Anti-HIV Drug Discovery and Development: Current Innovations and

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Anti-HIV Drug Discovery and Development: Current Innovations and Future Trends Miniperspective Peng Zhan,† Christophe Pannecouque,‡ Erik De Clercq,*,‡ and Xinyong Liu*,† †

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012, Jinan, Shandong, P. R. China ‡ Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium ABSTRACT: The early effectiveness of combinatorial antiretroviral therapy (cART) in the treatment of HIV infection has been compromised to some extent by rapid development of multidrug-resistant HIV strains, poor bioavailability, and cumulative toxicities, and so there is a need for alternative strategies of antiretroviral drug discovery and additional therapeutic agents with novel action modes or targets. From this perspective, we first review current strategies of antiretroviral drug discovery and optimization, with the aid of selected examples from the recent literature. We highlight the development of phosphate ester-based prodrugs as a means to improve the aqueous solubility of HIV inhibitors, and the introduction of the substrate envelope hypothesis as a new approach for overcoming HIV drug resistance. Finally, we discuss future directions for research, including opportunities for exploitation of novel antiretroviral targets, and the strategy of activation of latent HIV reservoirs as a means to eradicate the virus.

1. INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) is the main causative agent of acquired immunodeficiency syndrome (AIDS), which remains a serious public health problem throughout the world. At present, about 30 drugs in five main classes have been approved for the treatment of HIV/AIDS. These drugs target different steps of the viral life cycle: (i) viral entry (e.g., coreceptor antagonists and fusion inhibitors); (ii) reverse transcription (reverse transcriptase (RT) inhibitors); (iii) integration (integrase (IN) inhibitors); and (iv) viral maturation (protease (PR) inhibitors).1 Powerful combinatorial antiretroviral therapy (cART, a combination of anti-HIV drugs targeting different steps in the life cycle of the virus) has had considerable success in controlling HIV infection. However, there are still two key issues: first, the emergence of extensively cross-resistant strains of HIV-1 (partly because of poor compliance), and second, the adverse effects (poor tolerability, drug−drug interactions, toxicities) of long-term use of these drug regimens, leading to poor patient compliance (namely, failure of patients to adhere to the drug regimen).2−6 Thus, there is an urgent need for new anti-HIV drug candidates with increased potency, novel targets, improved pharmacokinetic properties, and reduced side effects. The traditional approach of random screening and subsequent optimization of lead compounds by systematic organic synthesis is highly resource- and time-consuming. Thus, more efficient and faster strategies that shorten and facilitate the discovery process would be extremely beneficial. Indeed, the discovery of antiHIV agents is moving on from trial-and-error approaches to © 2015 American Chemical Society

sophisticated methodologies. In recent years, several strategies have been employed to discover novel anti-HIV agents with novel scaffolds and better resistance profiles, including fragmentbased screening, privileged fragment-based reconstruction, dynamic ligation screening (DLS)-based drug discovery, rapid diversity-oriented synthesis combined with in situ screening, and hierarchical multiple-filter database searching. In this article, we present a critical survey of current structural modification strategies in the medicinal chemistry of anti-HIV agents, with the aid of selected examples from the recent literature. We also highlight a new strategy for improving drug efficacy and mitigating solubility-limited bioavailability by using phosphate ester-based prodrugs, and a novel structure-based drug design methodology to reduce drug susceptibility to resistance mutations, based on the substrate envelope hypothesis. Finally, we present an overview of structure biology-based exploration of novel targets in the HIV-1 replication cycle, as well as an alternative strategy, which has been described as the “shock-and-kill” (or “kick-and-kill”) strategy, based on the idea of activating the latent viral reservoirs in order to completely eradicate the virus.

2. NEW MEDICINAL CHEMISTRY STRATEGIES FOR LEAD DISCOVERY Orthodox drug discovery has usually relied on screening a significant number of druglike molecules to find lead compounds Received: March 27, 2015 Published: October 28, 2015 2849

DOI: 10.1021/acs.jmedchem.5b00497 J. Med. Chem. 2016, 59, 2849−2878

Journal of Medicinal Chemistry

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Figure 1. Binding of small molecules at novel sites of HIV PR. Protein Data Bank codes are the following: for 1 in the outside/top of the flap, 4EJ8; for 2 in the outside/top of flap, 4EJK; for 3 in the flap site, 4K4Q; for 4 in the exosite, 4K4R.12,13

overcomes this difficulty, as bromine can be located accurately through anomalous scattering. Consequently, in 2014, the same research group reported crystallographic fragment-based discovery of two more novel hits, 3-bromo-2,6-dimethoxybenzoic acid (3, Br6) (in the flap site) and 1-bromo-2-naphthoic acid (4, Br27) (in the exosite) from a brominated fragment library targeting HIV PR.12,13 It was indicated that fragment binding at the flap site favors a closed conformation of PR (Figure 1). Allosteric regulation of PR activity was suggested to be a potential approach to overcome the development of drug-resistance. These findings expanded the range of fragments available for development of higher-affinity allosteric inhibitors.12,13 We envision that future research will focus on elaborating these fragments into larger molecules that potently inhibit PR. In 2014, Deng et al. utilized the combination of AutoDock calculations with recently developed free energy methods (binding energy analysis method (BEDAM) and the standard double decoupling method (DDM)) to screen fragments targeting the allosteric site on the flap of HIV PR.14 In 2014, compounds 5 and 6a were identified as smallmolecular-weight fragments targeting dynamic pockets of HIV-1 PR by means of structure-based computational screening, and further derivatization afforded 6b,c, 7, and 8a,b.15 Among them, 6c, 7, and 8a,b are potential allosteric inhibitors (Figure 2). These compound pairs have high SAR information content and exhibit activity cliffs, exemplifying situations where small structural changes lead to significant differences in potency.16,17 HIV-1 RT is another central target for antiretroviral therapy. RT undergoes conformational changes during HIV replication, and its intrinsic flexibility provides potential allosteric sites for inhibition. In 2011, a novel scaffold bromoindanone 9 was identified as an inhibitor of wild-type (WT) and drug-resistant

that act at a putative target. Although high-throughput screening (HTS) and experimental screening of in-house chemical libraries have been successful in finding many “original hits”, leading ultimately to clinically useful drugs, the approach suffers from substantial drawbacks, including extremely low hit rates, many false positives, and the need for laborious lead modification, as the initial hit is often far from optimal for binding to the target.7,8 In contrast, innovations such as fragment-based drug design (FBDD), DLS methodology, high-speed diversity-oriented synthesis combined with in situ screening, and hierarchical multiple-filter database searching have enabled rapid and efficient progress in anti-HIV drug discovery. 2.1. Fragment-Based Screening. Fragment-based screening (FBS) by means of biophysical techniques, such as X-ray crystallography, NMR spectroscopy, or surface plasmon resonance (SPR), is quickly becoming a valuable technique for identifying new fragment hits and new target sites for drug discovery.9−11 It has been utilized in the development of novel antiretroviral agents bearing unprecedented scaffolds for several well-validated targets, such as HIV PR, RT, and IN, which are pivotal in HIV replication but mutate rapidly, leading to significant drug resistance. In 2013, the fragment indole-6-carboxylic acid (1, 1F1) was identified as a ligand binding to the flap site of HIV PR in a fragment-based screen. 3-Indolepropionic acid (2, 1F1-N) was then identified by means of computational docking (AutoDock) and a nucleation crystallization inhibition assay. It was demonstrated by X-ray crystallography and measurements of conformational stabilization and direct site occupancy that compounds 1 and 2 occupy an external site on PR both in the solid state and in solution.9 It is well-known that fragments are poor binders with only partial occupancy, resulting in weak, difficult-to-fit electron density. The employment of a brominated fragment library 2850



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Figure 2. Identification of fragments 5 and 6a targeting dynamic pockets of HIV-1 PR by virtual screening and the results of further derivatization to afford 6b,c, 7, and 8a,b.

of fragment 21, which show high ligand efficiency (IC50 = 295 μM), eventually leading to a more active molecule 22 (IC50 = 5 μM).21 A similar structure 23 was identified as a novel binder of the lens epithelium-derived growth factor/p75 (LEDGF/p75) binding site of the HIV catalytic core domain through fragmentbased screening and structure-based drug design (SBDD). It inhibited HIV infection in a cell-based assay with an EC50 of 29 μM. Examination of the crystallographic structure (PDB code 3ZSO) afforded further insights into the ligand-binding modes in IN and should provide a basis for the discovery of more active IN inhibitors.22 Drug development targeting the HIV-1 envelope glycoprotein gp41 fusion intermediate has been hindered by challenges inherent in the complexity of the underlying protein−protein interactions (PPIs). Using a specifically designed assay and a limited focused NMR screening of a 500-member fragment library (purchased from Chembridge), Chu and Gochin discovered thioenylaminopyrazole fragments (exemplified by 24, Kd (dissociation constant) of ∼500 μM) binding to a relatively shallow subpocket (at the C-terminal) adjacent to the deep hydrophobic site on the NHR coiled coil.23 Shape-based similarity searching identified additional phenylazole fragments in this library, revealing structural features required for activity and enriching the hit rate over serendipitous screening. These subpocket-binding fragments may exhibit cooperative binding when linked with hydrophobic pocket ligands.23 In addition, new antagonists that bind to known pockets within CCR5 have also been uncovered by fragment screening, and a fragment-based screening system to target the TAR-Tat interaction was reported.24 These examples show that fragment-based screening can be complementary to existing structure-based virtual screening approaches for identification of novel druggable sites and hits. It should be noted that different assay formats have their own advantages and disadvantages and are sensitive to the experimental conditions and assay design. For example, fragment screening by STD NMR allows the chemical authenticity of fragments to be established at the time of screening. But NMR screening was considerably prolonged and hindered by practical complications such as selection of fragments for clustering to avoid NMR signal overlap and issues of low solubility. On the other hand, SPR allows relatively straightforward measurement

HIV-1 RT by screening a library of 1040 fragments. It exhibits submillimolar Kd and IC50 values against WT and three drugresistant variants (K103N, Y181C, and L100I).18 Bauman et al. successfully utilized X-ray crystallography-based fragment screening to detect novel allosteric sites of HIV-1 RT that can be pursued for further drug design. Concretely, a total of 775 fragments were divided into 143 cocktails, and crystals of RT complexed with the non-nucleoside RT inhibitor (NNRTI) 14 (TMC278) were soaked in these cocktails. Finally, fragments binding to three druggable sites (a site adjacent to NNRTI, the Knuckles site, and the incoming nucleotide binding site) were found to show RT-inhibitory activity, opening up opportunities for discovery of new anti-AIDS drugs.19a,b An analysis of their binding modes based on X-ray crystallography data provided structural insights into the ligand-binding site in RT, and this should also be useful for the design of more active RT inhibitors (Figure 3a−e). Very recently, La et al. employed a combination of saturationtransfer difference (STD) NMR and in vitro inhibition assays to screen a fragment library for HIV-1 RT binders with novel scaffolds. Eventually, three molecules 15−17 were found to potently inhibit not only HIV-1 RT but also clinically relevant drug-resistant mutants (K103N, Y181C, or G190A).19c Notably, oxime 16 and p-hydroxyaniline 17 competitively inhibit HIV-1 RT with respect to the DNA template/primer (T/P) or the dNTP substrate and are thus mechanistically distinct from clinically used HIV-1 RT inhibitors (Figure 3f).19c Moreover, fragment-based screening has also resulted in the discovery of novel binders and binding sites in IN and gp41 (Figure 4).20−23 Superposition of the crystal structures of IN with fragments 18 (KM00835) and 19 (SB00942) led to discovery of the benzodioxole 20. Fragment 20 bound at novel site in IN, and the crystal structure of this complex (PDB code 3AO2) was employed by Wielens and his co-workers to design elaborated second-generation inhibitors that bind with higher affinity and good ligand efficiency.20 Meanwhile, a fragment-based screening employing NMR and SPR was successfully employed to probe interactions of various fragments with IN. A novel pocket in IN was defined by crystallography as a valid target site for rational design of allosteric IN inhibitors. Then, X-ray crystallographic data (PDB code 3ZT3) was used to guide the elaboration/linking 2851



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Figure 3. (a) Structures of new RT ligands 9−13 and the NNRTI 14 and (b−e) the results of crystallography of RT complexed with TMC278 and the corresponding ligands: (b) fragment 10 at the site adjacent to NNRTI (exemplified by TMC278) (PDB code 4KFB); (c, d) fragments 11,12 near the Knuckles site (PDB codes 4IG3, 4IFY); (e) fragment 13 at the incoming dNTP binding site (PDB code 4ICL). (f) HIV-1 RT binders identified by STD NMR and functional assays.

bonds of ≤3, and polar surface area of ≤60) is suggested to be useful for fragment selection.26 2.2. Privileged Fragment-Based Reconstruction Approach. A deconstruction−-reconstruction strategy utilizing privileged fragments of existing ligands (known as “privileged structure”-guided scaffold refining) has been reported.27,28 For example, compounds bearing the 8-hydroxyquinoline skeleton exhibit many kinds of biological activities, suggesting that this scaffold might be a privileged motif with great potential in drug discovery.29 In 2013, two 8-hydroxyquinolines 25 and 26 were identified as inhibitors of the HIV-1 IN-LEDGF/p75 via a privileged fragment-based reconstruction approach followed by diverse modifications (Figure 5); these compounds inhibited viral replication (EC50) in MT-4 cells at the low micromolar level.30

of binding affinity. Thus, a combination of complementary, orthogonal biophysical techniques is often employed to screen fragment libraries and validate hits.19c,25 Of course, fragment-based screening methods cannot identify the optimum molecule for synthesis, and the choice of the bestsuited fragment hit for further optimization, as well as elimination of chemically infeasible structures among the detected hits, is crucial for success. Fragments were selected to contain at least one aromatic group and some polar functionality. The most common approach is to apply multiple filters to remove molecules that do not meet all of the property parameters (such as small size, hydrophilic nature, and excellent ligand efficiency). In general, the so-called “rule of three” (molecular weight of 100 μM).35 The best fragments from each binding site of PR should react preferentially because of their spatial proximity to afford the corresponding dimeric binder displaying synergistic inhibition. Screening of the triazole library is very simple because triazole formation is easily detected by mass spectrometry (MS). Finally, 1,2,3-triazole 29 (IC50 = 6 nM, Ki = 1.7 nM) was picked up selectively by PR and was found to be a potent inhibitor of the WT and drug-resistant strains at the nanomolar level. This approach avoids labor-intensive organic synthesis, isolation, and purification and is considered as an efficient tool for drug discovery. The potential of DLS is certainly significant in the early stages of medicinal chemistry projects. For targets featuring ill-defined binding sites that cannot be addressed easily by SBDD, DLS represents a particularly elegant alternative. However, proteintemplated DLS has several shortcomings. For example, in most cases, the libraries employed remain of moderate size. In larger and more complex libraries, false negatives are usually ascribed to multiple and sometimes interdependent reasons, which can be tackled through multidisciplinary approaches (Table 1).31,32 The application of novel click or clicklike reactions is likely to offer additional opportunities to improve this methodology for accessing a wider range of anti-HIV drug targets. 2.4. Rapid Diversity-Oriented Synthesis and in Situ Screening. In current drug discovery programs, parallel highthroughput synthesis can be an effective method to exploit the chemical space and to rapidly probe structure−activity relationships (SARs). However, a key challenge is the availability and application of high-throughput chemical reactions that enable rapid synthesis, purification, and screening of diverse chemical libraries from privileged fragments. In recent years, diversityoriented high-speed synthesis of potent HIV-1 PR inhibitors has been achieved with Cu(I)-catalyzed azide−alkyne 1,3-dipolar cycloaddition (CuAAC) click chemistry in microtiter plates37,38 and with in situ aminocarbonylation reaction.39 This methodology usually requires synthetically available building blocks (i.e., diversely substituted azides and alkynes) or organic reactions (exemplified by CuAAC or aminocarbonylation). For CuAAC-based diversity-oriented high-speed library generation, diversely substituted azides and alkynes were subjected to CuAAC reaction in microtiter plates (solution-phase) to provide densely functionalized Huisgen’s 1,2,3-triazoles. The products can be assayed directly in situ without separation. On the basis of these preliminary protein inhibition data, the most potent library members were individually resynthesized on a larger scale for further biological screening and complete characterization. If necessary, further modifications including functionalization of

the triazoles at the 5-position, or replacement of the triazole with a range of alternative linkers, can be performed.38 This methodology, integrating high-throughput synthesis and screening, afforded highly active and bioavailable nonpeptidic HIV-1 PR inhibitors (e.g., 29, 30, and 31, with Ki values as low as 1.7, 4, and 8 nM, respectively; Figure 7) from two independent focused libraries.37,38 In general, a wide range of substituents can be readily installed by means of CuAAC, and the method has high functional group tolerance, as demonstrated by the prepared 1,2,3-triazole library. In addition, the commercial availability of diverse azide building blocks and improvements in the synthesis of the alkyne precursors have made this protocol suitable for rapid preparation of huge, diverse compound collections. Nevertheless, there is still a potential issue with false positives and negatives. Specifically, copper will be present in the samples after evaporation. To rule out possible undesirable effects in in situ screening, it will be important to examine the influence of diverse copper salts at the highest expected concentration. The introduction of new copper-free click chemistry, such as strainpromoted azide−alkyne cycloaddition, should circumvent this problem. In summary, DLS and rapid diversity-oriented lead generation are completely different, but as the Chinese proverb says, they allow us to achieve the same ultimate goal by dif ferent routes, as exemplified by the discovery of compound 29. DLS may be more useful as a pharmacological tool to pick up ligands with the best match from a focused pool of small fragments, whereas diversity-oriented synthesis and screening may be more useful as a novel HTS protocol for rapid drug discovery from large-scale compound collections.40 2.5. Hierarchical Virtual Screening. Hierarchical multiplefilter database searching is an economical and rapid approach that has had considerable success in numerous drug discovery campaigns. It can take advantage of available ligands and structural information to discover hits that are synthetically accessible or have favorable ADME or physicochemical properties and to reduce candidates from a large compound collection to a number small enough for experimental testing by applying multiple sequential virtual screening filters (such as Lipinski’s rule of five).41 Hierarchical virtual screening has become an important tool for lead discovery directed at specific anti-HIV targets. Two efficient approaches using hierarchical database screenings to seek novel NNRTIs were described several years ago.42,43 Nevertheless, it seems that there has not yet been any successful case of NNRTI drug discovery in which the hierarchical approach has been the major contributor. Even so, virtual screening methodology is likely to remain the focus of much interest. 2854



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Figure 7. Principle of the triazole-forming reaction to generate a library of HIV PR inhibitors, coupled with high-throughput screening (compounds 29, 30) and further modifications (compound 31).

resynthesized. Compound 37 (CX6) displayed significant potency against cell−cell fusion (IC50 = 1.9 μM for fusion elicited by X4 HIV-1NL 4-3 glycoprotein), inhibition of Ca2+-flux binding (IC50 = 92 nM, assayed with stromal cell-derived factor 1α (SDF-1α)), and anti-X4 HIV-1 activity (EC50 = 1.5 μM).46 Moreover, virtual screening approaches have played a significant role in the search for small-molecular inhibitors of PPIs and in changing the notion of nondruggability of PPIs.47 Several examples of virtual screening studies associated with PPIs have been disclosed, including HIV-1 IN-LEDGF/ p75 48−51 and HIV Nef-SH3.52 Christ et al. reported an example of the application of LBSB-hierarchical virtual screening for the discovery of inhibitors of the HIV-1 IN-LEDGF/p75 interaction.48 They successively employed pharmacophore-based screening, docking, and pharmacophore postfiltering to provide 25 diverse compounds for experimental verification. Medicinal chemistry optimization of initial hits resulted in the discovery of molecules showing inhibitory activity at low μM concentrations. Betzi et al. used a druglike filter, molecular docking, and a pharmacophoric filter to obtain inhibitors targeting HIV Nef-SH3 interaction with μM potency.52 Because hierarchical virtual screening requires simultaneous consideration of many filters, including biological activities and druglike properties, it involves subjective elements. For example, in situations where candidates are abundant, it would be appropriate to apply more filters. But where the cost of a missed opportunity is expensive, as is usually the case in drug discovery, the risk of incorrectly neglecting favorable molecules may be too great. Therefore, filters and a validation set should be treated with caution and compromises must be made.

A shift of binding energy contributions from highly mutable residues to more conserved residues is considered to be crucial to get improved drug resistance profiles for anti-HIV agents. On the basis of this hypothesis, a virtual screening protocol was devised including construction of combinatorial library (by modifying the two functional groups of 32 (lopinavir, LPV)), docking, rescoring of the docking results by MM/GBSA, and reranking based on the binding energy distribution (Figure 8a). Finally, 18 candidate molecules were picked up by means of this protocol for bioassay. Inhibitory activity measurements (IC50) and drug resistance predictions successfully uncovered two molecules (33 and 34) with HIV PR inhibitory potency, antiviral efficacy, and improved drug resistance profile against a set of HIV mutants (Figure 8b).44 An optimized computational search method, sequentially utilizing support vector machine (SVM), shape similarity, pharmacophore modeling, and molecular docking, was applied to screen the National Cancer Institute database (NCI), which contains 260 000 molecules (Figure 8c). Finally, compound 35 (NSC111887) was obtained, with an IC50 value of 62 μM against HIV-1 PR.45 Very recently, to identify potential CXCR4 antagonists, Das et al. carried out a hierarchical multistage virtual screening of a library containing about 604 000 molecules. First, compounds that were too flexible (>20 rotatable bonds) or did not lie within the desired molecular weight range (between 350 and 750 Da) were eliminated. Subsequent computational screening employed the shape of 36 (IT1t), a reported CXCR4 antagonist, as a reference and adopted various algorithms, including three-dimensional shape similarity (molecules with Tanimoto coefficients of at least 0.7 were retained), isomer generation, and docking into a CXCR4 crystal structure (Figure 9). Sixteen compounds were selected for bioassays on the basis of their high shape similarity to IT1t, and their putative binding modes including hydrogen bond interactions with at least two critical residues (Asp97 and Glu288, which are important for fusion elicited by HIV-1 envelope glycoprotein). Three molecules with piperidinylethanamine scaffolds demonstrated potency and were

3. MEDICINAL CHEMISTRY STRATEGIES IN THE SCAFFOLD EVOLUTION OF HIV INHIBITORS The frequent onset of resistance to approved anti-HIV drugs necessitates a continuing search for novel antiretroviral drugs and alternative drug design strategies. In this section, we will describe some successful case studies to illustrate the current state of the art in structural evolution (optimization) of HIV inhibitors by 2855



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Figure 8. Discovery of HIV PR inhibitors via a hierarchical computational strategy.

loop of the NNRTI binding pocket (NNIBP); this area is not directly related to RT binding, and some chemical variation could be tolerated there. For example, the phenyl moiety at the N-1 site of the 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) derivative 38 is amenable to structural alteration. IN binding requires at least a hydrophobic benzyl group and a metal(II) ion chelating motif (as exemplified by 39). Thus, this tolerant position in NNRTIs can be utilized to introduce a relatively hydrophilic IN chelating functionality, thereby generating a bifunctional scaffold.60a−f On the basis of the pharmacophoric profiles and SARs of existing molecules, several series of scaffolds capable of inhibiting both RT and IN were established via incorporation of an IN-inhibitory pharmacophore at this tolerant site of established NNRTIs (Figure 10). Remarkably, the obtained molecules 40a,b display robust anti-HIV activities in the nanomolar concentration range, highlighting the effectiveness of the MTDLs approach.55,56 Furthermore, in an extension of this research, 3-hydroxypyrimidine-2,4-dione (exemplified by 41a−c) featuring an N-OH

exploiting novel medicinal chemistry strategies to increase potency, to improve drug resistance profiles, to address pharmacokinetic issues, or even to acquire intellectual property rights. 3.1. Rationally Designed Multitarget-Directed Ligands (MTDLs). In recent years, the design of a single drug molecule that is able to simultaneously and specifically interact with multiple biological targets, namely, multitarget-directed ligands (MTDLs), has proven to be a promising anti-HIV drug discovery approach to improve drug resistance profile and patient compliance (poor compliance is a key reason for development of extensive cross-resistance).53,54 In particular, knowledge-based pharmacophore combination, namely, the rational construction of fused and highly merged MTDLs with minimal structural change in the scaffold of HIV-1 NNRTIs, has been a fruitful approach to acquire IN inhibitors (Figure 10).55−59 The key to rational design of MTDLs via a knowledge-based pharmacophore combination approach is to identify a tolerant domain in the target. Crystallographic investigations demonstrated that some NNRTI platforms contain structural groups located in the solvent-exposed area controlled by the Pro236 2856



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Figure 9. Flow diagram of discovery of piperidinylethanamine-base CXCR4 antagonist 37 via a hierarchical multiple-filter database searching protocol.

Figure 10. Discovery of fused or highly merged RT/IN dual inhibitors based on the combination of RT inhibitor 38 with IN inhibitor 39.

highly merged inhibitors is beneficial for the design of dual-acting ligands with more “druglike” properties. These concepts (fused or highly merged dual inhibitors) could be powerful tools for developing novel antiviral drugs to treat HIV infection. In addition, focused or random optimization of parent compounds is another important approach to generate MTDLs with multiple anti-HIV activities.61,62 Although compounds may already possess multiple potencies, structural decoration is required to balance or increase the activities. 3.2. Bisubstrate (Heterobivalent) Ligands. Bisubstrate (bivalent) inhibitors comprise two conjugated fragments, each

moiety was rationally derived from the HEPT skeleton as a novel molecular platform for RT/IN dual inhibition. The evolved scaffold includes a minimal 3-N hydroxylation in the pyrimidine core of HEPTs to give a chelating triad motif along with the existing benzyl moiety, meeting the basic structural requirements for IN binding. The newly introduced OH could potentially generate H-bonds with a consecutive lysine motif (Lys101Lys104) in the NNIBP.57−59 Thus, SAR investigations led to a detailed understanding of the optimal requirements for inhibition of RT and IN and laid the foundation for a novel class of anti-HIV agents. Furthermore, the low molecular weight of 2857



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Figure 11. Schematic representation of heterobivalent HIV-1 RT ligands consisting of NRTI and NNRTI.

surface of human immune cells. It is the primary target for antibody-mediated viral neutralization. Recently, Spiegel developed a series of ARMs that targets HIV gp120. These bifunctional ARMs are designed to bind simultaneously to HIV gp120 and anti-dinitrophenyl (anti-DNP) antibodies. Anti-DNP antibodies are present endogenously in the human bloodstream in high concentrations. By linking these two components together, ARMs (exemplified by 46, derived from the previously described compound 45 (BMS-378806)) could mediate the generation of a ternary complex, resulting in inhibition of virus entry as well as antibody-mediated immune clearance of envelope glycoprotein (Env)-expressing cells (Figure 12).67,68 In 2014, structure-based, computationally driven optimization of ARMs targeting HIV gp120 resulted in an optimized derivative 48 (derived from 47), which was almost 1000-fold more active than compound 46 in gp120-binding and antiviral assays in MT-2 cells. It was also active against multiple laboratory and clinical HIV pseudotypes (clades A, B, and C).68 In 2013, Sato et al. described the design and application of two N-acyl-β-lactam derivatives 49 and 50 (derived from 45) as chemically programmed antibodies to program the binding activity of aldolase antibody 38C2 and to inhibit the interaction between HIV-1 gp120 and T-cells based on the use of mAb 38C2, an aldolase antibody formed by reactive immunization with a 1,3-diketone hapten (Figure 9). This seems to be a promising new approach for advancing AIDS therapy.69 Certainly, the design of ARMs is a sophisticated game, requiring thoughtful selection and combination of drugs, linker, and antibody. As demonstrated by the above studies (exemplified by the discovery of 48), molecular modeling seems to be an indispensable tool to identify suitable linkage sites and structures in the design of ARMs and chemically programmed antibodies so that downstream chemical efforts can be well focused. 3.4. Multiobjective Optimization: Ligand Efficiency (LE), Ligand-Lipophilicity Efficiency (LLE), and Other Parameters. In the discovery of novel pharmaceutical agents, it is clear that multiple parameters should be optimized in parallel to find the best balance of potency and safety. Much recent work has focused on approaches to aid the simultaneous optimization of multiple parameters required for a successful drug, aiming to identify molecules with the highest possibilities of downstream success at an early stage of drug discovery.70−72 These methodologies have been described in various ways, such as multiobjective (-parameter or -dimensional) optimization or multicriteria decision-making (for convenience, herein we will refer to all

targeted to a binding site in a multisubstrate protein. The main advantage of bisubstrate inhibitors is their ability to form more contacts with the target, providing substantially increased affinity and selectivity, when compared with traditional single active sitetargeted inhibitors. This is also an attractive approach for the discovery of novel anti-HIV agents.63 The close proximity (10−15 Å) of the NRTI and NNRTI binding sites in RT and the crosstalk between these sites offer the opportunity for creating a single molecular “bifunctional” (chimeric) inhibitor that targets both sites simultaneously. In 2013, Anderson’s group64 used molecular modeling to guide focused chemical syntheses of heterobivalent ligands having nucleoside (d4T) and NNRTI (TMC derivative 42) moieties tethered via a flexible polyethylene glycol (PEG) linker. 43 (the triphosphate of d4T-4PEG-TMC conjugate), a representative compound of this series, maintains the potency of the original pharmacophores at both targets, being recognized as a substrate by RT and incorporated into double-stranded DNA in a basespecific manner.64 In addition, compound 43 demonstrated low nanomolar inhibition of RT polymerase inhibition in vitro, being 4300-fold and 4.3-fold more potent than d4T and 42, respectively; this result represents a proof-of-concept of this methodology for antiviral drug discovery.64 The same group also reported the preparation and biochemical assay of another bifunctional RT inhibitor 44 (d4T-6PEG-TIBO conjugate) utilizing d4T and a TIBO derivative linked by a PEG linker (Figure 11). However, its effectiveness was limited.65 Even so, this strategy should be applicable to other proteins with multiple binding sites. Moreover, these heterobivalent systems could also be utilized as pharmacological probes, for example, to estimate the distance between binding sites and the spatial distribution of binding sites. 3.3. Antibody-Recruiting Molecules Targeting HIV. Antibody-recruiting molecules (ARMs) are bifunctional compounds that mediate the generation of ternary complexes between disease-causing agents (such as proteins, viruses, or cells) and antibodies. ARM-induced antibody opsonization causes immune-mediated destruction of the target. In general, ARMs contain three structural domains: an antibody-binding terminus (ABT), a target-binding terminus (TBT), and a chemical linker. Modifications in the TBT domain and linker domain have resulted in the discovery of ARMs targeting bacteria, viruses, and cancer cells.66 In the initial step of HIV entry into host cells, the viral envelope protein gp120 interacts with CD4 glycoprotein expressed on the 2858



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Figure 12. Antibody-recruiting small molecules 46 and 48 that target HIV gp120 and the mechanism of action of chemically programmed antibodies 49 and 50.

Figure 13. Discovery of NNRTI candidate lersivirine via scaffold refining and LE/LLE-guided optimization. HLM = human liver microsomes. LE (binding energy per heavy atom, kcal mol−1) = −1.4 log(RT IC50)/number of heavy atoms. LLE = −log(RT IC50) − clog P. RT IC50 = Ki.

(see legend of Figure 13). Optimizing molecules has generally been based on their binding affinity and pharmacokinetic profile,73,74 but consideration of LLE is helpful for the improvement of bioactivity and metabolic stability. For instance,

such methods as multiobjective optimization). At present, ligand efficiency (LE) and ligand-lipophilicity efficiency (LLE) are considered to be important indicators of “druglikeness”, reflecting the ligand’s potency and physicochemical profile 2859



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Figure 14. Discovery of HIV-1 IN strand-transfer inhibitor 53 and indole-based attachment inhibitor 55 by means of LE/LLE-guided optimization.

early stage in this project was critical for ultimate success. In general, there appeared to be correlation between hERG inhibition and physiochemical properties such as lipophilicity or pKa. Therefore, unwanted hERG interactions could be abolished by altering the substitution patterns and/or orientation of the aromatic rings or by moderating the basicity/steric environment of the central nitrogen in the molecules. Besides, computational techniques also provide useful and feasible approaches to evaluate the hERG liability for large-scale compound collections.93 We envision the possibility that medicinal chemists could widely apply these strategies in early stages of drug discovery to eliminate hERG affinity associated with the desired molecules while retaining activity at the primary target. Moreover, it has been suggested that ligand−receptor residence time (the reciprocal of the dissociation rate constant koff), a useful binding kinetics parameters for the lifetime of the ligand−receptor complex, can be employed as a predictor of drug potency and safety.94,95 Darunavir (structure displayed in Figure 24), a clinically used HIV-1 PR inhibitor, is differentiated from its structural analogues by having an extremely long inhibitor-enzyme residence time. It was demonstrated that the binding kinetics of darunavir to HIV-1 PR well explain the potent antiviral activity and high genetic barrier to the development of resistance.96−101 In addition, a study of the molecular mechanism of binding of maraviroc indicated that its long residence time in the human CCR5 receptor is critical to its clinical success.102 The emergence of new techniques will greatly increase the utility of multiobjective optimization. For instance, the SPR technique allows both effective screening of molecules and more detailed mechanistic and kinetic analysis of specific interactions.103 To sum up, multiobjective optimization strategies were introduced into anti-HIV drug discovery about a decade ago and have steadily gained acceptance. Incorporation of suitable optimization parameters can decrease the attrition rate in later stages of drug development. We envision that further improvements in multiobjective optimization techniques contribute substantially to anti-HIV drug development, especially directed toward newer anti-HIV drug targets. 3.5. Structure-Guided (Crystallographic OverlayBased) Molecular Hybridization. Molecular hybridization is considered to be a very robust strategy for identification of bioactive compounds through combination of essential pharmacophore elements from diverse bioactive fragments to generate a novel hybrid entity endowed with significantly improved affinity and efficacy, compared to the parent compounds.104 The reported crystal structures of several important proteins in the HIV-l life cycle revealed critical factors determining the binding affinity of their ligands, providing a rational basis for structure-based drug

compounds that are relatively lipophilic generally suffered metabolic instability in human liver microsomes. Consequently, with aim to increase efficacy and improve metabolic stability, it is often necessary to improve the LLE value.75,76 Traditionally, structure optimizations of anti-HIV agents that lead to the improvement of one property, e.g., potency, often have a negative effect on other key properties, e.g., lipophilicity. Currently, the structure modification of anti-HIV agents is not solely affinity-driven, but instead LE/LLE values are generally employed to guide elaboration of the scaffold and substituent groups. For example, scaffold refinement of the abandoned NNRTI drug candidate 51 (capravirine) resulted in the discovery of pyrazole as a suitable heterocyclic scaffold for improved NNRTIs.77,78 Structure-based optimization with emphasis on LLE has led to the discovery of several novel chemotypes, which show robust antiviral potency, notably reduced clearance, and increased half-life (T1/2) (Figure 13).77,78 In particular, 52b (UK-453061, lersivirine) was selected for clinical trials on the basis of its potent and wide-spectrum antiviral activity against a broad panel of clinically relevant RT mutations, as well as its safety and its favorable pharmacokinetic properties.79−81 As anticipated, 52b was designed to be less lipophilic than 51, and it has greatly improved metabolic stability (T1/2 = 73 min).79 Moreover, LE and LLE have also been employed as filters (variables) in NNRTIs hit discovery via an in silico (virtual screening) approach82,83 and in the structure optimizations of HIV-1 IN inhibitors and attachment inhibitors (Figure 14).84,85 Notably, the impact of substitution on ligand efficiency was taken into account in the modifications of naphthyridinone HIV-1 IN strand-transfer inhibitors, which ultimately led to the discovery of naphthyridinone-based clinical candidate 53 (GSK-364735).84 In addition, optimization of the early lead molecule 54 with heteroaryl substitution at the C7 position guided by LE and/or LLE considerations afforded 1,2,4-oxadiazole analogue 55, which exhibits picomolar-level inhibition, as well as improved clearance and half-life characteristics and higher oral bioavailability in rats.85 Inhibition of the voltage-dependent ion channel encoded by human ether-a-go-go-related gene (hERG) may result in acquired long QT syndrome, which is a critical adverse effect of noncardiovasular therapeutic drugs. Thus, rejecting potential hERG channel blockers early in the drug discovery process will lower the risk of cardiotoxicity-related side effects in the later, more costly development phase.86 The discovery of 56 (maraviroc) provides a case history of the problems facing all practicing medicinal chemists in attempting multiparameter optimization of the original hit 57 (Figure 15).87−92 Notably, the focus on cardiovascular safety (hERG liability) and pharmacokinetic properties from a very 2860



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Figure 15. Overcoming hERG affinity in the discovery of the CCR5 antagonist 56.

Figure 16. Novel indazole NNRTIs 69a,b using crystallographic overlay-based molecular hybridization. Crystallographic overlay of the published RT cocrystal structures of 51 (green) (PDB code 1FKO) and 68 (pink) (PDB code 1EP4) (other residues removed for clarity).

displayed robust antiviral potency with satisfactory metabolic stability.105 Moreover, the crystal structure of the complex with 69a afforded structural insights into the inhibitory activity. As shown in Figure 17, compound 69a shows edge-to-face π-interactions with the highly conserved Trp229, and the indazole NH participates in a hydrogen bond with the main chain of Lys101.

design and valuable clues to ways of optimizing the scaffold and improving drug potency. In 2009, a novel class of indazoles with potency against both WT and key HIV-1 mutants was efficiently generated by employing crystallographic overlay-based molecular hybridization of the previously reported NNRTIs 51 (capravirine) and 68 (efavirenz) (Figure 16). In particular, compounds 69a,b 2861



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Figure 17. Crystallographic structure of 69a (pink) in the NNRTI binding site (PDB code 2JLE). Figure 19. Crystal structure of the prototype foamy virus (PFV) intasome (a homologous protein of HIV IN) complexed with 70 (pale) (PDB code 3OYF).

This case provides a proof-of-concept for the utility of structure-guided molecular hybridization in the discovery of novel molecules from substructures of pre-existing inhibitors that bind a common target. Given the increasingly availability of high-resolution X-ray cocrystal structures, the success rate of this strategy should be superior to that of more conventional de novo design methodologies. Such a crystallographic overlaybased molecular hybridization approach, taking full advantage of structural biology information, should become increasingly valuable.106 3.6. Conformational Restriction. Proteins are inherently dynamic and conformationally heterogeneous, existing as an ensemble of differently populated conformers in equilibrium. However, certain conformations may be playing key roles in biofunctions, including catalytic activity and molecular recognition. Cumulative evidence strongly supports the idea that conformational restriction (rigidity) of a flexible molecule to match the pharmacological target conformation is effective for drug discovery because it minimizes the entropic loss on ligand binding, resulting in increased affinity and increased isoform selectivity, as well as enhanced drug metabolic stability.107 70 (L-870810) was an early IN inhibitor that entered clinical trials.108 Because of its rotatable amide bond, there is a dynamic equilibrium between two rotational isomers of 70: conformational states A and B (Figure 18). State B was postulated to be the

that specifically target a certain conformational state of IN and “lock” IN into an inactive form. To prepay the conformational penalty in binding to IN, conformationally restricted platforms have been proposed. For example, tricyclic molecules 72−74 were synthesized to lock the metal coordination motif in the lead molecule 71 into the necessary coplanar orientation for effective metal binding. As expected, conformational rigidity resulted in increased inhibitory potency toward HIV and IN, compared with that of 71 (Figure 20).109 Compared with the N-methyl hydroxamate 75, the LLE value of the rigid rotamer N-hydroxydihydronaphthyridinone 76a was dramatically increased.110 Another derivative 76b (PF-4776548) was disclosed as a promising candidate with high activity and a high barrier to resistance (Figure 20).111 This research supports the value of conformational restriction as a strategy to generate novel IN inhibitors with improved potency and druglike profile. 3.7. Dismantling the Rigid Polycyclic Nucleus of Prototypes (Ring-Opening). It is well-known that an interesting option for further development of small-molecular drugs containing fused cyclic systems is to synthesize ring-opened analogues or to delete some of the rings. These derivatives often display potency similar to that of the parent analogues and have improved pharmacokinetic properties. Indeed, inhibitors incorporating conformational flexibility and positional adaptability can minimize the entropic penalty of binding and accommodating to mutated residues.112 As shown in Figure 21, the new-generation NNRTIs 1-[2-(diphenylmethoxy)ethyl]-2-methyl-5-nitroimidazole (78), 80, and 82−84 may be envisaged as “open models” of their parent compounds, since they retain the pharmacophoric elements and conformation of the classical NNRTIs necessary for efficient RT inhibition.113−119 These ring-opened derivatives seem to be promising starting points for further investigation.

4. NOVEL MEDICINAL CHEMISTRY STRATEGY FOR IMPROVING THE AQUEOUS SOLUBILITY OF HIV INHIBITORS: PHOSPHATE ESTER-BASED PRODRUG APPROACH Physiochemical properties, especially aqueous solubility, are a major factor determining the ultimate success or failure of drug candidates.120 The design of water-soluble prodrugs is therefore

Figure 18. Two rotational isomers of the IN inhibitor 70 arise from rotation around the amide bond.

bioactive form for metal chelation (Figure 19). The rotational energy barrier for conversion from A to B is approximately 5 kcal/mol. Consequently, it may be feasible to design molecules 2862



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Figure 20. Design of conformationally restricted HIV IN inhibitors.

Figure 21. Development of (a) 1-[2-(diphenylmethoxy)ethyl]-2-methyl-5-nitroimidazoles (DAMNIs) (exemplified by 78),113 (b) phenethylthiazolylthioureas (PETTs) (exemplified by 80),114 (c) 2,3-diaryl-1,3-thiazolidin-4-one (exemplified by 82),115 1,3-dihydro-2H-benzimidazol-2-one (exemplified by 84),116 and 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole (exemplified by 83)117−119 by dismantling parts of the rigid tricyclic scaffold of prototype leads.

exemplified by 93−96, also provided significantly increased aqueous solubility, high cleavage rates, and high plasma levels of their prototypes after oral administration to rats and dogs.125 In this prodrug approach, release of the parent compounds involves a two-step process. Hydrolytic dephosphorylation by alkaline phosphatase (primarily located in the brush border membranes of small intestinal epithelium) affords the hydroxymethyl intermediate 86, which is inherently unstable, spontaneously decomposing into formaldehyde and the parent compound 87 in the vicinity of the membrane, thus enabling favorable absorption (Figure 22).121,122 This seems to be a flexible strategy for improving exposure and addressing solubility limited absorption of anti-HIV agents.

expected to be an effective strategy in drug development. Recent examples from the literature illustrate the utility of phosphate ester introduction as a new strategy for improving drug efficacy and mitigating solubility-limited bioavailability of anti-HIV drug candidates.121−124 As shown in Figure 22, the phosphate ester prodrug 85 displays much greater water solubility (12 mg/mL) than the parent compound 87 (0.0002 mg/mL). This approach has been further validated by the development of phosphooxymethyl-based prodrugs 89 (BMS-663749) and 91 (BMS-663068) as HIV-1 attachment inhibitors, which are converted to the bioactive parent drugs 88 and 90 in vivo, respectively.122−124 Oxymethylphosphate (OMP) and oxyethylphosphate (OEP) prodrugs of HIV PR inhibitors 92 (lopinavir) and 32, 2863



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Figure 22. Illustration of the phosphooxymethyl prodrug approach (top, hydrolysis of 85 via an unstable intermediate 86 to liberate 87) and some examples of derivatization to obtain prodrugs.

5. SUBSTRATE ENVELOPE HYPOTHESIS: NOVEL STRUCTURE-BASED DRUG DESIGN METHODOLOGY TO REDUCE DRUG SUSCEPTIBILITY TO RESISTANCE MUTATIONS

As illustrated in Figure 23, in the WT drug target, an envelopeviolating inhibitor (a) occupies more of the binding site and forms more contacts than a substrate (d). But such an envelopeviolating molecule may bind only weakly to mutated proteins (mutants I and II) that differ from the WT target, either being forced away from the binding site via removal of a stabilizing interaction (consequently decreasing the binding affinity) (b, pentagram) or protruding further into the binding pocket and suffering a potential steric clash (c, polygon). On the contrary, the substrate does not show decreased affinity, as it does not contact the mutated residue (e and f). Therefore, if the inhibitor is designed to solely form interactions mimicking those of the substrate with invariant residues, HIV mutation would probably have little impact on the binding affinity. In fact, successful next-generation drug candidates tend to exploit more interactions with the substrate. For instance, in recent years, a general strategy of combining computational structure-based design with substrate-envelope constraints has been utilized to develop subnanomolar HIV-1 PR inhibitors active toward a panel of patient-derived multidrug-resistant (MDR) viruses; these inhibitors offer reduced structural complexity and good cost-effectiveness (Figure 24).129−131 Notably, the potency of 97 (darunavir, TMC114) against MDR PR variants is likely due to a combination of its extremely high affinity and its close fit within the substrate envelope.130 In 2012, a new series of HIV-1 PR inhibitors was designed using this combined strategy. Some derivatives exhibit antiviral inhibition at nanomolar level against clinically derived viruses from HIV-1 clades A, B, and C and two MDR mutants. Crystal structure analysis of representive compound 98 disclosed that the carbonyl groups in the newly introduced P2 moieties promote extensive H-bond interactions with the conserved

Drug resistance is a principal concern in drug design targeting rapidly evolving proteins, such as HIV-1 PR. Crystal structure analyses of HIV-1 PR complexed with its peptide substrates suggested that the specificity for substrates depends on matching a defined shape (envelope) within the binding pocket. This shape is termed the “substrate envelope”. Consequently, to obtain broad binding selectivity against a protein target and its mutants, a useful strategy has been to discover inhibitors that remain within the substrate envelope, since these should be less likely to induce resistance mutants than those that extend the envelope. This idea can be designated as the “substrate envelope hypothesis” (Figure 23).126−128

Figure 23. Schematic diagram illustrating the substrate envelope hypothesis. 2864



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site (allosteric) IN inhibitors,138 and PR dimerization inhibitors.138 From the HIV therapy point of view, an allosteric inhibitor could restore the potency of an active site inhibitor against multidrugresistant mutants, so combined therapy with an active site inhibitor and an allosteric inhibitor may be available as a new anti-HIV strategy to overcome drug resistance. Recently, a high-resolution crystal structure of human CCR5 bound to the approved drug 56 (Figure 25) revealed a ligand-binding pocket that is distinct from

Figure 24. Discovery of novel HIV-1 PR inhibitors 97 and 98 based on the substrate envelope hypothesis. Figure 25. Structure of 56 (pink carbons) complexed with CCR5 (PDB code 4MBS). (a) Key residues (blue carbons, in stick representation) in the CCR5 binding pocket involved in the interaction with 56. A water molecule is displayed as a red sphere. (b) Schematic representation of interactions between CCR5 and 56 for surface presentation.

Asp-29 residue of PR, which may be responsible for the impressive activities against MDR PR mutants and antiviral activities in cellular assays.132 In 2013, the effectiveness of substrate-envelope hypothesisguided drug design was further validated by the synthesis of designed pairs of compounds (envelope-respecting/-violating inhibitors, which are very similar to one another).133 It is noteworthy that the NRTI drugs zidovudine (AZT, structure not displayed) and lamivudine (3TC, structure not displayed) protrude beyond the corresponding elements of normal nucleotides (the consensus substrate envelope), creating an opportunity for HIV-1 RT to develop resistance. Hence, it might be beneficial to focus on the relationship between their structures and that of normal dNTPs and to design future NRTIs lying within the substrate envelope. Indeed, the substrate envelope hypothesis has already been applied in the discovery of nucleotide tenofovir.134 Tenofovir might elude drug resistance through locating itself within the substrate envelope, but it might still be vulnerable to excision and to resistance involving loss of key interactions. Further efforts to discover new NRTIs should also take fully into account the insight that if the ligand fits within the substrate envelope, certain interactions with the protein (especially the backbone atoms) may be lost, potentially decreasing its binding affinity. The substrate envelope hypothesis has also been applied to inhibitors of NS3/4A protease, a primary drug target for the hepatitis C virus (HCV).135,136 Application of this hypothesis may revolutionize future drug design.

the putative major binding sites for chemokines and HIV gp120, affording an unprecedented insight into the mechanism of allosteric modulation of chemokine signaling and viral entry.141 This structure may suggest potential new avenues for modifications of 56 that could further inhibit bioactivity of CCR5.141 In addition, a subpocket on the N-trimer of HIV-1 gp41 was identified, with implications for the development of anti-HIV entry inhibitors.142 Besides targeting an unconventional binding site, another rational design strategy to combat drug resistance has been to maximize highly conserved site interactions, especially to enhance extensive H-bond interactions with main-chain atoms.143 This strategy has been extensively employed to seek a variety of scaffolds for NNRTIs, HIV-1 PR inhibitors, and gp41 inhibitors.144−146 In addition, other targets have been functionally and structurally characterized as potentially interesting and druggable alternatives because they are crucial for key steps of viral replication, such as protein−protein and protein−nucleic acid interactions,147 DNA G-quadruplex formation,148 protein conformational transitions,149 the dimerization initiation site (DIS) of the HIV-1 genomic RNA,150,151 HIV-1 matrix protein,152,153 HIV-1 capsid,154−157 nuclear import of preintegration complex,158 Rev- mediated viral RNA biogenesis (compound 99 (ABX464) is a first-in-class anti-HIV drug candidate in phase II clinical trial, Figure 26),159 HIV assembly and release,160,161 retroviral

6. NEW INSIGHTS INTO THE CLINICALLY VALIDATED ANTIRETROVIRAL TARGETS AND BURGEONING TARGETS In spite of the availability of nearly 30 licensed anti-HIV drugs, the rapid emergence of MDR mutations requires the development of new, safe, and effective antiviral agents targeting alternative mechanisms or new binding sites on traditional targets or newly emerging targets.137,138 In theory, drugs that can target new sites should have different resistance profiles to existing drugs and would complement the current anti-HIV drugs used in cART. For the clinically validated HIV targets (RT, IN, PR, and CCR5), there is still great scope for further development of novel inhibitors with distinct mechanisms of action, such as RNase H inhibitors,139 nucleotide-competing RT inhibitors (NcRTIs),140 noncatalytic

Figure 26. Structure of 99, targeting Rev-mediated viral RNA biogenesis.

nucleocapsid zinc fingers,162,163 and many host antiviral restriction factors (such as Vif-dependent degradation of human APOBEC3G and mTORC-1/2).164−168 HIV requires host cellular factors for successful completion of its replication cycle. Host factors mutate less frequently than do 2865



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Figure 27. Function-oriented development of CXCR4 antagonists as selective HIV-1 entry inhibitors.

antagonists that selectively target critical residues in host factors governing viral replication.171 As another example, it was generally concluded that structural transformation on the oxalamide motif (region II) of 105 (NBD-556, a full CD4-agonist) was detrimental to binding and anti-HIV activity, which was considered to be intolerant of optimization previously.172 Very recently, through systematic structure-based modifications of the critical region II, synthesis, and functional and biophysical assays, Debnath et al. disclosed the successful conversion of 105 through 106 (NBD-09027, a partial CD4-agonist) to 107 (NBD-11021) as a full CD4antagonist with broad spectrum anti-HIV-1 activity (IC50 as low as 270 nM, for neutralization activity against a panel of HIV-1 Env pseudoviruses) (Figure 29). To our knowledge, this study unprecedentedly shows that optimization of the oxalamide motif is feasible and may be crucial to improving anti-HIV activity of this class of entry inhibitors.173 In summary, the availability of an enormous volume of structural biology information,174 revealing mechanisms of actions and resistance at the molecular and atomic levels and distinctive binding modes of inhibitors, will facilitate structure-based rational drug design and diminish the likelihood that drug resistance will develop.

viral proteins. They are very attractive targets, as fewer mutations mean a lower possibility of drug resistance. However, discovery of host antiviral factors-targeting compounds as HIV-1-selective inhibitors has always been problematic. Very recently, motivated by the key role of CXCR4 as an HIV entry co-receptor,169,170 Wu et al. reported a de novo function-oriented hit-to-lead effort for discovery of HIV-1-selective, subnanomolar purine-based CXCR4-specific antagonists with a broad therapeutic window (Figure 27).171 Interestingly, compound 103 (EC50 = 0.5 nM against HIV-1 entry into host cells; IC50 = 16.4 nM for inhibition of radioligand SDF-1α binding to CXCR4) exhibited a 130-fold improvement in anti-HIV potency compared to the approved CXCR4 antagonist, 104 (AMD3100, Plerixafor, Figure 28),

Figure 28. Structure of the approved CXCR4 antagonist 104.

though both molecules showed similar activity in mobilizing CXCR4(+)/CD34(+) stem cells at a high concentration. This investigation provides insight into the discovery of antiviral agents without major interference with SDF-1α function. It was rationalized that 102 complementarily interacted with key CXCR4 amino acids important for binding to the gp120 V3 loop and the subsequent HIV-1 entry process. Generally, this study offers tantalizing insights into the feasibility of designing

7. ACTIVATION OF LATENT HIV RESERVOIRS: “KICK AND KILL” STRATEGY Current cART provides potent suppression of HIV-1 replication and can maintain patients with undetectable viremia for a long time. However, cART does not eliminate latent viral reservoirs within cells so that HIV-infected individuals are not cured, and persistent infection is still a significant challenge.175

Figure 29. Conversion workflow of 105−106 (a partial CD4 agonist) to a full CD4 antagonist 107. 2866



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Figure 30. Disruption of HIV latency by HDAC inhibitors:185 (a) structures of representative HDAC inhibitors; (b) mechanisms involved in the reactivation of latent HIV-1 by HDAC inhibitors. LTR: long-terminal repeat.

expression by catalyzing deacetylation of histone tails and keeping the chromatin in a compacted form. Suppression of HDACs by HDAC inhibitors facilites histone acetylation by histone acetyl transferases (HATs), resulting in relaxation of the chromatin and initiation of transcription. Though HDAC inhibitors have demonstrated promise in invoking latency in humans, it will take many years to move from these initial findings to clinical application. One obstacle to repositioning HDAC inhibitors for therapy of HIV infection lies in intellectual property issues surrounding the original HDAC inhibitors. The number of drug analogues is far more than that of approved drugs, making “repositioning drug analogues (privileged structures)” a feasible way to find new leads for eliminating the latent HIV reservoir.27,187

Consequently, several therapeutic strategies are being evaluated to eliminate these viral reservoirs. One of these strategies, termed “kick (shock)-and-kill” strategy,176−179 is to employ smallmolecular compounds that stimulate viral replication (activate viral reservoirs), potentially allowing these reservoirs to be eliminated in conjunction with current cART.180,181 Recent studies have shown that histone deacetylase (HDAC) inhibitors (such as 108 (vorinostat/SAHA), 109 (entinostat), 110 (NCH51), 111 (M344), 112 (MC1239), and 113 (ITF2357)) can reactivate latent HIV in some patients, providing evidence that the shock-and-kill strategy may be feasible (Figure 30a).182−184 The mechanisms involved in the reactivation of latent HIV-1 by HDAC inhibitors have been identified (Figure 30b).185,186 In the latent state, HDACs block HIV-1 2867



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Figure 31. Representative NNRTIs as HIV microbicides.

Thus, combining agents with multiple mechanisms of action may improve microbicide efficiency.216−218

HIV latency is multifactorial, and thus, additional approaches are required for the eradication of HIV infection. Consequently, other potential targets, such as IκB proteins,188 lysine-specific demethylase 1 (LSD1),189 protein kinase C,190 P-TEFb,191 and various unidentified factors,192,193 have been recently studied. However, most of the mechanisms of action remain enigmatic. Various model systems, both in primary cells and in animal models, are beginning to be validated.194,195 Clearly, targeting of individual factors is not enough to eliminate latent virus, and so a combination of compounds that can reactivate HIV via diverse mechanisms will be needed.196−198 Rational design of agents to target HIV latency is not available at present, as better understanding of the molecular control of HIV-1 transcription is still deficient. Another obstacle to rational design for therapy lies in the requirement that the desired molecule should motivate viral reactivation without inducing global T cell activation. Improving the applicability of new models in investigating HIV-1 latency should be helpful for further research.199 Eventually, antilatency drugs should be used in parallel with other interventions, such as antiviral immune responses (immune augmentation strategies), antiretroviral drugs, or cytopathic effects, to eradicate persistent HIV infection.200−202

9. CONCLUSIONS There is a continuing demand for novel HIV-1 inhibitors due to the emergence of drug-resistant strains, as well as poor pharmacokinetic profiles and the cumulative toxicities of drugs currently used in cART. Tremendous progress in bioinformatics, structural biology, and understanding of the HIV life cycle over the past 2 decades has facilitated the rapid discovery of potent inhibitors for therapeutically established anti-HIV targets. But the discovery of new first-in-class drugs for the treatment of HIV infection is an expensive and lengthy process. Even an ideal follow-on anti-HIV drug (namely, a new antiretroviral for a known target) should meet rigorous requirements: (i) improved activity against WT and resistant virus; (ii) favorable oral bioavailability and metabolic stability; (iii) minimal side effects and good safety profile; (iv) lack of drug−drug interactions (ability to interact beneficially with other drugs); and (v) ease of preparation and formulation.219 Nevertheless, innovative medicinal chemistry methodologies, emerging techniques, and new concepts have been developed to address both drug resistance and poor aqueous solubility and also to promote the identification and optimization of leads with high potential for generating new antiviral therapeutic drugs. As Confucius said, “Consider the past, and you shall know the future”, and this gives us hope that the well-filled toolbox of contemporary medicinal chemists will indeed lead to the timely discovery of improved HIV inhibitors.

8. PRE-EXPOSURE PROPHYLAXIS AND TOPICAL MICROBICIDES Pre-exposure prophylaxis and topical microbicides have been regarded as essential approaches in the prevention of sexual transmission of HIV.203−205 Especially, many membranepermeable, tight-binding NNRTIs could inactivate cell-free and cell-based HIV-1 in semen without metabolic activation.206 Consequently, some NNRTIs are being investigated as experimental microbicides (Figure 31), including 114 (dapivirine, TMC120),207 115 (UAMC01398),207,208 116 (MC1220),209 117 (YML220),210 118 (UC781),211 119 (HI-443),212 120 (MIV-150),213 and dithiocarbamate−thiourea hybrid (121).214 Notably, 114 formulated as a vaginal ring has entered two phase III efficacy and safety studies.215 Recently, a diaryltriazine derivative 115 demonstrated a superior safety profile, favorable in vitro potency against dapivirine-resistant strains, and favorable biopharmaceutical profiles, thus warranting further investigation as a promising microbicide candidate.207,208 Equally important, a microbicide must be potent against multiple HIV-1 mutations.

10. DISCUSSION AND PROSPECTS In this review, we have briefly described the various methodologies and tactics that have been used to discover novel drug entities, to improve drug resistance profiles and solubility, and to promote eradication of latent HIV reservoirs (Figure 32). In recent years, fragment-based methodologies for lead discovery, such as fragment-based screening and reconstruction approaches, have gained increasing momentum in the pharmaceutical industry and in academia as complementary approaches to HTS. It is widely recognized that the challenge in the fragment evolution approach is to transform binders with low affinity into potent leads while maintaining high potency and reasonable druglike properties. In the future, the combination of 2868



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bioactivity with a “druglike” profile is a pivotal aspect of successful drug discovery. Therefore, the design of integrated MTDLs seems a very promising strategy for the discovery of new anti-HIV drugs. Bisubstrate (heterobivalent) ligands can interact simultaneously at two orthosteric binding sites of a heterodimer, potentially providing higher affinity, improved selectivity, and modified physiological response, with the possibility of enhanced administration compliance in patients. At present such compounds are useful as pharmacological probes, but their use as drugs is restricted by their relatively large molecular size. ARMs have the potential to interfere with HIV replication through multiple complementary mechanisms, viz., inhibition of virus entry and antibody-mediated immune clearance of Env-expressing cells. To design novel potent ARMs, conjugation strategies of bisubstrate (heterobivalent) ligands are available. This conjugation approach consists of two phases. The first is selection of an appropriate template molecule and identification of a suitable position for introducing a linker. The next is chemical optimization of promising inhibitors. A high quality drug must have a good balance of potency against its therapeutic target(s) with physicochemical properties, ADME properties, and safety. Multiparameter optimization approaches can guide the simultaneous optimization of multiple properties to find molecules with the highest success rate in downstream step. The application of efficiency indices (LE and LLE) has already proved highly effective to rapidly identify and improve the quality of molecules with anti-HIV potency and to rationally design derivatives with highly optimized chemical entities. In general, LE could be used as a decision-making parameter during the early stage of hit identification to maximize bioactivity versus molecular mass, whereas LLE could be adopted during the later stage of optimization to balance bioactivity and lipophilicity. In addition, the story of the discovery of CCR5 antagonist maraviroc as a drug for HIV treatment demonstrated that avoiding cardiac liability related to inhibition of hERG is key for successful antiviral drug discovery and development. As is well-known, drugs in vivo are situated in an open environment, in which their microenvironment and concentrations are continually variable. Aside from parameters of drug binding affinity, in recent years, the kinetics of drug binding (kon and koff of the drug−target interaction) and drug residence time have been increasingly recognized as vital parameters for therapeutic efficacy of anti-HIV drugs, such as HIV PR inhibitors and CCR5 antagonist maraviroc. Drugs with prolonged on-target residence times generally display superior efficacy, but so far, general strategies for optimizing drug−target residence time are lacking. It seems clear that integration of different ligand-binding assays (Kd, IC50, koff) will be important to enhance the efficiency of antiHIV drug discovery and development.220 Targeting of noncatalytic cysteines with reversible covalent inhibitors may serve as a broadly applicable approach that promotes “residence time by design”, i.e., the capacity of modulating and improving the duration of target engagement in vivo.221 The methodology could be generalizable to anti-HIV drug targets that contain such cysteine residues, such as Y181C mutated RT. From a medicinal chemistry point of view, crystallographic overlay-based molecular hybridization, conformational restriction, and ring-opening, three commonly used rational scaffoldhopping approaches, require the availability of structural biology information about the chemical space around the antiviral biotargets of interest so that the essential determinants of binding

Figure 32. Schematic illustration of key issues in anti-HIV therapy and innovative medicinal chemistry methodologies and approaches available for anti-HIV drug discovery and development.

fragment-based optimization with visualization of binding profiles by means of crystallographic studies is likely to be particularly effective in creating highly efficient inhibitors. New concepts and methodologies of combinatorial chemistry, including DLS and rapid diversity-oriented synthesis and in situ screening, have been proposed for the design and synthesis of structurally diverse or focused libraries.40 We wish to stress that design of molecular libraries for screening should take into account the need to obtain an appropriate balance of many diverse properties. In such solution-phase approaches, it is also important to minimize the usage of additional reagents that might adversely influence the subsequent in situ bioassays. This is important because the absence of a purification procedure in library assembly employing CuAAC reaction has allowed medicinal chemists to exploit the relevant chemical space around a candidate scaffold in a swift and efficient manner. However, despite the popularity of diversity-oriented synthesis via this widely used click chemistry, there is still a lack of highly concise and convergent synthesis strategies that give access to alkyne/ azide-containing fragments as key building blocks for click chemistry. The hierarchical multiple-filter database searching strategy, which combines many cheminformatic tools, is a fascinating approach to identify new anti-HIV leads with a high probability of achieving the required properties profile. However, it should be emphasized that issues such as the inaccuracy of scoring functions for estimating target−ligand binding free energy, the difficulty of considering target flexibility in docking, and the high false-positive rate still represent major challenges to current multiple-filters strategy. Besides, the uncertainties in in silico predictions mean that a computer software cannot discover a final candidate with complete confidence that it will gain all of the required druglike properties. Consequently, further optimization via chemically tractable synthesis is inevitable. Therefore, it is desirable to apply a synthetic accessibility score as a filter to remove molecules that would be hard to synthesize. A relatively new strategy in the scaffold evolution of HIV inhibitors is the development of MTDLs. The advantages of integrated MTDLs (also referred to as merged or fused designed multiple ligands or twin ligands) over linked MTDLs include reduction of molecular weight, clogP, and topological polar surface area, which are all key aspects of “druglike” profiles. It is broadly acknowledged that combining a desirable balanced 2869



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Figure 33. Structures of HIV-1 integrase inhibitors 122 and 123 and their acetal carbonate prodrugs 124 and 125 (also an acetal carbonate−phosphate double prodrug).

established agents, aiming to completely eradicate HIV from infected individuals. This approach is challenging and has been unsuccessful thus far. Although HDAC inhibitors are promising, an in-depth understanding of key mechanisms of HDAC isoforms in latency and feasible tools to translate these knowledge into practice are urgently needed. Moreover, a major concern with this strategy is that compounds should induce latent viruses without increasing the susceptibility of uninfected cells to HIV.180 Generally, as HIV latency and the persistence of infection are multifactorial, the thorough eradication of HIV infection may require a variety of approaches. Overall, the multidisciplinary coordination of methodologies from the usual approaches and the emerging alternatives will prompte anti-HIV drug discovery and development in the future.

affinity and the tolerant region for scaffold hybridization, fusion, or opening can be identified. For instance, in general, one of the benefits of conformationally restricted molecules is their ability to reduce conformational entropy by constraining themselves in conformations that can selectively bind to their target. However, for easily mutated targets, the relative flexibility of ring-opened compounds might allow them to adapt to a mutated drug pocket more readily, as compared to the rigid fused-ring structure. Decreasing the dosing frequency of anti-HIV therapies has been found to have a significant impact on improving patient adherence, which represents an important goal within antiretroviral drug discovery. Given that low aqueous solubility remains a primary issue in antiviral drug discovery, the development of structural analogues with improved solubility is highly desirable. The use of phosphate ester-based prodrugs is an important new approach to overcome solubility-limited bioavailability of anti-HIV agents. Besides, to overcome absorptionlimiting physicochemical properties, other innovative prodrug strategies (such as the acetal carbonate prodrug) were recently applied in the structural optimization of HIV-1 integrase inhibitors (including 122 (raltegravir) and 2-pyridinone 123 (MK-0536), Figure 33), which led to the identification of new drug candidates (exemplified by acetal carbonate prodrugs 124 and 125) with favorable antiviral efficacy, suitable physicochemical and preclinical pharmacokinetic profiles, and less frequent administration (to support a once-daily dosage).222,223 From a strategic point of view, the substrate−envelope hypothesis is a useful structure-based drug design methodology to reduce drug susceptibility to resistance mutations. While the means to achieve wide selectivity toward potential drug-resistance mutations depends on the system of interest, the idea of trying to mimic the static and dynamic molecular recognition features of the natural substrate (especially its shape and flexibility) is a powerful idea when dealing with proteins that are prone to resistance mutations. As the availability of high-resolution structures of viral particles is rapidly increasing, one of the most exciting fields of HIV research is the search for agents directed at novel targets, including new sites and/or alternative mechanisms interacting with the traditional targets. Due to the presence of latent viral reservoirs, another key issue is to develop new agents that induce activation of latent HIV reservoirs and to evaluate their use in combination with

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AUTHOR INFORMATION

Corresponding Authors

*E.D.C.: e-mail, [email protected]; phone, 3216337367. *X.L.: e-mail, [email protected]; phone, 086-531-88380270, 88382005. Notes

The authors declare no competing financial interest. Biographies Peng Zhan obtained his B.S. degree from Shandong University, China, in 2005. Then he earned his M.S. and Ph.D degrees in Medicinal Chemistry from Shandong University in 2008 and 2010, respectively. He subsequently joined the research group of Professor Xinyong Liu as a Lecturer (2010−2012). From 2012 to 2014, he worked as a Postdoctoral Fellow funded by JSPS (Japan Society for the Promotion of Science) in the Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan. He is now working as an Associate Professor in the laboratory of Professor Xinyong Liu. His research interests involve the discovery of novel antiviral, anticancer, and neurodegenerative disease-related agents based on rational drug design and combinatorial chemistry approaches. Christophe Pannecouque studied pharmaceutical sciences and obtained a Ph.D. in Medicinal Chemistry at the Rega Institute for Medical Research of the Katholieke Universiteit Leuven in 1990 and 1995, respectively. In 1995 he joined the group of Professor Erik De Clercq as a postdoctoral fellow. An Associate Professor since 2003, he teaches courses in cell biology and biochemistry at the Faculty of 2870



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NcRTI, nucleotide-competing reverse transcriptase inhibitor; NNIBP, non-nucleoside reverse transcriptase inhibitor binding pocket; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PR, protease; PPI, protein−protein interaction; RT, reverse transcriptase; SAR, structure−activity relationship; SBDD, structurebased drug design; SDF-1α, stromal-derived factor 1α; SPR, surface plasmon resonance; SVM, support vector machine; STD, saturation-transfer difference; TGS, target-guided synthesis; WT, wild-type

Pharmaceutical Sciences of the Katholieke Universiteit Leuven, Belgium. In the field of virological research, he has unraveled the mode of action of several classes of new products with anti-HIV activity. The last years of his research focused more on cellular targets interfering with HIV replication and research in the domain of the regulation of the (cyto)pathogenicity of the virus. Erik De Clercq has M.D. and Ph.D. degrees and has taught courses in cell biology, biochemistry, and microbiology at Katholieke Universiteit Leuven (and Kortrijk) Medical School, Belgium, where he was Chairman of the Department of Microbiology and Immunology until September 2006. He is currently Emeritus Professor of Katholieke Universiteit Leuven, Member of the Belgian (Flemish) Royal Academy of Medicine and the Academia Europaea, and Fellow of the American Association for the Advancement of Science. In 2008, he was elected European Inventor of the Year (Lifetime Achievement Award), and in 2010 he, together with Dr. A. S. Fauci, was Laureate of the Dr. Paul Janssen Award for Biomedical Research. He is the (co)inventor of a number of antiviral drugs (valaciclovir, brivudin, cidofovir, adefovir, and tenofovir).

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REFERENCES

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Xinyong Liu received his B.S. and M.S. degrees from School of Pharmaceutical Sciences, Shandong University, China, in 1984 and in 1991, respectively. From 1997 to 1999 he worked at Instituto de Quimica Medica (CSIC) in Spain as a senior visiting scholar. He obtained his Ph.D. from Shandong University in 2004. He is currently a Full Professor of the Institute of Medicinal Chemistry, Shandong University. His research interests include rational drug design and synthesis and antiviral evaluation of a variety of molecules that interact with specific enzymes and receptors in the viral life cycle (HIV, HBV, HCV, and FluV). Other ongoing programs include the molecular modification and structure−activity relationships study of some natural products to treat cardiovascular diseases, and drug delivery research using PEGylated small-molecular agents.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (NSFC Grants 81573347, 81102320, 81273354), Key Project of NSFC for International Cooperation (Grant 81420108027), Research Fund for the Doctoral Program of Higher Education of China (Grants 20110131130005, 20110131120037), The Science and Technology Development Project of Shandong Province (Grant 2014GSF118012), and the China Postdoctoral Science Foundation funded special project (Grant 2012T50584). All figures showing binding modes were generated using PyMol (www.pymol.org).

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ABBREVIATIONS USED AAC, azide−alkyne cycloaddition; AIDS, acquired immunodeficiency syndrome; ARM, antibody-recruiting molecule; BEAM, binding energy analysis method; cART, combinatorial antiretroviral therapy; CuAAC, Cu(I)-catalyzed azide−alkyne 1,3dipolar cycloaddition; DDM, double decoupling method; DLS, dynamic ligation screening; FBDD, fragment-based drug design; FBS, fragment-based screening; HAT, histone acetyl transferase; HCV, hepatitis C virus; HEPT, 1-[(2-hydroxyethoxy)methyl]-6(phenylthio)thymine; HIV-1, human immunodeficiency virus type 1; hERG, human ether-a-go-go-related gene; HDAC, histone deacetylase; HTS, high-throughput screening; IN, integrase; LE, ligand efficiency; LEDGF/p75, lens epitheliumderived growth factor/p75; LLE, ligand-lipophilicity efficiency; LSD1, lysine-specific demethylase 1; MDR, multidrug-resistant; MS, mass spectrometry; MTDL, multitarget-directed ligand; 2871



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