pharmaceuticals

Review Bioisosteric Replacement as a Tool in Anti-HIV Drug Design

Alexej Dick and Simon Cocklin * Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Rooms 10307, 10309, and 10315, 245 North 15th Street, Philadelphia, PA 19102, USA; [email protected] * Correspondence: [email protected]; Tel.: +215-762-7234 or +215-762-4979



Received: 3 February 2020; Accepted: 26 February 2020; Published: 28 February 2020


Abstract: Bioisosteric replacement is a powerful tool for modulating the drug-like properties, toxicity, and chemical space of experimental therapeutics. In this review, we focus on selected cases where bioisosteric replacement and scaffold hopping have been used in the development of new anti-HIV-1 therapeutics. Moreover, we cover field-based, computational methodologies for bioisosteric replacement, using studies from our group as an example. It is our hope that this review will serve to highlight the utility and potential of bioisosteric replacement in the continuing search for new and improved anti-HIV drugs.

Keywords: bioisosteres; HIV-1; antiviral; computer-aided drug design; envelope; reverse transcriptase; protease; integrase; tat; Vif

1. Introduction The design and development of a lead compound into a drug is a laborious and often costly process, with most candidates failing due to metabolism and pharmacokinetics issues rather than potency. Bioisosteric replacement is a strategy used by medicinal chemists to address these limitations while still retaining the potency/efficacy of the initial lead compound. The use of bioisosteres and the introduction of structural changes to the lead compound allows the chemist to alter the compound’s size, shape, electronic distribution, polarizability, dipole, polarity, lipophilicity, and pKa, while still retaining potent target engagement. Therefore, the bioisosteric approach can be used for the rational modification of a lead compound towards a more attractive therapeutic agent with improved potency, selectivity, altered physical, metabolic, toxicological properties with the bonus of generating novel intellectual property (IP). The objective of the present review is to provide a broad understanding of the principle of bioisosteric replacement underlined with cases from recent applications in anti-HIV drug design and optimization. Specific examples for lead compound development targeting a variety of HIV-1 targets, including the envelope (Env), reverse transcriptase (RT), protease, integrase (IN), Tat, and Vif, will be presented. Studies from our group that document the application of bioisosteric replacement to the ongoing progressive alteration of a dominant piperazine chemotype in the HIV-1 entry inhibitor field, and which suffers from bioavailability and breadth problems, are also covered. It is our hope that this review will serve to highlight the utility and potential of bioisosteric replacement in the continuing search for new and improved anti-HIV drugs.

2. Principle of Bioisosterism and Historical Background The term isosterism was first introduced by Irving Langmuir in 1919 during his studies on similarities of physicochemical properties of atoms, groups and molecules [1]. He described compounds or groups of atoms with the same number of atoms and electrons, such as N2 and CO, N2O and CO2, - - or N3 and NCO as isosteres, and, based on these similarities of the arrangement of electrons, he defined

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21 groups of isosteres. H. G. Grimm further developed this definition in the early 1920s. This early hypothesis of bioisosterism describes the ability of certain chemical groups to mimic other chemical groups [2,3]. Accordingly, the addition of a hydride to an atom gives to the resulting pseudoatom the properties of the atom with the next highest atomic number (Table1)[4].

Table 1. Grimm’s Hydride Displacement Law.

C N O F Ne Na+ CH NH OH FH - + CH2 NH2 OH2 FH2 + CH3 NH3 OH3 + CH4 NH4

Each vertical column represents an isostere, according to Grimm. In 1932, Hans Erlenmeyer extended Grimm’s definition of isosteres as atoms, ions, and molecules in which the peripheral layers of electrons (valence electrons) are considered as identical (Table2).

Table 2. Isosteres based on valence electron number.

Number of Valence Electrons 4 5 6 7 8 N+ P S Cl ClH P+ As Se Br BrH S+ Sb Te I IH + As PH SH SH2 + Sb PH2 PH3

Based on its application in biological systems, Harris Friedman introduced the term “bioisostere” in 1950 that included all atoms and molecules which fit the broadest definition for isosteres and have similar biological activity, either agonistic or antagonistic [5]. Today, the even more broadened definition of bioisosteres introduced by Alfred Burger in the early 1990s is in use. Accordingly, bioisosteres are “Compounds or groups that possess near-equal molecular shapes and volumes, approximately the same distribution of electrons, and similar physical properties” [6].

3. Classical and Non-Classical Bioisosteres In the 1970s, Alfred Bruger defined bioisosteres as either classical (atom number, number of valence electrons, and degree of unsaturation) or non-classical (similar pKa, electrostatic potentials, orbital occupation/HOMOs and LUMOs) [7]. Classical bioisosteres can be further subdivided into five classes: 1) monovalent atoms or groups (D and H; F and H; C and Si; Cl, Br, SH, and OH; NH and OH; RSH and ROH, –Cl, –PH2, –SH), 2) divalent atoms or groups (–CH2, –NH, –O, –S, –Se–, -COCH2-), 3) trivalent atoms or groups (–CH=, –N=, -P=, -As=), 4) tetravalent atoms or groups (>C<, >Si< and =C=, =N+=, =P+=), and 5) ring equivalents (-CH=CH-, -S- (e.g., benzene, thiophene), -CH=, -N= (e.g., benzene, pyridine), -O-, -S-, -CH2- (e.g., tetrahydrofuran, tetrahydrothiophene, cyclopentane) [4,8]. Non-classical bioisosteres are a more sophisticated mimicry of the emulated counterparts and they do not strictly obey the steric and electronic definition of classical isosteres. As non-classical bioisosteres can significantly differ in electronic distribution, physicochemical, steric and topological properties, they have found beneficial applications in drug discovery research. Non-classical bioisosteres are subdivided into two groups: 1) cyclic vs. non-cyclic and 2) exchangeable functional groups (Figure1)[4]. Pharmaceuticals 2020, 13, 36 3 of 16 Pharmaceuticals 2020, 13, 36 3 of 17

Figure 1.1. Non-ClassicalNon‐Classical Bioisosterism.

InIn thethe followingfollowing sections, sections, we we will will highlight highlight the the recent, recent, successful successful application application of both of both classical classical and non-classicaland non‐classical bioisosteres bioisosteres in the in design the design and redesignand redesign and developmentand development of new of anti-HIVnew anti‐ agents.HIV agents.

3.1. Recent Applications for Classical Bioisosteres in Anti-HIV Drug Design and Development 3.1. Recent Applications for Classical Bioisosteres in Anti‐HIV Drug Design and Development 3.1.1. Monovalent Bioisosteres 3.1.1. Monovalent Bioisosteres Deuterium asas aa HydrogenHydrogen IsostereIsostere inin HIV-1HIV‐1 Reverse Reverse Transcriptase transcriptase Inhibitorsinhibitors Deuterium ( 2H,H, heavy heavy hydrogen), hydrogen), as as one one of of two two isotopes isotopes of of hydrogen hydrogen with with an an isotopic isotopic mass mass of of2.014 2.014 u, u,is isthe the most most common common isostere isostere of of hydrogen. hydrogen. Both Both isotopes isotopes have have small small differences differences in theirtheir physicochemicalphysicochemical properties properties beneficial beneficial for for drug drug design. design. Deuterium Deuterium provides provides lower lipophilicity,lower lipophilicity, smaller molarsmaller volume, molar volume, and a shorterand a shorter (by 0.005Å) (by 0.005Å) C–D C–D bond bond as compared as compared to theto the C–H C–H bond. bond. DeuteriumDeuterium incorporationincorporation can can also also increase increase the the basicity basicity of amines of amines and decrease and decrease the acidity the of acidity phenols of and phenols carboxylic and acidscarboxylic [8]. As acids a result, [8]. As intermolecular a result, intermolecular interactions interactions with the target with canthe target be affected. can be Covalent affected. bonds, Covalent or adjacentbonds, or bonds, adjacent in which bonds, the in replaced which the deuterium replaced is deuterium involved, canis involved, display altered can display cleavage altered properties cleavage [9]. Non-covalentproperties [9]. interactions Non‐covalent can beinteractions changed as can well; be however, changed the as ewell;ffect ishowever, usually modest. the effect Nevertheless, is usually ifmodest. located Nevertheless, adjacent to modification if located adjacent sites, measurable to modification effects sites, are observable, measurable for effects example, are observable, in modulating for theexample, drug candidate’s in modulating pharmacokinetic the drug candidate’s properties pharmacokinetic such as its metabolism properties and such toxicity. as its metabolism and toxicity.Retroviruses encode an enzyme known as reverse transcriptase (RT) to convert their viral single-strandedRetroviruses RNA encode into an linear enzyme double-stranded known as reverse DNA transcriptase for integration (RT) to into convert the host their genome viral single and‐ subsequentstranded RNA transcription into linear and double replication.‐stranded This DNA process for integration is essential into for the viralhost genome replication and cycle subsequent and the targettranscription of one of and the replication. most successful This process inhibitor is class, essential the RT for inhibitors the viral replication (RTI) [10]. RTIcycle can and be the subdivided target of intoone of two the major most types,successful nucleoside inhibitor (nucleotide) class, the RT analog inhibitors reverse-transcriptase (RTI) [10]. RTI can inhibitors be subdivided (N(t)RTIs) into andtwo non-nucleosidemajor types, nucleoside reverse transcriptase (nucleotide) inhibitors analog reverse (NNRTIs).‐transcriptase inhibitors (N(t)RTIs) and non‐ nucleosideMutlib reverse and colleagues transcriptase showed inhibitors that the (NNRTIs). widely used HIV-1 NNRTI efavirenz (1) produced renal tubularMutlib epithelial and colleagues cell necrosis showed in rats that (Figure the widely2)[ 11]. used To understand HIV‐1 NNRTI the efavirenz biochemical (1) produced mechanisms renal of formingtubular epithelial toxic, reactive cell necrosis intermediates in rats and(Figure to characterize 2) [11]. To understand the safety of the efavirenz, biochemical Mutlib mechanisms et al. used of a forming toxic, reactive intermediates and to characterize the safety of efavirenz, Mutlib et al. used a hydrogen‐to‐deuterium exchange approach to improve the metabolic stability. Efavirenz undergoes

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Pharmaceuticals 2020, 13, 36 4 of 17 hydrogen-to-deuterium exchange approach to improve the metabolic stability. Efavirenz undergoes a complex metabolic transformation pathway that leads ultimately to the nephrotoxic glutathione a complex metabolic transformation pathway that leads ultimately to the nephrotoxic glutathione conjugate (3). Deuteration at the cyclopropyl moiety significantly reduced the formation of the conjugate (3). Deuteration at the cyclopropyl moiety significantly reduced the formation of the cyclopropylcarbinol intermediate (2) and the excretion of (3) in the urine of rats as quantified by cyclopropylcarbinol intermediate (2) and the excretion of (3) in the urine of rats as quantified by LC/MS. LC/MS.

Figure 2. Efavirenz metabolism in rats that leads to the nephrotoxic glutathione conjugate (3).

Silicon as a Carbon Isostere in HIV-1 Protease Inhibitors Silicon as a Carbon Isostere in HIV‐1 protease inhibitors Silicon has reawakened the interest of medicinal chemists over recent years as a bioisosteric Silicon has reawakened the interest of medicinal chemists over recent years as a bioisosteric replacement for carbon (so-called “silicon switching”). Carbon and silicon, despite both having a replacement for carbon (so‐called “silicon switching”). Carbon and silicon, despite both having a valency of 4, have the ability to form tetrahedral compounds with slightly different chemical properties. valency of 4, have the ability to form tetrahedral compounds with slightly different chemical Theproperties. most notable The most diff erencesnotable adifferencesfforded by afforded replacing by C replacing with Si are C the with increased Si are the covalent increased radius covalent of Si (50%),radius theof Si longer (50%), C–Si the longer bond (20%), C–Si bond decreased (20%), electronegativity decreased electronegativity according toaccording the Pauling to the scale Pauling of Si (1.74scale comparedof Si (1.74 tocompared 2.50 of C) to and 2.50 the of higherC) and lipophilicitythe higher lipophilicity of Si derivatives of Si [derivatives12,13]. These [12,13]. differences These candifferences result in can modest result shape in modest and conformation shape and conformation differences of differences silanols, crucial of silanols, for the protein–ligandcrucial for the π interaction.protein–ligand Silicon, interaction. as compared Silicon, to carbon, as compared also displays to carbon, a lower also tendency displays to a formlower stable tendencybonds, to form and the Si–Si σ bond (230 kJ mol 1) is weaker compared to the Si–O bond (368 kJ mol 1). Therefore, silicon stable π bonds, and the Si–Si− σ bond (230 kJ mol−1) is weaker compared to the− Si–O bond (368 kJ appears as silicates and silica in nature. The ability of Silanols to form hydrogen bonds, combined with mol−1). Therefore, silicon appears as silicates and silica in nature. The ability of Silanols to form theirhydrogen acidic bonds, character combined as compared with totheir carbinols, acidic character make them as an compared attractive to replacement carbinols, make for optimizing them an hydrogenattractive bondsreplacement in protein–ligand for optimizing interactions hydrogen [14 bonds]. To date, in protein–ligand no Si-related toxicityinteractions has been [14]. recorded, To date, whichno Si‐related makes toxicity Si an attractive has been candidate recorded, forwhich drug makes discovery. Si an attractive Silicon modified candidate derivatives for drug discovery. can alter metabolicSilicon modified pathways derivatives or improve can alter blood-brain metabolic barrier pathways penetration or improve due blood to its‐brain increased barrier lipophilicity. penetration Alterationsdue to its increased can vary lipophilicity. from simple Alterations alkyl replacement can vary from by asimple trialkylsilyl alkyl replacement to the more by sophisticated a trialkylsilyl silandiols (Si(OH)2). to the more sophisticated silandiols (Si(OH)2). Highly activeactive anti-retroviral anti‐retroviral therapy therapy (HAART) (HAART) is a veryis a everyffective effective treatment treatment for AIDS, for and AIDS, protease and inhibitorsprotease inhibitors (PIs) play (PIs) a crucial play rolea crucial in HAART. role in PIsHAART. block thePIs proteolyticblock the proteolytic cleavage of cleavage viral precursor of viral proteinsprecursor into proteins shorter into active shorter proteins active essential proteins for essential the viral for replication the viral cycle.replication Silinadiol cycle. represents Silinadiol arepresents very important a very class important of protease class inhibitors,of protease highlighting inhibitors, highlighting the potential the of Sipotential for this researchof Si for area.this Forresearch example, area. proteases For example, hydrolyze proteases the peptide hydrolyze amide the bond peptide with amide a tetrahedral bond with diol a intermediate tetrahedral thatdiol furtherintermediate reacts that to the further individual reacts peptides to the individual (Figure3). peptides (Figure 3). Mimicking this transition state is a powerful strategy for inhibitor design. Silicon, in fact, due to its preference to appear in sp3 hybridization over sp2, stabilizes the geminal transition state diol and prevents further hydrolysis into a silanone (Si=O) as pioneered by Sieburth [15]. The reduced electron-withdrawing character of Si disfavors the dehydration of the silanone moiety and stabilizes the transition state (Figure4, right equilibrium) [8].

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Figure 3. Proteolytic hydrolysis. The tetrahedral intermediate (in brackets) can be replaced by a non‐ hydrolyzable protease inhibitor (black dashed box).

Mimicking this transition state is a powerful strategy for inhibitor design. Silicon, in fact, due to

its preference to appear in sp3 hybridization over sp2, stabilizes the geminal transition state diol and preventsFigure further 3. ProteolyticProteolytic hydrolysis hydrolysis. hydrolysis. into Thea Thesilanone tetrahedral tetrahedral (Si=O) intermediate intermediate as pioneered (in brackets) (in brackets) by canSieburth canbe replaced be [15]. replaced byThe a non by reduced a‐ electronnon-hydrolyzablehydrolyzable‐withdrawing protease protease character inhibitor inhibitor of (black Si disfavors (black dashed dashed box). the box).dehydration of the silanone moiety and stabilizes the transition state (Figure 4, right equilibrium) [8]. Mimicking this transition state is a powerful strategy for inhibitor design. Silicon, in fact, due to its preference to appear in sp3 hybridization over sp2, stabilizes the geminal transition state diol and prevents further hydrolysis into a silanone (Si=O) as pioneered by Sieburth [15]. The reduced electron‐withdrawing character of Si disfavors the dehydration of the silanone moiety and stabilizes the transition state (Figure 4, right equilibrium) [8]. Figure 4. Hydration equilibrium for carbonyl (left) and silanone (right). Figure 4. Hydration equilibrium for carbonyl (left) and silanone (right).

UsingUsing a a high-pressure high‐pressure liquid liquid chromatographic chromatographic (HPLC) (HPLC) assay, assay, to quantify to quantify the rate the of HIV-1 rate of protease HIV‐1 inhibitionprotease inhibition based on thebased cleavage on the of acleavage HIV-1 Gag of peptidea HIV‐1 substrate, Gag peptide the silanediol substrate, (4) the inhibited silanediol HIV-1 (4) inhibited HIV‐1 protease with a Ki of 2.7 nM and in a plaque assay with human peripheral blood protease with a KiFigureof 2.7 4. nM Hydration and in a equilibrium plaque assay for carbonyl with human (left) and peripheral silanone blood (right). mononuclear cells mononuclear cells (PBMCs) an EC90 of 170 nM similar to the carbinol (5) (Ki = 0.37 nM, EC90 = 23 nM, (PBMCs) an EC90 of 170 nM similar to the carbinol (5) (Ki = 0.37 nM, EC90 = 23 nM, Figure3 green Figure 3 green dashed box) was determined. dashedUsing box) a was high determined.‐pressure liquid chromatographic (HPLC) assay, to quantify the rate of HIV‐1 Most of the HIV‐1 protease inhibitors are accompanied by side effects in long‐term treatment. proteaseMost inhibition of the HIV-1 based protease on the inhibitors cleavage are of accompanieda HIV‐1 Gag by peptide side e ffsubstrate,ects in long-term the silanediol treatment. (4) HIV protease inhibitor‐induced metabolic syndromes can include dyslipidemia, insulin resistance, HIVinhibited protease HIV inhibitor-induced‐1 protease with a metabolicKi of 2.7 nM syndromes and in a can plaque include assay dyslipidemia, with human insulin peripheral resistance, blood and lipodystrophy/lipoatrophy, as well as cardiovascular and cerebrovascular diseases [16]. andmononuclear lipodystrophy cells /(PBMCs)lipoatrophy, an asEC well90 of as170 cardiovascular nM similar to and the carbinol cerebrovascular (5) (Ki = diseases 0.37 nM, [16 EC].90 Therefore, = 23 nM, Therefore, safer and potentially promising protease inhibitor development is highly desirable. saferFigure and 3 green potentially dashed promising box) was determined. protease inhibitor development is highly desirable. Silicon-based Silicon‐based protease inhibitors provided a proof of principal for the use of this isostere and future proteaseMost inhibitors of the HIV provided‐1 protease a proof inhibitors of principal are foraccompanied the use of this by side isostere effects and in future long safety‐term evaluationtreatment. safety evaluation studies [15,17]. studiesHIV protease [15,17]. inhibitor‐induced metabolic syndromes can include dyslipidemia, insulin resistance, and lipodystrophy/lipoatrophy, as well as cardiovascular and cerebrovascular diseases [16]. 3.1.2.3.1.2. DivalentDivalent BioisosteresBioisosteres Therefore, safer and potentially promising protease inhibitor development is highly desirable. SiliconDivalentDivalent‐based protease isosteresisosteres inhibitors cancan bebe classifiedclassified provided intointo a twoprooftwo subgroups:subgroups: of principal (1)(1) for atomsatoms the use thatthat of areare this involvedinvolved isostere in inand aa double doublefuture bondsafetybond such suchevaluation as as C C=C,=C, studies CC=N,=N, C=O, C[15,17].=O, and and C=S C=S and and (2) (2) those those divalent divalent isosteres isosteres where where substitution of didifferentfferent atomsatoms resultsresults inin thethe alterationalteration ofof twotwo singlesingle bondsbonds suchsuch asas inin thethe series;series; C-C-C,C‐C‐C, C-NH-C,C‐NH‐C, C-O-C,C‐O‐C, andand C-S-C3.1.2.C‐S‐C Divalent [[4].4]. BothBoth Bioisosteres bioisostericbioisosteric classesclasses areare popularpopular andand havehave beenbeen usedused inin thethe past past extensively. extensively. Divalent isosteres can be classified into two subgroups: (1) atoms that are involved in a double EtherEther/Sulfone/Sulfone Substitution Substitution in in HIV-1 HIV‐1 Protease Protease Inhibitor Inhibitor Design Design bond such as C=C, C=N, C=O, and C=S and (2) those divalent isosteres where substitution of different atomsTheThe results highlyhighly in potentthepotent alteration HIV-1HIV‐1 protease ofprotease two single inhibitor inhibitor bonds saquinavir saquinavir such as in (IC (ICthe5050 series; = 0.23 CnM ‐C ‐in C, an C‐ NHin vitro‐C, C HIV-1HIV‐O‐C,‐1 GagandGag peptideCpeptide‐S‐C [4]. substrate Both bioisosteric cleavage assay)classes assay) suffers are suff popularers from from poorand poor have bioavailability bioavailability been used duein due the to past toits itspeptidic extensively. peptidic nature nature and and NH NHcontent. content. Absolute Absolute bioavailability bioavailability in healthy in healthy volunteers volunteers receiving receiving oral oral saquinavir saquinavir 600 600 mg waswas approximatelyEther/Sulfone Substitution 4% [18]. The in HIV reason‐1 Protease for its poor Inhibitor bioavailability Design is thought to be a combination of incomplete absorption and extensive first-pass metabolism. X-ray structures in complex with this The highly potent HIV‐1 protease inhibitor saquinavir (IC50 = 0.23 nM in an in vitro HIV‐1 Gag inhibitor class showed extensive interactions with the protease backbone, in particular, an extensive peptide substrate cleavage assay) suffers from poor bioavailability due to its peptidic nature and NH content. Absolute bioavailability in healthy volunteers receiving oral saquinavir 600 mg was

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approximately 4% [18]. The reason for its poor bioavailability is thought to be a combination of hydrogenincomplete bond network absorption was and shown extensive to be first crucial‐pass metabolism. for biological X‐ray activity. structures However, in complex as with drug this resistance inhibitor class showed extensive interactions with the protease backbone, in particular, an extensive emerged, Goshhydrogen and bond colleagues network was focused shown on to be the crucial retention for biological of a maximum activity. However, number as drug of contacts resistance of inhibitor with the proteinemerged, backbone Gosh and colleagues to combat focused mutant on the enzymes retention of [19 a maximum]. number of contacts of inhibitor A crucialwith the asparagine protein backbone moiety to combat in saquinavir mutant enzymes that contacts [19]. the backbone of Asp29 and Asp30 in HIV-1 proteaseA (Figurecrucial asparagine5A) was moiety replaced in saquinavir with 3(R)-tetrahydrofuranylglycine that contacts the backbone of Asp29 to and improve Asp30 in inhibitory HIV‐1 protease (Figure 5 A) was replaced with 3(R)‐tetrahydrofuranylglycine to improve inhibitory potency (Figure5B, IC = 0.05 nM in an in vitro HIV-1 Gag peptide substrate cleavage assay) [19,20]. potency (Figure50 5 B, IC50 = 0.05 nM in an in vitro HIV‐1 Gag peptide substrate cleavage assay) [19,20].

Figure 5. HIV-1Figure protease5. HIV‐1 inhibitorprotease inhibitor interaction. interaction. (A) Saquinavir (A) Saquinavir interacts interacts with with the the HIV-1 HIV‐1 protease protease backbone backbone residue Asp29 and Asp30. (B) Asparagine moiety in (A) was replaced by 3(R)‐ residue Asp29 and Asp30. (B) Asparagine moiety in (A) was replaced by 3(R)-tetrahydrofuranylglycine tetrahydrofuranylglycine (in pink) to obtain Amprenavir. (C) A cyclic sulfone group, in which the (in pink) tooxygen obtain can Amprenavir. accept H bonds (C from) A cyclicboth Asp29 sulfone and Asp30 group, and in retain which crucial the oxygenbackbone can interactions. accept H (D bonds) from both Asp29Darunavir and Asp30 backbone and interaction retain crucial with the backbone HIV‐1 protease interactions. (PDB code: ( 2F81)D) Darunavir. backbone interaction with the HIV-1 protease (PDB code: 2F81). To potentially improve bioavailability, Gosh and colleagues replaced this 3(R)‐ Totetrahydrofuranylglycine potentially improve moiety bioavailability,in amprenavir (Ki = 160 Gosh nM) with and a cyclic colleaguessulfone group, in replaced which this the oxygen can accept H bonds from both Asp29 and Asp30 (Figure 5 C) with retained potency (Ki = 3(R)-tetrahydrofuranylglycine1.4 nM). To retain the ability moiety to address in amprenavirboth asparagines, (K furtheri = 160 optimization nM) with did a lead cyclic to the sulfone design group, in which theof oxygen a more canlipophilic accept bicyclic H bonds ether as from a sulfone both isostere, Asp29 within and Asp30 the highly (Figure potent5C) protease with retainedinhibitor potency (Ki = 1.4 nM). To retain the ability to address both asparagines, further optimization did lead to the design of a more lipophilic bicyclic ether as a sulfone isostere, within the highly potent protease inhibitor darunavir (Presista®, Janssen Therapeutics), which has an absolute bioavailability of 37% [21] (as compared to 4% by saquinavir) and was licensed in the USA in 2006 (Figure5D) [19,20].

3.2. Non-Classical Bioisosteres in Anti-HIV-1 Drug Design and Development The following section will describe the major class of non-classical bioisosteric replacements and in silico methods for bioisostere identification with recent applications for HIV-1 entry inhibitor design and development. Non-classical bioisosteres are all replacements that are not defined by the classical definition. They usually are and can be very structurally different with an altered number of atoms, Pharmaceuticals 2020, 13, 36 7 of 16 Pharmaceuticals 2020, 13, 36 7 of 17 electronicsdarunavir or other (Presista physicochemical®, Janssen Therapeutics), properties which but their has an replacement absolute bioavailability mimics the of 37% spatial [21] arrangement(as compared to 4% by saquinavir) and was licensed in the USA in 2006 (Figure 5 D) [19,20]. of the original moiety such that biological activity is retained. As non-classical bioisosteres can be so different,3.2. theyNon‐Classical are most Bioisosteres often in employed Anti‐HIV‐1 Drug in “sca Designffold and Development hopping”, allowing the diversification of chemotypes forThe a following given target. section According will describe tothe Patani major class et al., of non non-classical‐classical bioisosteric bioisosteres replacements can be and subdivided into the replacementin silico methods of 1) for cyclic bioisostere groups identification by non-cyclic with groupsrecent applications and 2) replacement for HIV‐1 entry of functionalinhibitor groups with a varietydesign of and chemical development. moieties Non‐classical to retain bioisosteres biological are all activityreplacements (see that also are Figurenot defined1)[ by4]. the The utility and use ofclassical non-classical definition. bioisosteres They usually are have and been can be greatly very structurally furthered different by advances with an altered in novel number computation of atoms, electronics or other physicochemical properties but their replacement mimics the spatial representationsarrangement of compounds. of the original Computational moiety such that exploration biological activity of bioisosteric is retained. replacements As non‐classical for scaffold hopping canbioisosteres facilitate can be the so drug different, design they are and most development often employed process in “scaffold even hopping”, for smaller allowing companies the or academic groups,diversification who of typically chemotypes do for not a havegiven accesstarget. According to the large to Patani screening et al., non facilities‐classical of bioisosteres big pharma. At the end of thecan following be subdivided section, into the therefore, replacement we of describe 1) cyclic groups the advantages by non‐cyclic of groups this approachand 2) replacement with highlights of functional groups with a variety of chemical moieties to retain biological activity (see also Figure from our research1) [4]. The group utility and for use HIV-1 of non Env‐classical inhibitor bioisosteres design. have been greatly furthered by advances in novel computation representations of compounds. Computational exploration of bioisosteric 3.2.1. Heterocyclicreplacements Bioisosterism for scaffold hopping can facilitate the drug design and development process even for smaller companies or academic groups, who typically do not have access to the large screening Heterocyclesfacilities of are big pharma. important At the structural end of the following elements section, in drug therefore, design. we describe Variation the advantages in size of and shape can providethis approach substitution with highlights over a from wide our range, research while group for inherent HIV‐1 Env electronic inhibitor design. and physical properties are of importance in mediating drug–target interactions. If chosen carefully, they can modulate 3.2.1. Heterocyclic Bioisosterism crucial properties in drug design such as H-bond donor (NH, OH, CH) or acceptor properties, electron-withdrawingHeterocycles or are donating important structural effects, elements and the in drug potential design. toVariation engage in size in andπ– πshapeinteractions can [8]. provide substitution over a wide range, while inherent electronic and physical properties are of Tautomerism offers additional opportunities to optimize the topographical presentation of substituents importance in mediating drug–target interactions. If chosen carefully, they can modulate crucial and drug–targetproperties interactions. in drug design such The as azole H‐bond bioisosterism donor (NH, OH, will CH) beor acceptor briefly properties, described electron for‐ the HIV-1 integrase inhibition.withdrawing or donating effects, and the potential to engage in π–π interactions [8]. Tautomerism Integrationoffers additional of the viral opportunities DNA into to the optimize host genomethe topographical is a crucial presentation step in theof substituents HIV-1 and and other drug– retroviruses target interactions. The azole bioisosterism will be briefly described for the HIV‐1 integrase life cycle and is performed by the virus-encoded integrase [22]. The catalytic process involves the inhibition. 2+ coordination ofIntegration Mg by of the the HIV-1 viral DNA integrase. into the Azoles, host genome considered is a crucial as amide step in surrogates,the HIV‐1 and were other extensively studied asretroviruses HIV-1 integrase life cycle inhibitors and is performed [23–27 ].by In the contrast virus‐encoded to six-membered integrase [22]. heterocycles,The catalytic process azoles are able to adopt ainvolves coplanar the arrangementcoordination of Mg with2+ by additional the HIV‐1 integrase. metal-chelating Azoles, considered elements as amide presented surrogates, by a series of were extensively studied as HIV‐1 integrase inhibitors [23–27]. In contrast to six‐membered pyrido [1,2-a]pyrimidineheterocycles, azoles and are 1,6-naphthyridine-basedable to adopt a coplanar arrangement integrase with inhibitors additional (Figure metal‐chelating6). Of particular importanceelements is the presented nitrogen by arrangement a series of pyrido in [1,2 the‐a]pyrimidine 1,2,4-oxadiazoles and 1,6‐naphthyridine (Figure6B,C).‐based The integrase nitrogens lone electron pairinhibitors is crucial (Figure for 6). the Of coordinationparticular importance of a second is the nitrogen Mg2+ ionarrangement and activity in the against1,2,4‐oxadiazoles resistant viruses. In the pyrido(Figure [1,2-a]pyrimidine 6B and C). The nitrogens series, lone the electron thiazole pair (Figureis crucial 6forA) the was coordination chosen for of a future second Mg optimization2+ ion and activity against resistant viruses. In the pyrido [1,2‐a]pyrimidine series, the thiazole (Figure against clinically6A) was chosen relevant for future resistant optimization mutants. against clinically relevant resistant mutants.

2+ Figure 6. Coordination of Mg ions by azole-substituted pyrido[1,2-a]pyrimidines (A) and 1,6-naphthyridines (B) of HIV-1 integrase. In the 1,2,4-oxadiazole (C) the nitrogens lone electron 2+ pair is missing, preventing coordination by a second Mg ion. IC50 values were determined by using a recombinant HIV-1 integrase strand transfer assay.

3.2.2. Bioisosterism of Functional Groups Replacement of functional groups does not always result in the retention of biological activity. Nevertheless, a few examples will be highlighted in the following sections, with a focus on guanidine and amidine replacements. Guanidine and amidines are basic functionalities and protonated at Pharmaceuticals 2020, 13, 36 8 of 17

Figure 6. Coordination of Mg2+ ions by azole‐substituted pyrido[1,2‐a]pyrimidines (A) and 1,6‐ naphthyridines (B) of HIV‐1 integrase. In the 1,2,4‐oxadiazole (C) the nitrogens lone electron pair is

missing, preventing coordination by a second Mg2+ ion. IC50 values were determined by using a recombinant HIV‐1 integrase strand transfer assay.

3.2.2. Bioisosterism of Functional Groups Pharmaceuticals 2020, 13, 36 8 of 16 Replacement of functional groups does not always result in the retention of biological activity. Nevertheless, a few examples will be highlighted in the following sections, with a focus on guanidine physiologicaland amidine pH. replacements. This basicity Guanidine and protonation and amidines can reduce are basic membrane functionalities permeability, and protonated reducing at active compoundphysiological concentrations pH. This basicity in target and cells, protonation and contributing can reduce to membrane leads to poorpermeability, bioavailability. reducing Besides active the compound concentrations in target cells, and contributing to leads to poor bioavailability. Besides replacement of the entire guanidine or amidine entity (pKa of 13–14), the adjacent CH2 group can be replacedthe replacement with a carbonyl of the (pKaentire ofguanidine 8) or an or oxygen amidine atom entity (pKa (pKa of of 7–7.5) 13–14), to the reduce adjacent basicity CH2 group [8]. can be replaced with a carbonyl (pKa of 8) or an oxygen atom (pKa of 7–7.5) to reduce basicity [8]. Guanidine Bioisosteres Guanidine Bioisosteres Upon HIV-1 entry, genomic RNA is reverse transcribed and integrated into the host cell Upon HIV‐1 entry, genomic RNA is reverse transcribed and integrated into the host cell chromosome. Subsequently, the hijacked host RNA polymerase II (RNAP II) initiates transcription chromosome. Subsequently, the hijacked host RNA polymerase II (RNAP II) initiates transcription of of thisthis proviral proviral genome back back into into genomic genomic RNA, RNA, and elongation and elongation is stimulated is stimulated by the HIV by‐1 Tat the protein HIV-1 Tat protein[28]. [28 This]. Thismechanism mechanism involves involves the binding the bindingof Tat to ofa cis Tat‐regulatory to a cis-regulatory sequence in sequence the viral 5 in′ Long the viral 50 LongTerminal Terminal Repeat Repeat (LTR) (LTR)[29]. This [29 stem]. This‐loop stem-loop RNA element RNA is elementtermed TAR is termed RNA [30]. TAR This RNA activation [30]. This activationhas implications has implications for HIV for replication HIV replication and activation and activation of latent virus of latent reservoirs. virus reservoirs. The inhibition The of inhibition this of thisinteraction interaction represents represents a very a very attractive attractive approach approach for AIDS for treatment AIDS treatment and cure. and cure. In anIn RNA-targeted an RNA‐targeted SAR SAR study, study, Lee Lee and and colleaguescolleagues discovered discovered a novel a novel peptidomimetic peptidomimetic that that blocksblocks this interaction,this interaction, by replacing by replacing the guanidine the guanidine group group with awith squaryldiamine a squaryldiamine as a potent as a potent bioisostere bioisostere (Figure 7). Although the new squaric acid diamide analog showed a 4‐fold decrease in (Figure7). Although the new squaric acid diamide analog showed a 4-fold decrease in a ffinity (from a KD affinity (from a KD of 1.8 uM to 7.7 uM), as measured using an in vitro competition assay based on of 1.8fluorescence uM to 7.7 uM), resonance as measured energy transfer using an (FRET),in vitro it competitionwas the first assaybioisostere based to on mimic fluorescence the guanidine resonance energygroup transfer successfully (FRET), [31]. it was the first bioisostere to mimic the guanidine group successfully [31].

FigureFigure 7. Diaminosquarate 7. Diaminosquarate bioisostere bioisostere for thefor guanidinethe guanidine moiety moiety as a as novel a novel HIV-1 HIV Tat-TAR‐1 Tat‐TAR RNA RNA inhibitor. inhibitor. Amide and Ester Bioisosterism Amide and Ester Bioisosterism The amide bioisosterism is gaining popularity nowadays because of its implications for peptide The amide bioisosterism is gaining popularity nowadays because of its implications for peptide chemistry and development of peptide mimetics. It is of particular interest for medicinal chemists to chemistry and development of peptide mimetics. It is of particular interest for medicinal chemists to replace peptide bonds into chemically more stable and bioavailable entities. Esters, however, are usually replace peptide bonds into chemically more stable and bioavailable entities. Esters, however, are replacedusually to increasereplaced metabolic to increase stability, metabolic because stability, esters because are rapidly esters cleaved are rapidlyin vivo cleaved. Heterocyclic in vivo. rings suchHeterocyclic as 1,2,3-oxadiazoles rings such (6), as 1,3,4-oxadiazoles1,2,3‐oxadiazoles (6), (7), 1,3,4 triazoles‐oxadiazoles such as(7), 1,2,4-triazoles triazoles such (8),as 1,2,4 2-isoxazoline‐triazoles (9), and imidazoline(8), 2‐isoxazoline entities (9), (10)and imidazoline are very popular entities for (10) amide are very or ester popular replacements for amide or (Figure ester replacements8)[ 4]. In light of the diversity(Figure 8) of [4]. amide In light and of ester the bioisosterism,diversity of amide only and one ester example bioisosterism, using heterocycles only one example for the using design of HIV-1heterocycles accessory proteinfor the design viral infectivityof HIV‐1 accessory factor (Vif) protein will viral be discussedinfectivity factor here. (Vif) will be discussed Pharmaceuticalshere. 2020, 13, 36 9 of 17

FigureFigure 8. 8. ExamplesExamples of of heterocyclic heterocyclic rings rings as replacements as replacements for amide for amide and ester and groups. ester groups. The 1,2,3 The‐ 1,2,3-oxadiazolesoxadiazoles (6), (6),1,3,4 1,3,4-oxadiazoles‐oxadiazoles (7), (7),triazoles triazoles such such as 1,2,4 as 1,2,4-triazoles‐triazoles (8), (8),2‐isoxazoline 2-isoxazoline (9), (9),and and imidazolineimidazoline entities entities (10). (10).

The HIV‐1 Vif is an accessory protein and crucial for viral replication by antagonizing the host’s innate immunity [32,33]. Vif directly targets the human DNA‐editing enzyme APOBEC3G (A3G), which catalyzes hypermutations in viral DNA as a defense mechanism [34]. HIV‐1 Vif has no cellular homologs and is, therefore, a very attractive target for antiviral intervention. The RN‐18 class of Vif inhibitors enhances A3G‐dependent Vif degradation, increasing A3G incorporation into virions, and enhance cytidine deamination of the viral genome [35–37]. However, this class of inhibitors suffers from low potency and metabolic stability. Mohammed and colleagues successfully used bioisosteres of the central amide bond in RN‐18 (Figure 9, pink moiety) such as 1,3,4‐oxadiazole (11), 1,2,4‐ oxadiazole (12), 1,4‐disubstituted‐1,2,3‐triazole (13), and 1,5‐disubstituted‐1,2,3‐triazole (14) to develop new chemotypes of HIV‐1 Vif inhibitors for further optimization [38].

Figure 9. Heterocyclic bioisosteres of the RN‐18 HIV‐1 Vif inhibitor class and IC50 values obtained from a multi‐round infection assay (HIV‐1 LAI) using H9 and MT‐4 cells.

4. In silico Molecular Field‐Based Scaffold Hopping for Non‐Classical Bioisostere Identification Scaffold hopping using bioisosteric replacement has always been an integral part of the conventional drug development process but has expanded in recent years due to the availability of facile computational methods. High throughput screening (HTS), which tests thousands or millions of compounds, is a mainstay in the pharmaceutical industry. However, by applying computationally driven bioisosteric replacement to such initial hits, chemotype diversification can be readily achieved, which in turn increases the probability of successfully carrying one of these chemotypes through to the clinic. Of these computational methods of non‐classical bioisostere identification, the use of molecular field points has gained increasing recognition over recent years and has been very successfully utilized by our group in our initial efforts to redesign a first in‐class entry inhibitor to address non‐ optimal drug‐like properties and bioavailability. Typical approaches that do not use molecular field points tend to view the bioisosteric fragment in a vacuum, taking only fragment comparison into account and not factoring in the effect of the replacement across the whole molecule. Using molecular fields to facilitate a comparison of the entire spatial physicochemical properties across the entire

Pharmaceuticals 2020, 13, 36 9 of 17

Figure 8. Examples of heterocyclic rings as replacements for amide and ester groups. The 1,2,3‐ Pharmaceuticalsoxadiazoles2020 , (6),13, 36 1,3,4‐oxadiazoles (7), triazoles such as 1,2,4‐triazoles (8), 2‐isoxazoline (9), and9 of 16 imidazoline entities (10).

TheThe HIV-1HIV‐1 Vif Vif is is an an accessory accessory proteinprotein and and crucialcrucial forfor viralviral replicationreplication byby antagonizingantagonizing thethe host’shost’s innateinnate immunityimmunity [[32,33].32,33]. Vif directlydirectly targetstargets thethe humanhuman DNA-editingDNA‐editing enzymeenzyme APOBEC3GAPOBEC3G (A3G),(A3G), whichwhich catalyzes catalyzes hypermutations hypermutations in in viral viral DNA DNA asas aa defensedefense mechanismmechanism [[34].34]. HIV-1HIV‐1 VifVif hashas nono cellularcellular homologshomologs and is, therefore, therefore, a a very very attractive attractive target target for for antiviral antiviral intervention. intervention. The The RN RN-18‐18 class class of Vif of Vifinhibitors inhibitors enhances enhances A3G A3G-dependent‐dependent Vif Vifdegradation, degradation, increasing increasing A3G A3G incorporation incorporation into intovirions, virions, and andenhance enhance cytidine cytidine deamination deamination of the of viral the viralgenome genome [35–37]. [35 However,–37]. However, this class this of class inhibitors of inhibitors suffers sufromffers low from potency low potency and metabolic and metabolic stability. stability.Mohammed Mohammed and colleagues and colleaguessuccessfully successfully used bioisosteres used bioisosteresof the central of theamide central bond amide in RN bond‐18 in(Figure RN-18 9, (Figure pink 9moiety), pink moiety) such as such 1,3,4 as‐oxadiazole 1,3,4-oxadiazole (11), 1,2,4 (11),‐ 1,2,4-oxadiazoleoxadiazole (12), (12), 1,4‐ 1,4-disubstituted-1,2,3-triazoledisubstituted‐1,2,3‐triazole (13), (13), and and 1,5 1,5-disubstituted-1,2,3-triazole‐disubstituted‐1,2,3‐triazole (14) (14) toto developdevelop newnew chemotypeschemotypes ofof HIV-1HIV‐1 Vif Vif inhibitors inhibitors for for further further optimization optimization [ 38[38].].

Figure 9. Heterocyclic bioisosteres of the RN‐18 HIV‐1 Vif inhibitor class and IC50 values obtained Figure 9. Heterocyclic bioisosteres of the RN-18 HIV-1 Vif inhibitor class and IC50 values obtained from afrom multi-round a multi‐round infection infection assay (HIV-1assay (HIV LAI)‐1 using LAI) H9using and H9 MT-4 and cells.MT‐4 cells.

4.4. In Silicosilico Molecular Field Field-Based‐Based Scaffold Scaffold Hopping for Non-ClassicalNon‐Classical BioisostereBioisostere IdentificationIdentification ScaScaffoldffold hoppinghopping usingusing bioisostericbioisosteric replacementreplacement hashas alwaysalways beenbeen anan integralintegral partpart ofof thethe conventionalconventional drugdrug developmentdevelopment process butbut hashas expandedexpanded inin recentrecent yearsyears duedue toto thethe availabilityavailability ofof facilefacile computationalcomputational methods. methods. High High throughput throughput screening screening (HTS), (HTS), which which tests tests thousands thousands or millionsor millions of compounds,of compounds, is ais mainstay a mainstay in in the the pharmaceutical pharmaceutical industry. industry. However, However, by by applying applying computationally computationally drivendriven bioisostericbioisosteric replacementreplacement toto suchsuch initialinitial hits, chemotype diversificationdiversification can be readily achieved, whichwhich inin turnturn increasesincreases thethe probabilityprobability ofof successfullysuccessfully carryingcarrying oneone ofof thesethese chemotypeschemotypes throughthrough toto thethe clinic.clinic. OfOf thesethese computationalcomputational methodsmethods ofof non-classicalnon‐classical bioisosterebioisostere identification,identification, thethe useuse ofof molecularmolecular fieldfield points points has has gained gained increasing increasing recognition recognition over over recent recent years years and has and been has very been successfully very successfully utilized byutilized our group by our in group our initial in our eff initialorts to efforts redesign to redesign a first in-class a first entryin‐class inhibitor entry inhibitor to address to address non-optimal non‐ drug-likeoptimal drug properties‐like properties and bioavailability. and bioavailability Typical approaches. Typical approaches that do not that use moleculardo not use field molecular points tendfield topoints view tend the bioisosteric to view the fragment bioisosteric in a vacuum,fragment taking in a vacuum, only fragment taking comparison only fragment into comparison account and into not factoringaccount and in the not e fffactoringect of the in replacement the effect of across the replacement the whole molecule. across the Using whole molecular molecule. fields Using to molecular facilitate afields comparison to facilitate of the a entirecomparison spatial of physicochemical the entire spatial properties physicochemical across the entireproperties molecule, across rather the entire than the structure alone, can and has led to discoveries of non-intuitive bioisosteric replacements that have positively modulated drug-like properties while retaining target engagement [39–43]. However, molecular fields are continuous and vary with the conformation of the compound, and sampling at defined points in the three-dimensional grid can be computationally expensive (Figure 10A). Therefore, program collections such as Cresset (Cresset, Litlington, UK) use only extrema of these fields, the so-called field point, where intermolecular interactions most likely occur (Figure 10B). Pharmaceuticals 2020, 13, 36 10 of 17

molecule, rather than the structure alone, can and has led to discoveries of non‐intuitive bioisosteric replacements that have positively modulated drug‐like properties while retaining target engagement [39–43]. However, molecular fields are continuous and vary with the conformation of the compound, and sampling at defined points in the three‐dimensional grid can be computationally expensive (Figure 10A). Therefore, program collections such as Cresset (Cresset, Litlington, UK) use only

Pharmaceuticalsextrema of these2020, 13fields,, 36 the so‐called field point, where intermolecular interactions most likely10 occur of 16 (Figure 10B).

Figure 10. Electrostatic (blue/red), van der Waals (yellow) and hydrophobic (beige) contours (A) and Figure 10. Electrostatic (blue/red), van der Waals (yellow) and hydrophobic (beige) contours (A) and Field Point maxima (B) of an HIV‐1 entry inhibitor. Field Point maxima (B) of an HIV-1 entry inhibitor. This calculation and comparison of field points is based on the eXtended Electron Density Force This calculation and comparison of field points is based on the eXtended Electron Density Force Field Theory (XED) [44,45]. XED takes into account positive and negative electrostatic, Van der Waals, Field Theory (XED) [44,45]. XED takes into account positive and negative electrostatic, Van der Waals, and hydrophobic field points. In contrast to most molecular mechanics force field methods that apply and hydrophobic field points. In contrast to most molecular mechanics force field methods that atom‐centered partial charges, XED generates negative pseudo‐orbitals surrounding the positive apply atom-centered partial charges, XED generates negative pseudo-orbitals surrounding the positive nuclei and distributes partial charges according to the orbital location. XED, therefore, mimics the nuclei and distributes partial charges according to the orbital location. XED, therefore, mimics the electron distribution in a more accurate way by taking π‐clouds and lone pairs into account, which electron distribution in a more accurate way by taking π-clouds and lone pairs into account, which play play important roles in biological systems. In practice, Field Points of two or more compounds can important roles in biological systems. In practice, Field Points of two or more compounds can be be compared by sampling conformations to maximize the fields for optimal alignment, and in compared by sampling conformations to maximize the fields for optimal alignment, and in contrast to contrast to a structural alignment, new compounds with different structures but similar numbers and a structural alignment, new compounds with different structures but similar numbers and distribution distribution of Field Points can be found. This so‐called “templating” of molecules can also be used of Field Points can be found. This so-called “templating” of molecules can also be used to generate to generate a high‐content pharmacophore for therapeutically interesting targets for which there is a high-content pharmacophore for therapeutically interesting targets for which there is no known no known information regarding the small molecule binding site. The following sections will describe information regarding the small molecule binding site. The following sections will describe recent recent applications using such computational approaches from our lab for the design of novel HIV‐1 applications using such computational approaches from our lab for the design of novel HIV-1 entry entry inhibitors with improved potency, specificity, and ADME properties. inhibitors with improved potency, specificity, and ADME properties.

Sca4.1.ff Scaffoldold Hopping Hopping for Potency for Potency and ADMEand ADME Improvement Improvement of HIV-1 of HIV Entry‐1 Entry Leads Leads The inhibitioninhibition of of HIV-1 HIV‐ entry1 entry is an is attractive,an attractive, yet underexploited yet underexploited therapeutic therapeutic approach approach for several for reasons.several reasons. First, much First, of themuch entry of processthe entry occurs process in water-soluble occurs in water compartments‐soluble compartments easily accessible easily to drugs.accessible Second, to drugs. both Second, viral and both host viral cell and components host cell components involved in involved HIV-1 entry in HIV have‐1 entry been have identified been andidentified can be and targeted. can be Third, targeted. because Third, inhibition because inhibition of virus entry of virus prevents entry the prevents host cell the from host becoming cell from permanentlybecoming permanently infected, such infected, inhibitors such areinhibitors potentially are potentially useful in many useful di infferent many modalities, different modalities, including pre-exposureincluding pre prophylactics,‐exposure prophylactics, prophylactics, prophylactics, and microbicides and microbicides [46]. [46]. InIn anan eeffortffort toto bringbring thisthis classclass ofof inhibitorinhibitor outout ofof thethe realmrealm ofof salvagesalvage therapy, wewe havehave appliedapplied both high-content,high‐content, field-point field‐point pharmacophores pharmacophores to perform to perform virtual screening,virtual screening, and iterative and bioisosteric iterative replacements,bioisosteric replacements, to identify novel to identify leads for novel HIV-1 leads entry for inhibitors. HIV‐1 entry The inhibitors. most potent The and most broadly potent acting and HIV-1broadly inhibitors acting HIV to‐1 date inhibitors are piperazine-based to date are piperazine introduced‐based by introduced Bristol-Myers by Bristol Squibb‐Myers [47]. Squibb However, [47]. thisHowever, class has this low class bioavailability, has low bioavailability, resulting from resulting low solubility from low and solubility poor dissolution. and poor Bristol-Myers dissolution. SquibbBristol‐ hasMyers partially Squibb overcome has partially these overcome limitations these by adopting limitations a prodrug by adopting approach a prodrug with BMS-663068 approach with that shows increased solubility in the gut. However, after removal of the phosphonooxymethyl-solubilizing group in the gut, the properties of the active compound, BMS-626529, dominate and, despite its potency, 1.2 g per day is required to achieve an effective plasma concentration [48]. Clearly, an entry inhibitor with more intrinsic drug-like properties would be preferable [49]. Therefore, we used field-based three-dimensional similarity virtual screening experiments using Blaze (Cresset, Litlington, Pharmaceuticals 2020, 13, 36 11 of 17

BMS‐663068 that shows increased solubility in the gut. However, after removal of the phosphonooxymethyl‐solubilizing group in the gut, the properties of the active compound, BMS‐ 626529,Pharmaceuticals dominate2020 and,, 13, 36despite its potency, 1.2 g per day is required to achieve an effective plasma 11 of 16 concentration [48]. Clearly, an entry inhibitor with more intrinsic drug‐like properties would be preferable [49]. Therefore, we used field‐based three‐dimensional similarity virtual screening experimentsUK) with using a high-content Blaze (Cresset, field-based Litlington, pharmacophore UK) with a high template‐content derived field‐based from pharmacophore BMS-626529 and two templateof its predecessors, derived from BMS-488043BMS‐626529 and and two BMS-378806, of its predecessors, to identify BMS novel‐488043 scaff oldsand BMS that‐ could378806, function to as identifyentry inhibitorsnovel scaffolds [50]. Fieldthat templatingcould function was implemented,as entry inhibitors as at the[50]. time Field of thistemplating study, no was structural implemented,information as regarding at the time the of bindingthis study, site no nor structural the bioactive information conformation regarding of the the binding BMS compounds site nor was theavailable. bioactive FieldTemplaterconformation of (Forge) the BMS allows compounds the individual was available. to generate FieldTemplater a bioactive (Forge) conformation allows hypothesis,the individualand a field-point to generate pattern a pharmacophore,bioactive conformation in the absence hypothesis, of structural and information, a field‐point as longpattern as multiple, pharmacophore,distinct compounds in the absence are known of structural and are supposed information, to interactas long as with multiple, the same distinct site oncompounds the target. are Figure 11 knownshows and the are field-point supposed templateto interact generated with the same and usedsite on in the our target. HIV-1 Figure entry 11 inhibitor shows the studies field‐ (Figurepoint 11A), templatein addition generated to a comparison and used in of our the HIV template‐1 entry to theinhibitor experimentally studies (Figure determined 11A), in bioactive addition conformation to a comparisonof BMS-626529 of the template (Figure 11 toB) the [51 experimentally,52]. determined bioactive conformation of BMS‐626529 (Figure 11B) [51,52].

FigureFigure 11. 11. (A)( AField) Field-based‐based template template containing containing a single a single conformation conformation of compounds of compounds BMS‐378806 BMS-378806 (pink),(pink), BMS BMS-488043‐488043 (lime (lime green), green), and and BMS BMS-626529‐626529 (teal(teal green)green) aligned aligned based based on on their their three-dimensional three‐ dimensionalfield point field patterns. point patterns. Negatively Negatively charged charged field points field arepoints shown are shown in blue; in positively blue; positively charged field chargedpoints field are points red; van are der red; Waals van /dershape Waals/shape field points field are points displayed are displayed in yellow; in centers yellow; ofcenters hydrophobicity of hydrophobicity are shown in orange. The dodecahedral size of the field points is proportional to the are shown in orange. The dodecahedral size of the field points is proportional to the magnitude of magnitude of the extrema. (B) Structural superimposition of BMS‐62529 conformer generated with the extrema. (B) Structural superimposition of BMS-62529 conformer generated with FieldTemplater FieldTemplater (pink) and experimentally obtained crystal structure in complex with HIV‐1 BG505 (pink) and experimentally obtained crystal structure in complex with HIV-1 BG505 SOSIP.664 (yellow, SOSIP.664 (yellow, PDB code: 5U7O). PDB code: 5U7O). From this field‐point pharmacophore screen using Blaze (Cresset UK), we identified hits with From this field-point pharmacophore screen using Blaze (Cresset UK), we identified hits with piperazine‐based cores similar to the BMS chemotypes but with potencies in the low micromolar piperazine-based cores similar to the BMS chemotypes but with potencies in the low micromolar range, range, ranking from 13 to 153 μM using a HIV‐1 YU‐2 Env pseudotyped virus in a single‐round ranking from 13 to 153 µM using a HIV-1 YU-2 Env pseudotyped virus in a single-round infection assay infection assay (SRIA). Therefore, to obtain a truly novel core scaffold, we chose to conduct (SRIA). Therefore, to obtain a truly novel core scaffold, we chose to conduct bioisosteric replacement bioisosteric replacement (scaffold hopping) with Spark (Cresset, UK). The results of this indicated that(sca replacingffold hopping) the piperazine with Spark group (Cresset, with UK).a dipyrrolidine The results moiety of this would indicated be thatviable. replacing The resulting the piperazine group with a dipyrrolidine moiety would be viable. The resulting compound (SC04) showed lower compound (SC04) showed lower potency than the piperazine‐based compounds (IC50 HIV‐1 YU‐2 = potency than the piperazine-based compounds (IC HIV-1 YU-2 = 70 6 µM; IC HIV-1 JR-CSF = 100 70 ± 6 μM; IC50 HIV‐1 JR‐CSF = 100 ± 30 μM using a pseudotyped50 virus in an± SRIA) but50 demonstrated 30 µM using a pseudotyped virus in an SRIA) but demonstrated specificity, making it perfect for specificity,± making it perfect for further optimization in potency (Figure 12). further optimization in potency (Figure 12). Using Spark (Cresset, UK) and a fragment library generated from PubChem by fragmenting compounds with similarities to BMS-488043, we researched whether the dipyrrolidine could support nanomolar potency, whilst retaining specificity. Sequentially, we generated SC07 (IC50 HIV-1 JR-CSF = 0.98 0.06 µM in an SRIA), SC08 (IC HIV-1 JR-CSF = 0.09 0.01 µM in an SRIA), and SC11 (IC HIV-1 ± 50 ± 50 JR-CSF values of 0.0008 0.0004 µM in an SRIA). This was the first time that the piperazine core in this ± class of inhibitors had been successfully changed whilst retaining high potency. Finally, the replacement of the terminal phenyl in SC11 by cyclohexene in SC26 resulted in the first dipyrolrolodine-scaffolded highly potent HIV-1 entry inhibitor (IC HIV-1 JR-CSF of 2.0 0.1 nM; IC HIV-1 HxBc2 of 0.6 0.01 50 ± 50 ± nM in an SRIA), with significantly improved predicted ADME properties (as indicated by an increase in the Oral Non-CNS Drug-like Score in StarDrop, Optibrium, UK; Figure 13). Following this initial success, we then used similar methods to extend the core chemotypes in the HIV-1 entry inhibitor class. This resulted in the creation of five distinct core chemotypes from Pharmaceuticals 2020, 13, 36 12 of 17

Pharmaceuticals 2020, 13, 36 12 of 16

the original piperazine BMS-626529 that support specificity and nanomolar potencies (Figure 14 and TablePharmaceuticals3)[53]. 2020, 13, 36 12 of 17

Figure 12. Evolution (from left to right) of HIV‐1 entry inhibitors to improve potency and ADME properties. Structural changes between each compound are highlighted in pink.

Using Spark (Cresset, UK) and a fragment library generated from PubChem by fragmenting compounds with similarities to BMS‐488043, we researched whether the dipyrrolidine could support nanomolar potency, whilst retaining specificity. Sequentially, we generated SC07 (IC50 HIV‐1 JR‐CSF = 0.98 ± 0.06 μM in an SRIA), SC08 (IC50 HIV‐1 JR‐CSF = 0.09 ± 0.01 μM in an SRIA), and SC11 (IC50 HIV‐1 JR‐CSF values of 0.0008 ± 0.0004 μM in an SRIA). This was the first time that the piperazine core in this class of inhibitors had been successfully changed whilst retaining high potency. Finally, the replacement of the terminal phenyl in SC11 by cyclohexene in SC26 resulted in the first dipyrolrolodine‐scaffolded highly potent HIV‐1 entry inhibitor (IC50 HIV‐1 JR‐CSF of 2.0 ± 0.1 nM; IC50 HIV‐1 HxBc2 of 0.6 ± 0.01 nM in an SRIA), with significantly improved predicted ADME propertiesFigure (as 12.12. indicated Evolution by (from an increase left to right) in the of Oral HIV-1HIV Non‐1 entryentry‐CNS inhibitorsinhibitors Drug‐like toto improve improveScore in potency potencyStarDrop, andand Optibrium, ADMEADME UK; Figureproperties.properties. 13). Structural changes between eacheach compoundcompound areare highlightedhighlighted inin pink.pink.

Using Spark (Cresset, UK) and a fragment library generated from PubChem by fragmenting compounds with similarities to BMS‐488043, we researched whether the dipyrrolidine could support nanomolar potency, whilst retaining specificity. Sequentially, we generated SC07 (IC50 HIV‐1 JR‐CSF = 0.98 ± 0.06 μM in an SRIA), SC08 (IC50 HIV‐1 JR‐CSF = 0.09 ± 0.01 μM in an SRIA), and SC11 (IC50 HIV‐1 JR‐CSF values of 0.0008 ± 0.0004 μM in an SRIA). This was the first time that the piperazine core in this class of inhibitors had been successfully changed whilst retaining high potency. Finally, the replacement of the terminal phenyl in SC11 by cyclohexene in SC26 resulted in the first dipyrolrolodine‐scaffolded highly potent HIV‐1 entry inhibitor (IC50 HIV‐1 JR‐CSF of 2.0 ± 0.1 nM; IC50 HIV‐1 HxBc2 of 0.6 ± 0.01 nM in an SRIA), with significantly improved predicted ADME properties (as indicated by an increase in the Oral Non‐CNS Drug‐like Score in StarDrop, Optibrium, UK; Figure 13).

Figure 13. Improved ADME properties of SC26. Plot showing the StarDrop (Optibrium, Ltd., FigureCambridge, 13. Improved UK)–derived ADME logS properties versus the of score SC26 from. Plot a multimetric showing the oral StarDrop non-CNS (Optibrium, profile for the Ltd., BMS Cambridge,and SC compounds. UK)–derived Scores logS rangeversus from the score 0 to from 1, with a multimetric 0 suggesting oral extremely non‐CNS non-drug-like, profile for the BMS and 1 andsuggesting SC compounds. the perfect Scores drug. range from 0 to 1, with 0 suggesting extremely non‐drug‐like, and 1 suggesting the perfect drug. Stemming from these initial successes, we have since applied this workflow to identify new bioisosteres for the methyltriazole-azaindole moiety, as the main potency determinant of this entry inhibitor class and identified a compound (SC56), wherein the methyltriazole in SC28 was replaced with an amine-oxadiazole (Figure 15)[54]. This compound displayed a 1000-fold increased affinity for the B41 SOSIP Env, as judged by surface plasmon resonance (SPR).

Figure 13. Improved ADME properties of SC26. Plot showing the StarDrop (Optibrium, Ltd., Cambridge, UK)–derived logS versus the score from a multimetric oral non‐CNS profile for the BMS and SC compounds. Scores range from 0 to 1, with 0 suggesting extremely non‐drug‐like, and 1 suggesting the perfect drug.

Pharmaceuticals 2020, 13, 36 13 of 17

Following this initial success, we then used similar methods to extend the core chemotypes in the HIV‐1 entry inhibitor class. This resulted in the creation of five distinct core chemotypes from the Pharmaceuticalsoriginal piperazine2020, 13, 36 BMS‐626529 that support specificity and nanomolar potencies (Figure 14 and13 of 16 Table 3) [53].

FigureFigure 14. 14.Sca Scaffoldffold hopping hopping inin the core region of of an an HIV HIV-1‐1 entry entry inhibitor. inhibitor. Structural Structural changes changes in the in the core region between each compound are highlighted in pink. core region between each compound are highlighted in pink.

TableStemming 3. Specificity from these and potency initial successes, (in µM) of SCwe compounds have since againstapplied HIV-1J this workflow R-CSF, HIV-1 to identify B41, HIV-1 new bioisosteresHxBc2 Env, for and the HIV-1 methyltriazole AMLV pseudotyped‐azaindole HIV-1 moiety, using as the a single-round main potency infection determinant assay. of this entry inhibitor class and identified a compound (SC56), wherein the methyltriazole in SC28 was replaced with anCompound amine‐oxadiazole (Figure IC50 JR-CSF 15) [54]. This compound IC50 B41 displayed IC 50a 1000HxBc2‐fold increased IC50 AMLV affinity for the B41SC11 SOSIP Env, as 0.0008judged by0.0001 surface plasmon 0.002 resonance0.0002 (SPR). 0.001 0.0001 N.A. ± ± ± SC12 0.008 0.002 0.006 0.003 0.080 0.020 N.A. ± ± ± SC15 0.003 0.001 0.007 0.001 0.009 0.001 N.A. Table 3. Specificity and potency± (in μM) of SC compounds± against HIV‐1J R±‐CSF, HIV‐1 B41, HIV‐1 SC28 0.096 0.019 0.085 0.03 0.069 0.014 N.A. HxBc2 Env, and HIV‐1 AMLV ±pseudotyped HIV‐1 using± a single‐round infection± assay. SC45 0.224 0.017 0.350 0.030 0.380 0.030 N.A. ± ± ± Compound IC50 JR‐CSFN.A. = not active; N.D.IC50 =B41not determined.IC50 HxBc2 IC50 AMLV SC11 0.0008 ± 0.0001 0.002 ± 0.0002 0.001 ± 0.0001 N.A. Pharmaceuticals 2020, 13, 36 14 of 17 SC12 0.008 ± 0.002 0.006 ± 0.003 0.080 ± 0.020 N.A. SC15 0.003 ± 0.001 0.007 ± 0.001 0.009 ± 0.001 N.A. SC28 0.096 ± 0.019 0.085 ± 0.03 0.069 ± 0.014 N.A. SC45 0.224 ± 0.017 0.350 ± 0.030 0.380 ± 0.030 N.A. N.A. = not active; N.D. = not determined.

Finally, in a soon to be published study, we have also demonstrated that SC28, with the azabicyclo‐hexane core scaffold, has twice the metabolic stability of BMS‐626529. This approach highlights bioisosteric replacement as a powerful tool that can be utilized to rapidly diversify chemotypes but can improve drug–target kinetic profiles and metabolism in the drug development process.

FigureFigure 15. 15. ModulationModulation of of binding binding kinetics kinetics using using bioisosteric bioisosteric replacement replacement in in HIV HIV-1‐1 entry entry inhibitor inhibitor design.design. The The structural structural change change between between the the two two compounds compounds is is highlighted highlighted in in pink. pink. Affinity Affinity and and kinetic kinetic parametersparameters were were determined using a surface plasmon resonance assay.

5. ConclusionFinally, in a soon to be published study, we have also demonstrated that SC28, with the azabicyclo-hexane core scaffold, has twice the metabolic stability of BMS-626529. In this review, we demonstrated, using a few selected examples, the advantages and progress This approach highlights bioisosteric replacement as a powerful tool that can be utilized to rapidly that have been made using bioisosteric replacement strategies to create new, more potent therapies diversify chemotypes but can improve drug–target kinetic profiles and metabolism in the drug to combat the AIDS pandemic. In the absence of a viable cure or vaccine, the demand for drugs development process. targeting new therapeutic targets, in addition to new, optimized drugs to old targets is paramount. As such, we believe that the use of bioisosterism will only increase in the development of such HIV therapies in the future.

Author Contributions: All authors contributed to the writing, editing and review of this article. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NIH, grant number GM125396 (Cocklin, PI). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Langmuir, I. Isomorphism, Isosterism and Covalence. J. Am. Chem. Soc. 1919, 41, 1543–1559. 2. Grimm, H.G. Structure and Size of the Non‐metallic Hydrides. Z. Electrochem. 1925, 31, 474–480. 3. Grimm, H.G. On the Systematic Arrangement of Chemical Compounds from the Perspective of Research on Atomic Composition; and on Some Challenges in Experimental Chemistry. Naturwissenschaften 1929, 17, 557–564. 4. Patani, G.A.; LaVoie, E.J. Bioisosterism: A Rational Approach in Drug Design. Chem. Rev. 1996, 96, 3147– 3176. 5. Friedman, H.L. Influence of Isosteric Replacements upon Biological Activity. Nasnrs 1951, 206, 295–358. 6. Burger, A. Isosterism and Bioisosterism in Drug Design. In Progress in Drug Research/Fortschritte der Arzneimittelforschung/Progrès des Recherches Pharmaceutiques; Birkhäuser, Basel, 1991. 7. Medicinal Chemistry, 3rd ed.; Wiley‐Interscience: New York, NY, USA, 1970. 8. Meanwell, N.A. Synopsis of some recent tactical application of bioisosteres in drug design. J. Med. Chem. 2011, 54, 2529–2591. 9. Wade, D. Deuterium isotope effects on noncovalent interactions between molecules. Chem. Biol. Interact. 1999, 117, 191–217. 10. Hu, W.S.; Hughes, S.H. HIV‐1 reverse transcription. Cold Spring Harb. Perspect. Med. 2012, 2, a006882.

Pharmaceuticals 2020, 13, 36 14 of 16

5. Conclusions In this review, we demonstrated, using a few selected examples, the advantages and progress that have been made using bioisosteric replacement strategies to create new, more potent therapies to combat the AIDS pandemic. In the absence of a viable cure or vaccine, the demand for drugs targeting new therapeutic targets, in addition to new, optimized drugs to old targets is paramount. As such, we believe that the use of bioisosterism will only increase in the development of such HIV therapies in the future.

Author Contributions: All authors contributed to the writing, editing and review of this article. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NIH, grant number GM125396 (Cocklin, PI). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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