyn, M., Plx4032, a potent 39. Fransen, S.; Gupta, S.; Danovich, R.; Hazuda, D.; inhibitor of the b-raf v600e oncogene, selectively inhibits Miller, M.; Witmer, M.; Petropoulos, C. J.; Huang, W., Loss v600e-positive melanomas. Pigment Cell Melanoma Res. of raltegravir susceptibility by human immunodeficiency 2010, 23 (6), 820-827. virus type 1 is conferred via multiple nonoverlapping genetic 53. Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; pathways. J Virol. 2009, 83 (22), 11440-11446. Mamo, S.; Bremer, R.; Gillette, S.; Kong, J.; Haass, N. K.; 40. Metifiot, M.; Vandegraaff, N.; Maddali, K.; Sproesser, K.; Li, L.; Smalley, K. S.; Fong, D.; Zhu, Y. L.; Naumova, A.; Zhang, X.; Rhodes, D.; Marchand, C.; Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Pommier, Y., Elvitegravir overcomes resistance to Rice, J.; Suzuki, Y.; Luu, C.; Settachatgul, C.; Shellooe, R.; raltegravir induced by integrase mutation y143. AIDS. 2011, Cantwell, J.; Kim, S. H.; Schlessinger, J.; Zhang, K. Y.; 25 (9), 1175-1178. West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. 41. Wensing, A. M.; Calvez, V.; Gunthard, H. F.; N.; Hirth, P.; Artis, D. R.; Herlyn, M.; Bollag, G., Discovery Johnson, V. A.; Paredes, R.; Pillay, D.; Shafer, R. W.; of a selective inhibitor of oncogenic b-raf kinase with potent Richman, D. D., 2014 update of the drug resistance antimelanoma activity. Proc Natl Acad Sci U S A. 2008, 105 mutations in HIV-1. Top Antivir Med. 2014, 22 (3), 642-650. (8), 3041-3046. 42. Clotet, B.; Feinberg, J.; van Lunzen, J.; Khuong- 54. Wang, Y. S.; Strickland, C.; Voigt, J. H.; Kennedy, Josses, M. A.; Antinori, A.; Dumitru, I.; Pokrovskiy, V.; M. E.; Beyer, B. M.; Senior, M. M.; Smith, E. M.; Nechuta, Fehr, J.; Ortiz, R.; Saag, M.; Harris, J.; Brennan, C.; T. L.; Madison, V. S.; Czarniecki, M.; McKittrick, B. A.; Fujiwara, T.; Min, S.; Team, I. N. G. S., Once-daily Stamford, A. W.; Parker, E. M.; Hunter, J. C.; Greenlee, W. dolutegravir versus darunavir plus ritonavir in antiretroviral- J.; Wyss, D. F., Application of fragment-based NMR naive adults with HIV-1 infection (flamingo): 48 week screening, x-ray crystallography, structure-based design, and results from the randomised open-label phase 3b study. focused chemical library design to identify novel microm Lancet. 2014, 383 (9936), 2222-2231. leads for the development of nm bace-1 (beta-site app 43. Blas-Garcia, A.; Esplugues, J. V.; Apostolova, N., cleaving enzyme 1) inhibitors. J Med Chem. 2010, 53 (3), Twenty years of HIV-1 non-nucleoside reverse transcriptase 942-950. inhibitors: Time to reevaluate their toxicity. Curr Med Chem. 55. Zhu, Z.; Sun, Z. Y.; Ye, Y.; Voigt, J.; Strickland, 2011, 18 (14), 2186-2195. C.; Smith, E. M.; Cumming, J.; Wang, L.; Wong, J.; Wang, 44. Kuo, L. C., Fragment-based drug design - tools, Y. S.; Wyss, D. F.; Chen, X.; Kuvelkar, R.; Kennedy, M. E.; practical approaches, and examples. Preface. Methods Favreau, L.; Parker, E.; McKittrick, B. A.; Stamford, A.; Enzymol. 2011, 493, xxi-xxii. Czarniecki, M.; Greenlee, W.; Hunter, J. C., Discovery of 45. Rees, D. C.; Congreve, M.; Murray, C. W.; Carr, cyclic acylguanidines as highly potent and selective beta-site R., Fragment-based lead discovery. Nat Rev Drug Discov. amyloid cleaving enzyme (bace) inhibitors: Part i--inhibitor 2004, 3 (8), 660-672. design and validation. J Med Chem. 2010, 53 (3), 951-965. 46. Erlanson, D. A., Introduction to fragment-based 56. Rader, J.; Russell, M. R.; Hart, L. S.; Nakazawa, M. drug discovery. Top Curr Chem. 2012, 317, 1-32. S.; Belcastro, L. T.; Martinez, D.; Li, Y.; Carpenter, E. L.; 47. Peironcely, J. E.; Reijmers, T.; Coulier, L.; Bender, Attiyeh, E. F.; Diskin, S. J.; Kim, S.; Parasuraman, S.; A.; Hankemeier, T., Understanding and classifying Caponigro, G.; Schnepp, R. W.; Wood, A. C.; Pawel, B.; metabolite space and metabolite-likeness. PLoS One. 2011, 6 Cole, K. A.; Maris, J. M., Dual cdk4/cdk6 inhibition induces (12), e28966. cell-cycle arrest and senescence in neuroblastoma. Clin 48. Murray, C. W.; Verdonk, M. L.; Rees, D. C., Cancer Res. 2013, 19 (22), 6173-6182. Experiences in fragment-based drug discovery. Trends 57. Bembenek, S. D.; Tounge, B. A.; Reynolds, C. H., Pharmacol Sci. 2012, 33 (5), 224-232. Ligand efficiency and fragment-based drug discovery. Drug 49. Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, Discov Today. 2009, 14 (5-6), 278-283. S. W., Discovering high-affinity ligands for proteins: Sar by 58. Hopkins, A. L.; Groom, C. R.; Alex, A., Ligand NMR. Science. 1996, 274 (5292), 1531-1534. efficiency: A useful metric for lead selection. Drug Discov 50. Hajduk, P. J.; Sheppard, G.; Nettesheim, D. G., Today. 2004, 9 (10), 430-431. Discovery of potent nonpeptide inhibitors of stromelysin 59. Hajduk, P. J., Fragment-based drug design: How using sar by NMR. Journal of the …. 1997. big is too big? J Med Chem. 2006, 49 (24), 6972-6976. 51. Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. 60. Abad-Zapatero, C.; Metz, J. T., Ligand efficiency N.; Cho, H.; Spevak, W.; Zhang, C.; Zhang, Y.; Habets, G.; indices as guideposts for drug discovery. Drug Discov Burton, E. A.; Wong, B.; Tsang, G.; West, B. L.; Powell, B.; Today. 2005, 10 (7), 464-469. Shellooe, R.; Marimuthu, A.; Nguyen, H.; Zhang, K. Y.; Artis, D. R.; Schlessinger, J.; Su, F.; Higgins, B.; Iyer, R.; Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 11

61. Mortenson, P. N.; Murray, C. W., Assessing the K.; Parker, M. W.; Peat, T. S.; Scanlon, M. J., Parallel lipophilicity of fragments and early hits. J Comput Aided screening of low molecular weight fragment libraries: Do Mol Des. 2011, 25 (7), 663-667. differences in methodology affect hit identification? J 62. Nissink, J. W., Simple size-independent measure of Biomol Screen. 2013, 18 (2), 147-159. ligand efficiency. J Chem Inf Model. 2009, 49 (6), 1617- 78. Maple, H. J.; Garlish, R. A.; Rigau-Roca, L.; Porter, 1622. J.; Whitcombe, I.; Prosser, C. E.; Kennedy, J.; Henry, A. J.; 63. Murray, C. W.; Erlanson, D. A.; Hopkins, A. L.; Taylor, R. J.; Crump, M. P.; Crosby, J., Automated protein- Keseru, G. M.; Leeson, P. D.; Rees, D. C.; Reynolds, C. H.; ligand interaction screening by mass spectrometry. J Med Richmond, N. J., Validity of ligand efficiency metrics. ACS Chem. 2012, 55 (2), 837-851. Med Chem Lett. 2014, 5 (6), 616-618. 79. Chen, X.; Qin, S.; Chen, S.; Li, J.; Li, L.; Wang, Z.; 64. Shultz, M. D., Setting expectations in molecular Wang, Q.; Lin, J.; Yang, C.; Shui, W., A ligand-observed optimizations: Strengths and limitations of commonly used mass spectrometry approach integrated into the fragment composite parameters. Bioorg Med Chem Lett. 2013, 23 based lead discovery pipeline. Sci Rep. 2015, 5, 8361. (21), 5980-5991. 80. Ciulli, A.; Williams, G.; Smith, A. G.; Blundell, T. 65. Shultz, M. D., Improving the plausibility of success L.; Abell, C., Probing hot spots at protein-ligand binding with inefficient metrics. ACS Med Chem Lett. 2014, 5 (1), 2- sites: A fragment-based approach using biophysical 5. methods. J Med Chem. 2006, 49 (16), 4992-5000. 66. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; 81. Kobe, A.; Caaveiro, J. M.; Tashiro, S.; Kajihara, D.; Feeney, P. J., Experimental and computational approaches to Kikkawa, M.; Mitani, T.; Tsumoto, K., Incorporation of estimate solubility and permeability in drug discovery and rapid thermodynamic data in fragment-based drug discovery. development settings. Adv Drug Deliv Rev. 1997, 23, 323- J Med Chem. 2013, 56 (5), 2155-2159. 325. 82. Mashalidis, E. H.; Sledz, P.; Lang, S.; Abell, C., A 67. Leeson, P. D.; St-Gallay, S. A., The influence of the three-stage biophysical screening cascade for fragment-based 'organizational factor' on compound quality in drug drug discovery. Nat Protoc. 2013, 8 (11), 2309-2324. discovery. Nat Rev Drug Discov. 2011, 10 (10), 749-765. 83. Meiby, E.; Knapp, S.; Elkins, J. M.; Ohlson, S., 68. Doak, B. C.; Morton, C. J.; Simpson, J. S.; Scanlon, Fragment screening of cyclin g-associated kinase by weak M. J., Design and evaluation of the performance of an NMR affinity chromatography. Anal Bioanal Chem. 2012, 404 (8), screening fragment library. Australian Journal of Chemistry. 2417-2425. 2013, 66 (12), 1465-1472. 84. Meiby, E.; Simmonite, H.; le Strat, L.; Davis, B.; 69. Boyd, S. M.; Turnbull, A. P.; Walse, B., Fragment Matassova, N.; Moore, J. D.; Mrosek, M.; Murray, J.; library design considerations. WIREs Comput Mol Sci. 2012, Hubbard, R. E.; Ohlson, S., Fragment screening by weak 2, 868-885. affinity chromatography: Comparison with established 70. Klages, J.; Coles, M.; Kessler, H., NMR-based techniques for screening against hsp90. Anal Chem. 2013, 85 screening: A powerful tool in fragment-based drug (14), 6756-6766. discovery. Analyst. 2007, 132 (7), 693-705. 85. Duong-Thi, M. D.; Meiby, E.; Bergstrom, M.; Fex, 71. Navratilova, I.; Hopkins, A. L., Emerging role of T.; Isaksson, R.; Ohlson, S., Weak affinity chromatography surface plasmon resonance in fragment-based drug as a new approach for fragment screening in drug discovery. discovery. Future Med Chem. 2011, 3 (14), 1809-1820. Anal Biochem. 2011, 414 (1), 138-146. 72. Perspicace, S.; Banner, D.; Benz, J.; Muller, F.; 86. Duong-Thi, M. D.; Bergstrom, M.; Fex, T.; Schlatter, D.; Huber, W., Fragment-based screening using Svensson, S.; Ohlson, S.; Isaksson, R., Weak affinity surface plasmon resonance technology. J Biomol Screen. chromatography for evaluation of stereoisomers in early 2009, 14 (4), 337-349. drug discovery. J Biomol Screen. 2013, 18 (6), 748-755. 73. Chilingaryan, Z.; Yin, Z.; Oakley, A. J., Fragment- 87. Goff, S. P., Retroviral reverse transcriptase: based screening by protein crystallography: Successes and Synthesis, structure, and function. J Acquir Immune Defic pitfalls. Int J Mol Sci. 2012, 13 (10), 12857-12879. Syndr. 1990, 3 (8), 817-831. 74. Davies, T. G.; Tickle, I. J., Fragment screening 88. Kohlstaedt, L. A.; Wang, J.; Friedman, J. M.; Rice, using x-ray crystallography. Top Curr Chem. 2012, 317, 33- P. A.; Steitz, T. A., Crystal structure at 3.5 a resolution of 59. HIV-1 reverse transcriptase complexed with an inhibitor. 75. Caliandro, R.; Belviso, D. B.; Aresta, B. M.; de Science. 1992, 256 (5065), 1783-1790. Candia, M.; Altomare, C. D., Protein crystallography and 89. Jacobo-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A. fragment-based drug design. Future Med Chem. 2013, 5 D., Jr.; Lu, X.; Tantillo, C.; Williams, R. L.; Kamer, G.; (10), 1121-1140. Ferris, A. L.; Clark, P.; et al., Crystal structure of human 76. Tsai, Y.; McPhillips, S. E.; Gonzalez, A.; immunodeficiency virus type 1 reverse transcriptase McPhillips, T. M.; Zinn, D.; Cohen, A. E.; Feese, M. D.; complexed with double-stranded DNA at 3.0 a resolution Bushnell, D.; Tiefenbrunn, T.; Stout, C. D.; Ludaescher, B.; shows bent DNA. Proc Natl Acad Sci U S A. 1993, 90 (13), Hedman, B.; Hodgson, K. O.; Soltis, S. M., Autodrug: Fully 6320-6324. automated macromolecular crystallography workflows for 90. Sluis-Cremer, N., The emerging profile of cross- fragment-based drug discovery. Acta Crystallogr D Biol resistance among the nonnucleoside HIV-1 reverse Crystallogr. 2013, 69 (Pt 5), 796-803. transcriptase inhibitors. Viruses. 2014, 6 (8), 2960-2973. 77. Wielens, J.; Headey, S. J.; Rhodes, D. I.; Mulder, R. 91. Das, K.; Bauman, J. D.; Clark, A. D., Jr.; Frenkel, J.; Dolezal, O.; Deadman, J. J.; Newman, J.; Chalmers, D. Y. V.; Lewi, P. J.; Shatkin, A. J.; Hughes, S. H.; Arnold, E., 12 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

High-resolution structures of HIV-1 reverse 103. Wang, L. Z.; Kenyon, G. L.; Johnson, K. A., Novel transcriptase/tmc278 complexes: Strategic flexibility mechanism of inhibition of HIV-1 reverse transcriptase by a explains potency against resistance mutations. Proc Natl new non-nucleoside analog, km-1. J Biol Chem. 2004, 279 Acad Sci U S A. 2008, 105 (5), 1466-1471. (37), 38424-38432. 92. Zhan, P.; Liu, X.; Li, Z.; Pannecouque, C.; De 104. Sheen, C. W.; Alpturk, O.; Sluis-Cremer, N., Novel Clercq, E., Design strategies of novel NNRTIs to overcome high-throughput screen identifies an HIV-1 reverse drug resistance. Curr Med Chem. 2009, 16 (29), 3903-3917. transcriptase inhibitor with a unique mechanism of action. 93. Chen, X.; Liu, X.; Meng, Q.; Wang, D.; Liu, H.; De Biochem J. 2014, 462 (3), 425-432. Clercq, E.; Pannecouque, C.; Balzarini, J.; Liu, X., Novel 105. Brown, P. O., Integration of retroviral DNA. Curr piperidinylamino-diarylpyrimidine derivatives with dual Top Microbiol Immunol. 1990, 157, 19-48. structural conformations as potent HIV-1 non-nucleoside 106. Cherepanov, P.; Devroe, E.; Silver, P. A.; reverse transcriptase inhibitors. Bioorg Med Chem Lett. Engelman, A., Identification of an evolutionarily conserved 2013, 23 (24), 6593-6597. domain in human lens epithelium-derived growth 94. Bahar, I.; Erman, B.; Jernigan, R. L.; Atilgan, A. R.; factor/transcriptional co-activator p75 (LEDGF/p75) that Covell, D. G., Collective motions in HIV-1 reverse binds HIV-1 integrase. J Biol Chem. 2004, 279 (47), 48883- transcriptase: Examination of flexibility and enzyme 48892. function. J Mol Biol. 1999, 285 (3), 1023-1037. 107. Cherepanov, P.; Ambrosio, A. L.; Rahman, S.; 95. Geitmann, M.; Elinder, M.; Seeger, C.; Brandt, P.; Ellenberger, T.; Engelman, A., Structural basis for the de Esch, I. J.; Danielson, U. H., Identification of a novel recognition between HIV-1 integrase and transcriptional scaffold for allosteric inhibition of wild type and drug coactivator p75. Proc Natl Acad Sci U S A. 2005, 102 (48), resistant HIV-1 reverse transcriptase by fragment library 17308-17313. screening. J Med Chem. 2011, 54 (3), 699-708. 108. McKee, C. J.; Kessl, J. J.; Shkriabai, N.; Dar, M. J.; 96. Elinder, M.; Geitmann, M.; Gossas, T.; Kallblad, P.; Engelman, A.; Kvaratskhelia, M., Dynamic modulation of Winquist, J.; Nordstrom, H.; Hamalainen, M.; Danielson, U. HIV-1 integrase structure and function by cellular lens H., Experimental validation of a fragment library for lead epithelium-derived growth factor (LEDGF) protein. J Biol discovery using spr biosensor technology. J Biomol Screen. Chem. 2008, 283 (46), 31802-31812. 2011, 16 (1), 15-25. 109. Kessl, J. J.; Li, M.; Ignatov, M.; Shkriabai, N.; 97. Brandt, P.; Geitmann, M.; Danielson, U. H., Eidahl, J. O.; Feng, L.; Musier-Forsyth, K.; Craigie, R.; Deconstruction of non-nucleoside reverse transcriptase Kvaratskhelia, M., Fret analysis reveals distinct inhibitors of human immunodeficiency virus type 1 for conformations of in tetramers in the presence of viral DNA exploration of the optimization landscape of fragments. J or LEDGF/p75. Nucleic Acids Res. 2011, 39 (20), 9009- Med Chem. 2011, 54 (3), 709-718. 9022. 98. Bauman, J. D.; Patel, D.; Dharia, C.; Fromer, M. 110. Desimmie, B. A.; Weydert, C.; Schrijvers, R.; Vets, W.; Ahmed, S.; Frenkel, Y.; Vijayan, R. S.; Eck, J. T.; Ho, S.; Demeulemeester, J.; Proost, P.; Paron, I.; De Rijck, J.; W. C.; Das, K.; Shatkin, A. J.; Arnold, E., Detecting Mast, J.; Bannert, N.; Gijsbers, R.; Christ, F.; Debyser, Z., allosteric sites of HIV-1 reverse transcriptase by x-ray HIV-1 in/pol recruits LEDGF/p75 into viral particles. crystallographic fragment screening. J Med Chem. 2013, 56 Retrovirology. 2015, 12 (1), 16. (7), 2738-2746. 111. Christ, F.; Voet, A.; Marchand, A.; Nicolet, S.; 99. Bauman, J. D.; Das, K.; Ho, W. C.; Baweja, M.; Desimmie, B. A.; Marchand, D.; Bardiot, D.; Van der Himmel, D. M.; Clark, A. D., Jr.; Oren, D. A.; Boyer, P. L.; Veken, N. J.; Van Remoortel, B.; Strelkov, S. V.; De Hughes, S. H.; Shatkin, A. J.; Arnold, E., Crystal Maeyer, M.; Chaltin, P.; Debyser, Z., Rational design of engineering of HIV-1 reverse transcriptase for structure- small-molecule inhibitors of the LEDGF/p75-integrase based drug design. Nucleic Acids Res. 2008, 36 (15), 5083- interaction and HIV replication. Nat Chem Biol. 2010, 6 (6), 5092. 442-448. 100. La, J.; Latham, C. F.; Tinetti, R. N.; Johnson, A.; 112. Engelman, A.; Kessl, J. J.; Kvaratskhelia, M., Tyssen, D.; Huber, K. D.; Sluis-Cremer, N.; Simpson, J. S.; Allosteric inhibition of HIV-1 integrase activity. Curr Opin Headey, S. J.; Chalmers, D. K.; Tachedjian, G., Chem Biol. 2013, 17 (3), 339-345. Identification of mechanistically distinct inhibitors of HIV-1 113. Jurado, K. A.; Engelman, A., Multimodal reverse transcriptase through fragment screening. Proc Natl mechanism of action of allosteric HIV-1 integrase inhibitors. Acad Sci U S A. 2015, 112 (22), 6979-6984. Expert Rev Mol Med. 2013, 15, e14. 101. Freisz, S.; Bec, G.; Radi, M.; Wolff, P.; Crespan, 114. Metifiot, M.; Marchand, C.; Pommier, Y., HIV E.; Angeli, L.; Dumas, P.; Maga, G.; Botta, M.; Ennifar, E., integrase inhibitors: 20-year landmark and challenges. Adv Crystal structure of HIV-1 reverse transcriptase bound to a Pharmacol. 2013, 67, 75-105. non-nucleoside inhibitor with a novel mechanism of action. 115. Christ, F.; Shaw, S.; Demeulemeester, J.; Angew Chem Int Ed Engl. 2010, 49 (10), 1805-1808. Desimmie, B. A.; Marchand, A.; Butler, S.; Smets, W.; 102. Yamazaki, S.; Tan, L.; Mayer, G.; Hartig, J. S.; Chaltin, P.; Westby, M.; Debyser, Z.; Pickford, C., Small- Song, J. N.; Reuter, S.; Restle, T.; Laufer, S. D.; Grohmann, molecule inhibitors of the LEDGF/p75 binding site of D.; Krausslich, H. G.; Bajorath, J.; Famulok, M., Aptamer integrase block HIV replication and modulate integrase displacement identifies alternative small-molecule target multimerization. Antimicrob Agents Chemother. 2012, 56 sites that escape viral resistance. Chem Biol. 2007, 14 (7), (8), 4365-4374. 804-812. Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 13

116. Jurado, K. A.; Wang, H.; Slaughter, A.; Feng, L.; terminal domains: A model for viral DNA binding. Proc Kessl, J. J.; Koh, Y.; Wang, W.; Ballandras-Colas, A.; Patel, Natl Acad Sci U S A. 2000, 97 (15), 8233-8238. P. A.; Fuchs, J. R.; Kvaratskhelia, M.; Engelman, A., 128. Wielens, J.; Headey, S. J.; Deadman, J. J.; Rhodes, Allosteric integrase inhibitor potency is determined through D. I.; Le, G. T.; Parker, M. W.; Chalmers, D. K.; Scanlon, the inhibition of HIV-1 particle maturation. Proc Natl Acad M. J., Fragment-based design of ligands targeting a novel Sci U S A. 2013, 110 (21), 8690-8695. site on the integrase enzyme of human immunodeficiency 117. Feng, L.; Larue, R. C.; Slaughter, A.; Kessl, J. J.; virus 1. ChemMedChem. 2011, 6 (2), 258-261. Kvaratskhelia, M., HIV-1 integrase multimerization as a 129. Goldgur, Y.; Dyda, F.; Hickman, A. B.; Jenkins, T. therapeutic target. Curr Top Microbiol Immunol. 2015. M.; Craigie, R.; Davies, D. R., Three new structures of the 118. Fenwick, C.; Amad, M.; Bailey, M. D.; Bethell, R.; core domain of HIV-1 integrase: An active site that binds Bos, M.; Bonneau, P.; Cordingley, M.; Coulombe, R.; Duan, magnesium. Proc Natl Acad Sci U S A. 1998, 95 (16), 9150- J.; Edwards, P.; Fader, L. D.; Faucher, A. M.; Garneau, M.; 9154. Jakalian, A.; Kawai, S.; Lamorte, L.; LaPlante, S.; Luo, L.; 130. Rhodes, D. I.; Peat, T. S.; Vandegraaff, N.; Mason, S.; Poupart, M. A.; Rioux, N.; Schroeder, P.; Jeevarajah, D.; Le, G.; Jones, E. D.; Smith, J. A.; Coates, J. Simoneau, B.; Tremblay, S.; Tsantrizos, Y.; Witvrouw, M.; A.; Winfield, L. J.; Thienthong, N.; Newman, J.; Lucent, D.; Yoakim, C., Preclinical profile of bi 224436, a novel HIV-1 Ryan, J. H.; Savage, G. P.; Francis, C. L.; Deadman, J. J., non-catalytic-site integrase inhibitor. Antimicrob Agents Structural basis for a new mechanism of inhibition of HIV-1 Chemother. 2014, 58 (6), 3233-3244. integrase identified by fragment screening and structure- 119. Fader, L. D.; Malenfant, E.; Parisien, M.; Carson, based design. Antivir Chem Chemother. 2011, 21 (4), 155- R.; Bilodeau, F.; Landry, S.; Pesant, M.; Brochu, C.; Morin, 168. S.; Chabot, C.; Halmos, T.; Bousquet, Y.; Bailey, M. D.; 131. De Luca, L.; Morreale, F.; Christ, F.; Debyser, Z.; Kawai, S. H.; Coulombe, R.; LaPlante, S.; Jakalian, A.; Ferro, S.; Gitto, R., New scaffolds of natural origin as Bhardwaj, P. K.; Wernic, D.; Schroeder, P.; Amad, M.; integrase-LEDGF/p75 interaction inhibitors: Virtual Edwards, P.; Garneau, M.; Duan, J.; Cordingley, M.; Bethell, screening and activity assays. Eur J Med Chem. 2013, 68, R.; Mason, S. W.; Bos, M.; Bonneau, P.; Poupart, M. A.; 405-411. Faucher, A. M.; Simoneau, B.; Fenwick, C.; Yoakim, C.; 132. Serrao, E.; Debnath, B.; Otake, H.; Kuang, Y.; Tsantrizos, Y., Discovery of bi 224436, a noncatalytic site Christ, F.; Debyser, Z.; Neamati, N., Fragment-based integrase inhibitor (ncini) of HIV-1. ACS Med Chem Lett. discovery of 8-hydroxyquinoline inhibitors of the HIV-1 2014, 5 (4), 422-427. integrase-lens epithelium-derived growth factor/p75 (in- 120. Dyda, F.; Hickman, A. B.; Jenkins, T. M.; LEDGF/p75) interaction. J Med Chem. 2013, 56 (6), 2311- Engelman, A.; Craigie, R.; Davies, D. R., Crystal structure 2322. of the catalytic domain of HIV-1 integrase: Similarity to 133. Mobley, D. L.; Liu, S.; Lim, N. M.; Wymer, K. L.; other polynucleotidyl transferases. Science. 1994, 266 Perryman, A. L.; Forli, S.; Deng, N.; Su, J.; Branson, K.; (5193), 1981-1986. Olson, A. J., Blind prediction of HIV integrase binding from 121. Wang, J. Y.; Ling, H.; Yang, W.; Craigie, R., the sampl4 challenge. J Comput Aided Mol Des. 2014, 28 Structure of a two-domain fragment of HIV-1 integrase: (4), 327-345. Implications for domain organization in the intact protein. 134. Wielens, J.; Headey, S. J.; Jeevarajah, D.; Rhodes, EMBO J. 2001, 20 (24), 7333-7343. D. I.; Deadman, J.; Chalmers, D. K.; Scanlon, M. J.; Parker, 122. Cai, M.; Zheng, R.; Caffrey, M.; Craigie, R.; Clore, M. W., Crystal structure of the HIV-1 integrase core domain G. M.; Gronenborn, A. M., Solution structure of the n- in complex with sucrose reveals details of an allosteric terminal zinc binding domain of HIV-1 integrase. Nat Struct inhibitory binding site. FEBS Lett. 2010, 584 (8), 1455-1462. Biol. 1997, 4 (7), 567-577. 135. Lubkowski, J.; Yang, F.; Alexandratos, J.; 123. Cai, M.; Huang, Y.; Caffrey, M.; Zheng, R.; Wlodawer, A.; Zhao, H.; Burke, T. R., Jr.; Neamati, N.; Craigie, R.; Clore, G. M.; Gronenborn, A. M., Solution Pommier, Y.; Merkel, G.; Skalka, A. M., Structure of the structure of the his12 --> cys mutant of the n-terminal zinc catalytic domain of avian sarcoma virus integrase with a binding domain of HIV-1 integrase complexed to cadmium. bound HIV-1 integrase-targeted inhibitor. Proc Natl Acad Protein Sci. 1998, 7 (12), 2669-2674. Sci U S A. 1998, 95 (9), 4831-4836. 124. Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; 136. Peat, T. S.; Rhodes, D. I.; Vandegraaff, N.; Le, G.; Cherepanov, P., Retroviral intasome assembly and inhibition Smith, J. A.; Clark, L. J.; Jones, E. D.; Coates, J. A.; of DNA strand transfer. Nature. 2010, 464 (7286), 232-236. Thienthong, N.; Newman, J.; Dolezal, O.; Mulder, R.; Ryan, 125. Johnson, B. C.; Metifiot, M.; Ferris, A.; Pommier, J. H.; Savage, G. P.; Francis, C. L.; Deadman, J. J., Small Y.; Hughes, S. H., A homology model of HIV-1 integrase molecule inhibitors of the LEDGF site of human and analysis of mutations designed to test the model. J Mol immunodeficiency virus integrase identified by fragment Biol. 2013, 425 (12), 2133-2146. screening and structure based design. PLoS One. 2012, 7 (7), 126. Krishnan, L.; Li, X.; Naraharisetty, H. L.; Hare, S.; e40147. Cherepanov, P.; Engelman, A., Structure-based modeling of 137. Al-Mawsawi, L. Q.; Christ, F.; Dayam, R.; the functional HIV-1 intasome and its inhibition. Proc Natl Debyser, Z.; Neamati, N., Inhibitory profile of a LEDGF/p75 Acad Sci U S A. 2010, 107 (36), 15910-15915. peptide against HIV-1 integrase: Insight into integrase-DNA 127. Chen, J. C.; Krucinski, J.; Miercke, L. J.; Finer- complex formation and catalysis. FEBS Lett. 2008, 582 (10), Moore, J. S.; Tang, A. H.; Leavitt, A. D.; Stroud, R. M., 1425-1430. Crystal structure of the HIV-1 integrase catalytic core and c- 14 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

138. Perryman, A. L.; Santiago, D. N.; Forli, S.; Santos- into extracellular space like maraviroc. J Int AIDS Soc. 2014, Martins, D.; Olson, A. J., Virtual screening with autodock 17 (4 Suppl 3), 19531. vina and the common pharmacophore engine of a low 150. Jacobson, J. M.; Lalezari, J. P.; Thompson, M. A.; diversity library of fragments and hits against the three Fichtenbaum, C. J.; Saag, M. S.; Zingman, B. S.; allosteric sites of HIV integrase: Participation in the sampl4 D'Ambrosio, P.; Stambler, N.; Rotshteyn, Y.; Marozsan, A. protein-ligand binding challenge. J Comput Aided Mol Des. J.; Maddon, P. J.; Morris, S. A.; Olson, W. C., Phase 2a 2014, 28 (4), 429-441. study of the ccr5 monoclonal antibody pro 140 administered 139. Sharma, A.; Slaughter, A.; Jena, N.; Feng, L.; intravenously to HIV-infected adults. Antimicrob Agents Kessl, J. J.; Fadel, H. J.; Malani, N.; Male, F.; Wu, L.; Chemother. 2010, 54 (10), 4137-4142. Poeschla, E.; Bushman, F. D.; Fuchs, J. R.; Kvaratskhelia, 151. Dierynck, I.; Van Marck, H.; Van Ginderen, M.; M., A new class of multimerization selective inhibitors of Jonckers, T. H.; Nalam, M. N.; Schiffer, C. A.; Raoof, A.; HIV-1 integrase. PLoS Pathog. 2014, 10 (5), e1004171. Kraus, G.; Picchio, G., Tmc310911, a novel human 140. Woolfson, A. D.; Malcolm, R. K.; Morrow, R. J.; immunodeficiency virus type 1 protease inhibitor, shows in Toner, C. F.; McCullagh, S. D., Intravaginal ring delivery of vitro an improved resistance profile and higher genetic the reverse transcriptase inhibitor tmc 120 as an HIV barrier to resistance compared with current protease microbicide. Int J Pharm. 2006, 325 (1-2), 82-89. inhibitors. Antimicrob Agents Chemother. 2011, 55 (12), 141. Feng, M.; Wang, D.; Grobler, J. A.; Hazuda, D. J.; 5723-5731. Miller, M. D.; Lai, M. T., In vitro resistance selection with 152. Hoetelmans, R. M.; Dierynck, I.; Smyej, I.; doravirine (mk-1439), a novel nonnucleoside reverse Meyvisch, P.; Jacquemyn, B.; Marien, K.; Simmen, K.; transcriptase inhibitor with distinct mutation development Verloes, R., Safety and pharmacokinetics of the HIV-1 pathways. Antimicrob Agents Chemother. 2015, 59 (1), 590- protease inhibitor tmc310911 coadministered with ritonavir 598. in healthy participants: Results from 2 phase 1 studies. J 142. Gaffney, M. M.; Belliveau, P. P.; Spooner, L. M., Acquir Immune Defic Syndr. 2014, 65 (3), 299-305. Apricitabine: A nucleoside reverse transcriptase inhibitor for 153. Cha, Y. J.; Lim, K. S.; Park, M. K.; Schneider, S.; HIV infection. Ann Pharmacother. 2009, 43 (10), 1676- Bray, B.; Kang, M. C.; Chung, J. Y.; Yoon, S. H.; Cho, J. Y.; 1683. Yu, K. S., Pharmacokinetics and tolerability of the new 143. Cahn, P.; Altclas, J.; Martins, M.; Losso, M.; second-generation nonnucleoside reverse- transcriptase Cassetti, I.; Cooper, D. A.; Cox, S., Antiviral activity of inhibitor km-023 in healthy subjects. Drug Des Devel Ther. apricitabine in treatment-experienced HIV-1-infected 2014, 8, 1613-1619. patients with M184V who are failing combination therapy. 154. Van Wesenbeeck, L.; Rondelez, E.; Feyaerts, M.; HIV Med. 2011, 12 (6), 334-342. Verheyen, A.; Van der Borght, K.; Smits, V.; Cleybergh, C.; 144. Li, Z.; Zhou, N.; Sun, Y.; Ray, N.; Lataillade, M.; De Wolf, H.; Van Baelen, K.; Stuyver, L. J., Cross- Hanna, G. J.; Krystal, M., Activity of the HIV-1 attachment resistance profile determination of two second-generation inhibitor bms-626529, the active component of the prodrug HIV-1 integrase inhibitors using a panel of recombinant bms-663068, against cd4-independent viruses and HIV-1 viruses derived from raltegravir-treated clinical isolates. envelopes resistant to other entry inhibitors. Antimicrob Antimicrob Agents Chemother. 2011, 55 (1), 321-325. Agents Chemother. 2013, 57 (9), 4172-4180. 155. Bar-Magen, T.; Sloan, R. D.; Donahue, D. A.; Kuhl, 145. Bruno, C. J.; Jacobson, J. M., Ibalizumab: An anti- B. D.; Zabeida, A.; Xu, H.; Oliveira, M.; Hazuda, D. J.; cd4 monoclonal antibody for the treatment of HIV-1 Wainberg, M. A., Identification of novel mutations infection. J Antimicrob Chemother. 2010, 65 (9), 1839-1841. responsible for resistance to mk-2048, a second-generation 146. Xie, D.; Yao, C.; Wang, L.; Min, W.; Xu, J.; Xiao, HIV-1 integrase inhibitor. J Virol. 2010, 84 (18), 9210-9216. J.; Huang, M.; Chen, B.; Liu, B.; Li, X.; Jiang, H., An 156. Jegede, O.; Khodyakova, A.; Chernov, M.; Weber, albumin-conjugated peptide exhibits potent anti-HIV activity J.; Menendez-Arias, L.; Gudkov, A.; Quinones-Mateu, M. and long in vivo half-life. Antimicrob Agents Chemother. E., Identification of low-molecular weight inhibitors of HIV- 2010, 54 (1), 191-196. 1 reverse transcriptase using a cell-based high-throughput 147. Radzio, J.; Spreen, W.; Yueh, Y. L.; Mitchell, J.; screening system. Antiviral Res. 2011, 91 (2), 94-98. Jenkins, L.; Garcia-Lerma, J. G.; Heneine, W., The long- 157. Ehteshami, M.; Scarth, B. J.; Tchesnokov, E. P.; acting integrase inhibitor gsk744 protects macaques from Dash, C.; Le Grice, S. F.; Hallenberger, S.; Jochmans, D.; repeated intravaginal sHIV challenge. Sci Transl Med. 2015, Gotte, M., Mutations M184V and Y115F in HIV-1 reverse 7 (270), 270ra275. transcriptase discriminate against "nucleotide-competing 148. Mosi, R. M.; Anastassova, V.; Cox, J.; Darkes, M. reverse transcriptase inhibitors". J Biol Chem. 2008, 283 C.; Idzan, S. R.; Labrecque, J.; Lau, G.; Nelson, K. L.; Patel, (44), 29904-29911. K.; Santucci, Z.; Wong, R. S.; Skerlj, R. T.; Bridger, G. J.; 158. Zhang, Z.; Walker, M.; Xu, W.; Shim, J. H.; Huskens, D.; Schols, D.; Fricker, S. P., The molecular Girardet, J. L.; Hamatake, R. K.; Hong, Z., Novel pharmacology of amd11070: An orally bioavailable cxcr4 nonnucleoside inhibitors that select nucleoside inhibitor HIV entry inhibitor. Biochem Pharmacol. 2012, 83 (4), 472- resistance mutations in human immunodeficiency virus type 479. 1 reverse transcriptase. Antimicrob Agents Chemother. 2006, 149. Kramer, V.; Hassounah, S.; Colby-Germinario, S.; 50 (8), 2772-2781. Mesplede, T.; Lefebvre, E.; Wainberg, M., Cenicriviroc 159. Jochmans, D.; Deval, J.; Kesteleyn, B.; Van Marck, blocks HIV entry but does not lead to redistribution of HIV H.; Bettens, E.; De Baere, I.; Dehertogh, P.; Ivens, T.; Van Ginderen, M.; Van Schoubroeck, B.; Ehteshami, M.; Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 15

Wigerinck, P.; Gotte, M.; Hertogs, K., Indolopyridones B., RNase H active site inhibitors of human inhibit human immunodeficiency virus reverse transcriptase immunodeficiency virus type 1 reverse transcriptase: Design, with a novel mechanism of action. J Virol. 2006, 80 (24), biochemical activity, and structural information. J Med 12283-12292. Chem. 2009, 52 (19), 5781-5784. 160. Rajotte, D.; Tremblay, S.; Pelletier, A.; Salois, P.; 170. Williams, P. D.; Staas, D. D.; Venkatraman, S.; Bourgon, L.; Coulombe, R.; Mason, S.; Lamorte, L.; Sturino, Loughran, H. M.; Ruzek, R. D.; Booth, T. M.; Lyle, T. A.; C. F.; Bethell, R., Identification and characterization of a Wai, J. S.; Vacca, J. P.; Feuston, B. P.; Ecto, L. T.; Flynn, J. novel HIV-1 nucleotide-competing reverse transcriptase A.; DiStefano, D. J.; Hazuda, D. J.; Bahnck, C. M.; inhibitor series. Antimicrob Agents Chemother. 2013, 57 (6), Himmelberger, A. L.; Dornadula, G.; Hrin, R. C.; Stillmock, 2712-2718. K. A.; Witmer, M. V.; Miller, M. D.; Grobler, J. A., Potent 161. Xu, H. T.; Colby-Germinario, S. P.; Quashie, P. K.; and selective HIV-1 ribonuclease H inhibitors based on a 1- Bethell, R.; Wainberg, M. A., Subtype-specific analysis of hydroxy-1,8-naphthyridin-2(1h)-one scaffold. Bioorg Med the K65R substitution in HIV-1 that confers Chem Lett. 2010, 20 (22), 6754-6757. hypersusceptibility to a novel nucleotide-competing reverse 171. Yanagita, H.; Urano, E.; Matsumoto, K.; Ichikawa, transcriptase inhibitor. Antimicrob Agents Chemother. 2015. R.; Takaesu, Y.; Ogata, M.; Murakami, T.; Wu, H.; Chiba, 162. Hang, J. Q.; Rajendran, S.; Yang, Y.; Li, Y.; In, P. J.; Komano, J.; Hoshino, T., Structural and biochemical W.; Overton, H.; Parkes, K. E.; Cammack, N.; Martin, J. A.; study on the inhibitory activity of derivatives of 5-nitro- Klumpp, K., Activity of the isolated HIV RNase H domain furan-2-carboxylic acid for RNase H function of HIV-1 and specific inhibition by n-hydroxyimides. Biochem reverse transcriptase. Bioorg Med Chem. 2011, 19 (2), 816- Biophys Res Commun. 2004, 317 (2), 321-329. 825. 163. Klumpp, K.; Hang, J. Q.; Rajendran, S.; Yang, Y.; 172. Fuji, H.; Urano, E.; Futahashi, Y.; Hamatake, M.; Derosier, A.; Wong Kai In, P.; Overton, H.; Parkes, K. E.; Tatsumi, J.; Hoshino, T.; Morikawa, Y.; Yamamoto, N.; Cammack, N.; Martin, J. A., Two-metal ion mechanism of Komano, J., Derivatives of 5-nitro-furan-2-carboxylic acid rna cleavage by HIV RNase H and mechanism-based design carbamoylmethyl ester inhibit RNase H activity associated of selective HIV RNase H inhibitors. Nucleic Acids Res. with HIV-1 reverse transcriptase. J Med Chem. 2009, 52 (5), 2003, 31 (23), 6852-6859. 1380-1387. 164. Parkes, K. E.; Ermert, P.; Fassler, J.; Ives, J.; 173. Distinto, S.; Esposito, F.; Kirchmair, J.; Cardia, M. Martin, J. A.; Merrett, J. H.; Obrecht, D.; Williams, G.; C.; Gaspari, M.; Maccioni, E.; Alcaro, S.; Markt, P.; Wolber, Klumpp, K., Use of a pharmacophore model to discover a G.; Zinzula, L.; Tramontano, E., Identification of HIV-1 new class of influenza endonuclease inhibitors. J Med Chem. reverse transcriptase dual inhibitors by a combined shape-, 2003, 46 (7), 1153-1164. 2d-fingerprint- and pharmacophore-based virtual screening 165. Himmel, D. M.; Maegley, K. A.; Pauly, T. A.; approach. Eur J Med Chem. 2012, 50, 216-229. Bauman, J. D.; Das, K.; Dharia, C.; Clark, A. D., Jr.; Ryan, 174. Gong, Q.; Menon, L.; Ilina, T.; Miller, L. G.; Ahn, K.; Hickey, M. J.; Love, R. A.; Hughes, S. H.; Bergqvist, S.; J.; Parniak, M. A.; Ishima, R., Interaction of HIV-1 reverse Arnold, E., Structure of HIV-1 reverse transcriptase with the transcriptase ribonuclease H with an acylhydrazone inhibitor. inhibitor beta-thujaplicinol bound at the RNase H active site. Chem Biol Drug Des. 2011, 77 (1), 39-47. Structure. 2009, 17 (12), 1625-1635. 175. Himmel, D. M.; Sarafianos, S. G.; Dharmasena, S.; 166. Chung, S.; Himmel, D. M.; Jiang, J. K.; Wojtak, K.; Hossain, M. M.; McCoy-Simandle, K.; Ilina, T.; Clark, A. Bauman, J. D.; Rausch, J. W.; Wilson, J. A.; Beutler, J. A.; D., Jr.; Knight, J. L.; Julias, J. G.; Clark, P. K.; Krogh- Thomas, C. J.; Arnold, E.; Le Grice, S. F., Synthesis, Jespersen, K.; Levy, R. M.; Hughes, S. H.; Parniak, M. A.; activity, and structural analysis of novel alpha- Arnold, E., HIV-1 reverse transcriptase structure with RNase hydroxytropolone inhibitors of human immunodeficiency H inhibitor dihydroxy benzoyl naphthyl hydrazone bound at virus reverse transcriptase-associated. J Med Chem. 2011, 54 a novel site. ACS Chem Biol. 2006, 1 (11), 702-712. (13), 4462-4473. 176. Chung, S.; Wendeler, M.; Rausch, J. W.; Beilhartz, 167. Budihas, S. R.; Gorshkova, I.; Gaidamakov, S.; G.; Gotte, M.; O'Keefe, B. R.; Bermingham, A.; Beutler, J. Wamiru, A.; Bona, M. K.; Parniak, M. A.; Crouch, R. J.; A.; Liu, S.; Zhuang, X.; Le Grice, S. F., Structure-activity McMahon, J. B.; Beutler, J. A.; Le Grice, S. F., Selective analysis of vinylogous urea inhibitors of human inhibition of HIV-1 reverse transcriptase-associated immunodeficiency virus-encoded ribonuclease H. ribonuclease H activity by hydroxylated tropolones. Nucleic Antimicrob Agents Chemother. 2010, 54 (9), 3913-3921. Acids Res. 2005, 33 (4), 1249-1256. 177. Wendeler, M.; Lee, H. F.; Bermingham, A.; Miller, 168. Lansdon, E. B.; Liu, Q.; Leavitt, S. A.; J. T.; Chertov, O.; Bona, M. K.; Baichoo, N. S.; Ehteshami, Balakrishnan, M.; Perry, J. K.; Lancaster-Moyer, C.; Kutty, M.; Beutler, J.; O'Keefe, B. R.; Gotte, M.; Kvaratskhelia, M.; N.; Liu, X.; Squires, N. H.; Watkins, W. J.; Kirschberg, T. Le Grice, S., Vinylogous ureas as a novel class of inhibitors A., Structural and binding analysis of pyrimidinol carboxylic of reverse transcriptase-associated ribonuclease H activity. acid and n-hydroxy quinazolinedione HIV-1 RNase H ACS Chem Biol. 2008 3 (10), 635-644. inhibitors. Antimicrob Agents Chemother. 2011, 55 (6), 178. Masaoka, T.; Chung, S.; Caboni, P.; Rausch, J. W.; 2905-2915. Wilson, J. A.; Taskent-Sezgin, H.; Beutler, J. A.; Tocco, G.; 169. Kirschberg, T. A.; Balakrishnan, M.; Squires, N. H.; Le Grice, S. F., Exploiting drug-resistant enzymes as tools to Barnes, T.; Brendza, K. M.; Chen, X.; Eisenberg, E. J.; Jin, identify thienopyrimidinone inhibitors of human W.; Kutty, N.; Leavitt, S.; Liclican, A.; Liu, Q.; Liu, X.; immunodeficiency virus reverse transcriptase-associated Mak, J.; Perry, J. K.; Wang, M.; Watkins, W. J.; Lansdon, E. ribonuclease H. J Med Chem. 2013, 56 (13), 5436-5445. 16 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

179. Esposito, F.; Kharlamova, T.; Distinto, S.; Zinzula, of 1,3-diarylpropenones as dual inhibitors of HIV-1 reverse L.; Cheng, Y. C.; Dutschman, G.; Floris, G.; Markt, P.; transcriptase. ChemMedChem. 2014, 9 (8), 1869-1879. Corona, A.; Tramontano, E., Alizarine derivatives as new 190. Meleddu, R.; Distinto, S.; Corona, A.; Bianco, G.; dual inhibitors of the HIV-1 reverse transcriptase-associated Cannas, V.; Esposito, F.; Artese, A.; Alcaro, S.; Matyus, P.; DNA polymerase and RNase H activities effective also on Bogdan, D.; Cottiglia, F.; Tramontano, E.; Maccioni, E., the RNase H activity of non-nucleoside resistant reverse (3z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3- transcriptases. FEBS J. 2011, 278 (9), 1444-1457. dihydro-1h-indol-2-o ne derivatives as dual inhibitors of 180. Himmel, D. M.; Myshakina, N. S.; Ilina, T.; Van HIV-1 reverse transcriptase. Eur J Med Chem. 2015, 93, Ry, A.; Ho, W. C.; Parniak, M. A.; Arnold, E., Structure of a 452-460. dihydroxycoumarin active-site inhibitor in complex with the 191. Kessl, J. J.; Jena, N.; Koh, Y.; Taskent-Sezgin, H.; RNase H domain of HIV-1 reverse transcriptase and Slaughter, A.; Feng, L.; de Silva, S.; Wu, L.; Le Grice, S. F.; structure-activity analysis of inhibitor analogs. J Mol Biol. Engelman, A.; Fuchs, J. R.; Kvaratskhelia, M., Multimode, 2014, 426 (14), 2617-2631. cooperative mechanism of action of allosteric HIV-1 181. Morris, M. C.; Robert-Hebmann, V.; Chaloin, L.; integrase inhibitors. J Biol Chem. 2012, 287 (20), 16801- Mery, J.; Heitz, F.; Devaux, C.; Goody, R. S.; Divita, G., A 16811. new potent HIV-1 reverse transcriptase inhibitor. A synthetic 192. Hayouka, Z.; Rosenbluh, J.; Levin, A.; Loya, S.; peptide derived from the interface subunit domains. J Biol Lebendiker, M.; Veprintsev, D.; Kotler, M.; Hizi, A.; Loyter, Chem. 1999, 274 (35), 24941-24946. A.; Friedler, A., Inhibiting HIV-1 integrase by shifting its 182. Depollier, J.; Hourdou, M. L.; Aldrian-Herrada, G.; oligomerization equilibrium. Proc Natl Acad Sci U S A. Rothwell, P.; Restle, T.; Divita, G., Insight into the 2007, 104 (20), 8316-8321. mechanism of a peptide inhibitor of HIV reverse 193. Rhodes, D. I.; Peat, T. S.; Vandegraaff, N.; transcriptase dimerization. Biochemistry. 2005, 44 (6), 1909- Jeevarajah, D.; Newman, J.; Martyn, J.; Coates, J. A.; Ede, 1918. N. J.; Rea, P.; Deadman, J. J., Crystal structures of novel 183. Agopian, A.; Gros, E.; Aldrian-Herrada, G.; allosteric peptide inhibitors of HIV integrase identify new Bosquet, N.; Clayette, P.; Divita, G., A new generation of interactions at the LEDGF binding site. Chembiochem. 2011, peptide-based inhibitors targeting HIV-1 reverse 12 (15), 2311-2315. transcriptase conformational flexibility. J Biol Chem. 2009, 194. Kessl, J. J.; Eidahl, J. O.; Shkriabai, N.; Zhao, Z.; 284 (1), 254-264. McKee, C. J.; Hess, S.; Burke, T. R., Jr.; Kvaratskhelia, M., 184. Das, K.; Bauman, J. D.; Rim, A. S.; Dharia, C.; An allosteric mechanism for inhibiting HIV-1 integrase with Clark, A. D., Jr.; Camarasa, M. J.; Balzarini, J.; Arnold, E., a small molecule. Mol Pharmacol. 2009, 76 (4), 824-832. Crystal structure of tert-butyldimethylsilyl- 195. Rogolino, D.; Carcelli, M.; Compari, C.; De Luca, spiroaminooxathioledioxide-thymine (tsao-t) in complex L.; Ferro, S.; Fisicaro, E.; Rispoli, G.; Neamati, N.; Debyser, with HIV-1 reverse transcriptase (rt) redefines the elastic Z.; Christ, F.; Chimirri, A., Diketoacid chelating ligands as limits of the non-nucleoside inhibitor-binding pocket. J Med dual inhibitors of HIV-1 integration process. Eur J Med Chem. 2011, 54 (8), 2727-2737. Chem. 2014, 78, 425-430. 185. Sluis-Cremer, N.; Dmitrienko, G. I.; Balzarini, J.; 196. Billamboz, M.; Bailly, F.; Barreca, M. L.; De Luca, Camarasa, M. J.; Parniak, M. A., Human immunodeficiency L.; Mouscadet, J. F.; Calmels, C.; Andreola, M. L.; virus type 1 reverse transcriptase dimer destabilization by 1- Witvrouw, M.; Christ, F.; Debyser, Z.; Cotelle, P., Design, [spiro[4"-amino-2",2" -dioxo-1",2" -oxathiole-5",3'-[2', 5'- synthesis, and biological evaluation of a series of 2- bis-o-(tert-butyldimethylsilyl)-beta-d-ribofuranosyl]]]-3- hydroxyisoquinoline-1,3(2h,4h)-diones as dual inhibitors of ethylthy mine. Biochemistry. 2000, 39 (6), 1427-1433. human immunodeficiency virus type 1 integrase and the 186. Borkow, G.; Fletcher, R. S.; Barnard, J.; Arion, D.; reverse transcriptase RNase H domain. J Med Chem. 2008, Motakis, D.; Dmitrienko, G. I.; Parniak, M. A., Inhibition of 51 (24), 7717-7730. the ribonuclease H and DNA polymerase activities of HIV-1 197. Costi, R.; Metifiot, M.; Chung, S.; Cuzzucoli reverse transcriptase by n-(4-tert-butylbenzoyl)-2-hydroxy-1- Crucitti, G.; Maddali, K.; Pescatori, L.; Messore, A.; Madia, naphthaldehyde hydrazone. Biochemistry. 1997, 36 (11), V. N.; Pupo, G.; Scipione, L.; Tortorella, S.; Di Leva, F. S.; 3179-3185. Cosconati, S.; Marinelli, L.; Novellino, E.; Le Grice, S. F.; 187. Cruchaga, C.; Anso, E.; Font, M.; Martino, V. S.; Corona, A.; Pommier, Y.; Marchand, C.; Di Santo, R., Basic Rouzaut, A.; Martinez-Irujo, J. J., A new strategy to inhibit quinolinonyl diketo acid derivatives as inhibitors of HIV the excision reaction catalysed by HIV-1 reverse integrase and their activity against RNase H function of transcriptase: Compounds that compete with the template- reverse transcriptase. J Med Chem. 2014, 57 (8), 3223-3234. primer. Biochem J. 2007, 405 (1), 165-171. 198. Cuzzucoli Crucitti, G.; Metifiot, M.; Pescatori, L.; 188. Tatyana, K.; Francesca, E.; Luca, Z.; Giovanni, F.; Messore, A.; Madia, V. N.; Pupo, G.; Saccoliti, F.; Scipione, Cheng, Y. C.; Ginger, E. D.; Enzo, T., Inhibition of HIV-1 L.; Tortorella, S.; Esposito, F.; Corona, A.; Cadeddu, M.; ribonuclease H activity by novel frangula-emodine Marchand, C.; Pommier, Y.; Tramontano, E.; Costi, R.; Di derivatives. Med Chem. 2009, 5 (5), 398-410. Santo, R., Structure-activity relationship of pyrrolyl diketo 189. Meleddu, R.; Cannas, V.; Distinto, S.; Sarais, G.; acid derivatives as dual inhibitors of HIV-1 integrase and Del Vecchio, C.; Esposito, F.; Bianco, G.; Corona, A.; reverse transcriptase ribonuclease H domain. J Med Chem. Cottiglia, F.; Alcaro, S.; Parolin, C.; Artese, A.; Scalise, D.; 2015, 58 (4), 1915-1928. Fresta, M.; Arridu, A.; Ortuso, F.; Maccioni, E.; 199. Tramontano, E.; Esposito, F.; Badas, R.; Di Santo, Tramontano, E., Design, synthesis, and biological evaluation R.; Costi, R.; La Colla, P., 6-[1-(4-fluorophenyl)methyl-1h- Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 17 pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester a novel 201. Corona, A.; Di Leva, F. S.; Thierry, S.; Pescatori, diketo acid derivative which selectively inhibits the HIV-1 L.; Cuzzucoli Crucitti, G.; Subra, F.; Delelis, O.; Esposito, viral replication in cell culture and the ribonuclease H F.; Rigogliuso, G.; Costi, R.; Cosconati, S.; Novellino, E.; Di activity in vitro. Antiviral Res. 2005, 65 (2), 117-124. Santo, R.; Tramontano, E., Identification of highly conserved 200. Shaw-Reid, C. A.; Munshi, V.; Graham, P.; Wolfe, residues involved in inhibition of HIV-1 RNase H function A.; Witmer, M.; Danzeisen, R.; Olsen, D. B.; Carroll, S. S.; by diketo acid derivatives. Antimicrob Agents Chemother. Embrey, M.; Wai, J. S.; Miller, M. D.; Cole, J. L.; Hazuda, 2014, 58 (10), 6101-6110. D. J., Inhibition of HIV-1 ribonuclease H by a novel diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid. J Biol Chem. 2003, 278 (5), 2777-2780.

18 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

Table 1. New antiretroviral drugs in clinical trialsa.

Drug Class Target Drug Name Alternate Name/s Company References Phase III NNRTI HIV-1 RT Dapivirineb TMC-120 Tibotec Pharmaceuticals/Janssen; [140] Johnson & Johnson NNRTI HIV-1 RT Doravirine MK-1439 Merck & Co [141] NRTI HIV-1 RT Apricitabineh AVX-754; BCH-10618; Avexa [142-143] BCH-10619; BCH- 10652; SPD-754 Entry HIV-1 gp120 Fostemsavir BMS-663068; BMS- Bristol-Myers Squibb [144] 626529 Entry CD4 receptor Ibalizumabc TNX-355; Hu5A8 Tanox; Biogen Idec; Taimed Biologies [145] Fusion HIV-1 gp41 Albuvirtided FB006M Chongqing Frontier Biotechnologies [146] Phase II INSTI HIV-1 IN Cabotegravirf GSK1265744 GlaxoSmithKline [147] Entry CXCR4 receptor AMD-070 AMD-11070 Genzyme Corporation [148] Entry CCR5 receptor Cenicriviroc TBR-652; TAK-652 Tobira Therapeutics [149] Entry CCR5 receptor PRO-140e Progenics Pharmaceuticals; CytoDyn Inc [150] Entry CCR5 receptor PF-00232798 Viiv Healthcare - Entry CCR5 receptor SB-728-T CCR5-specific zinc Sangamo BioSciences - (gene) finger nuclease PI HIV-1 PR TMC-310911 ASC-09 Tibotec/Janssen; Ascletis [151-152] Maturation HIV-1 Gag BMS-955176 Bristol-Myers Squibb Phase I NNRTI HIV-1 RT KM-023 Kainos Medicine [153] INSTI HIV-1 IN MK-2048g Merck & Co [154-155] NCINI HIV-1 IN GS-9883 BI-224436 Boehringer Ingelheim; Gilead [118-119] Maturation HIV-1 Gag GSK 2838232 GlaxoSmithKline - aAs of 18 June, 2015, http://www.clinicaltrials.gov and company press releases available publicly online; bHIV prevention (intravaginal ring) currently under evaluation in the Phase III “ASPIRE” Trial and “The Ring Study”; cCD4-specific humanized IgG4 monoclonal antibody; dsynthetic peptide; ehumanised anti-CCR5 monoclonal antibody; fFor both Treatment (oral) and HIV prevention (long-acting injectable packaged into nanoparticles); gHIV prevention (intravaginal ring) in formulation with Vicriviroc; hAnnouncement that funding for Phase III trial has been raised (July 2014); NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor.

Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 19

Table 2. New classes of HIV-1 RT and IN inhibitors described in the literature but not yet in clinical trials or clinical use.

Drug Classa Inhibitory Description Example Inhibitors References Mechanism HIV-1 REVERSE TRANSCRIPTASE (RT) Nucleotide-competing Allosteric Compete with indolopyridones [156-159] RT Inhibitors (NcRTI) nucleotides for RT e.g. INDOPY-1 binding benzo[4,5]furo[3,2,d]pyrimidin-2-one [160-161] series DAVP-1 [101] RNase H inhibitors Active Site Complex with the N-hydroxyimides [162-164] divalent metal cofactor hydroxylated tropolones e.g. β- [165-167] 2+ 2+ (Mg or Mn ) thujaplicinol required for catalysis pyrimidinol carboxylic acids [168-169] naphthyridinones [170] nitrofuran-2-carboxylic acid [171-172] carbamoylmethyl esters (NACMEs). Allosteric Inhibit RNase H N-acyl hydrazones [173-175] activity by binding to a thiophene-3-carboxamides [176-177] secondary site thienopyrimidinones [178] naphthyridinones [179]

dihydroxycoumarins [180] e.g. F3284-8495 (33) RT dimerization Allosteric Stabilise or destabilize peptide-based inhibitors [181-183] inhibitors RT dimer by binding at e.g. PAW, Pep-7 p66/p51interface TSAO-Tb [184-185] BBNH [186] T/P inhibitors Allosteric Inhibits T/P binding APEX57219 [104] 2GP [187] SY-3E4 [102] KM-1 [103] Dual inhibitors Inhibits polymerase anthraquinones [188] and RNase H activities propenones [189-190] HIV-1 INTEGRASE (IN) Allosteric IN Allosteric Multi-modal: inhibit quinolines & quinolones [112, 191] Inhibitors LEDGF binding to IN (ALLINI/LEDGIN) LEDGF/p75 binding peptides pocket affecting IN [137, 192- multimerisation, 193] LEDGF binding and virion maturation Multimerisation Allosteric tetra-acetylated inhibitor [194] Selective Inhibitors [139] Dual inhibitors Allosteric Inhibit both strand diketo acids [195] transfer and the LEDGF/IN interaction Non-catalytic site IN Allosteric distinct allosteric site BI 224436 [119] Inhibitors (NCINI)** from ALLINIs RT/IN DUAL INHIBITORS IN/RT-RNase H diketo acids [196-201] inhibition aNovel sites or inhibitors identified by fragment screening are not listed in this table and described in Sections 4.3-4.5, 5.3-5.5; b TSAO-T binds to the NNRTI-binding pocket [184], but has a mechanism and chemical structure distinct from other inhibitors classed as NNRTIs. cAn NCINI, BI 224436, has recently entered Phase I clinical trials (2014). 20 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

Table 3. Compounds discovered by fragment-based drug discovery that bind to and/or inhibit HIV-1 reverse transcriptase (RT).

Compounda Drug class/ RT b binding site IC50 (µM) LE 1 Br NNRTI 4 0.43 (NNIBP)

O 2 NNRTI- 350 0.34 O O adjacent

N N

3 O Incoming 200 0.31 N N Nucleotide OH binding site 4 F F Knuckles No N/A H N 2 O F inhibition at 1mM F F 5 HO Knuckles 600 0.37

O F

6 + Knuckles ND ND N

O O O O O O 7 HO O RNase H N/A N/A adjacent grip

N N

8 HO ND 259 0.42

Cl

S HO 9 N NcRTI 222 0.43

10 OH T/P competing 222 0.51 Cl Cl & RNase H inhibitor

NH 2 11 Cl T/P competing 70 0.58 Cl Cl

NH 2 aFragment 1 is from the SPR fragment screen [95], fragments 2-7 were discovered by X-ray crystallographic fragment screening [98] and fragments 8-10 by STD-NMR screening [100]. bLE, ligand efficiency, values as published in Geitmann et al [95] for 1, Bauman et al [98] for 2-7, and calculated for fragments 8-11 using the formula LE = -RTln(IC50)/HAC where R is the gas constant, T is temperature and HAC is the heavy atom, or non-hydrogen, atom count ND, not determined; NNRTI, non- nucleoside reverse transcriptase inhibitor; NNIBP; NNRTI binding pocket; NcRTI, nucleotide-competing reverse transcriptase inhibitor; T/P, template/primer. Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 21

Table 4. Compounds discovered by fragment-based drug discovery that bind to and/or inhibit HIV-1 integrase (IN).

a b Compound Drug class/ INCCD c d binding site Kd (µM) IC50 (µM) LE 12 FBP 2800 ND 0.3 N N

H2N S

13 O FBP 5500 ND 0.32

O HO 14 FBP 170 ND 0.31 N N

H2N O

O Br O OH 15 O Y3 site 259 295 0.21

N O

O

O OH 16 O Y3 site ND 5 0.24

O O N

O

O OH O 17 O ALLINI 1570 ND 0.17 O

O

O

O OH 18 O ALLINI 1375 200 0.17

O

O

19 R1 R2 ALLINI 7.6 8.1 0.16

O NH O OH

O N

R3 O R1 = R2 = p-MeO-Ph, R3 = allyl aCompounds 12-14 are from the Wielens et al [128] NMR fragment screen, compound 15-16 were discovered by STD- NMR/SPR fragment screening [130] and fragments 17-19 were described in Peat et al [136]; bSee original studies for construct c of IN central core domain (INCCD) that was used for each assay; Inhibitory concentrations at 50% (IC50) values refer to inhibition of LEDGF binding to HIV-1 IN for ALLINIs, determined using the AlphaScreen [137]; dLE, ligand efficiency, either as published for 15, or calculated for compounds 12-14, 16-19 using the equation LE = (-RTlnKd)/(HAC) where R is the universal gas constant, T is temperature, “HAC” is the heavy atom or non-hydrogen atom count and Kd is the dissociation constant; ND, not determined; FBP, fragment binding pocket; ALLINI, allosteric Integrase inhibitor that binds to the LEDGF interaction site on HIV-1 integrase. 22 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

Figure 1. Three new allosteric inhibitory pockets in HIV-1 RT identified using FBDD [98]. (A) Crystal structures of HIV- 1 RT:rilpivirine with fragment 2 (Table 3), green, bound in the NNRTI-adjacent pocket (PDB code 4KFB), p66 subunit in blue and p51 in grey, and HIV-1 RT:rilpivirine (PDB code 2ZD1), red. Rilpivirine bound in the NNRTI pocket is shown in yellow. (B) Crystal structures HIV-1 RT:rilpivirine with fragment 3 (Table 3), green, bound in the Incoming Nucleotide Binding Site (PDB code 4ICL), blue, superimposed on HIV-1 RT with rilpivirine only (PDB code 2ZD1), red. (C) Crystal structures HIV-1 RT:rilpivirine with fragment 6 (Table 3), green, bound in the Knuckles pocket (PDB code 4IG3), p66 subunit in blue and p51 in grey, and HIV-1 RT:rilpivirine with fragment 4 (Table 3), yellow, also bound in the Knuckles pocket (PDB code 4IFY), light orange. Side chains are shown in A, B and C for residues interacting with bound fragment molecules. Images were generated using Pymol (Schrödinger).

Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 23

Figure 2. Fragment-based discovery of inhibitory fragments of HIV-1 IN central core domain [128, 136]. (A) left panel, Crystal structure of HIV-1 INCCD, dark green, bound to compound 12 (Table 4) (PDB code 3OVN) showing the interaction with Ser195 via a key water molecule (red) in the fragment binding pocket (FBP); right panel, crystal structure of HIV-1 INCCD, orange, bound to optimised compound 14 (Table 4) (PDB code 3AO2) showing a direct interaction with Ser195 in the FBP. (B) Crystal structure of HIV-1 INCCD, dark green, bound to compound 15 (Table 4) (PDB code 3NF6), and key interacting residues His183, Gln62 and Ser147.