"Old Dogs with New Tricks": exploiting alternative mechanisms of action and new drug design strategies for clinically validated HIV targets.

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‘‘Old Dogs with New Tricks’’: exploiting alternative mechanisms of action and new drug design strategies for clinically validated HIV targets Dongwei Kang,a Yu’ning Song,b Wenmin Chen,a Peng Zhan*a and Xinyong Liu*a HIV-1 reverse transcriptase, protease and integrase have been recognized as clinically validated but still underexploited targets for antiretroviral treatment. Although a large number of inhibitors have been used in clinical trials, the rapid emergence of multiple drug-resistant strains requires the identification of not

Received 11th March 2014, Accepted 11th May 2014

only novel classes of antiretroviral drugs that act via the unprecedented mechanism of action but also innovative drug discovery strategies towards these three important targets. This review summarizes and

DOI: 10.1039/c4mb00147h

discusses current endeavours towards the discovery and development of novel inhibitors with alternative mechanisms of action, and also provides examples illustrating new methodologies in medicinal chemistry

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that contribute to the identification of novel antiretroviral agents.

1. Introduction The genome of human immunodeficiency virus-type 1 (HIV-1) encodes 15 different proteins. Among them, reverse transcriptase (RT), protease (PR), and integrase (IN) possess essential enzymatic functions, which are vital for viral replication and a

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. E-mail: [email protected], [email protected]; Fax: +86-531-88382731; Tel: +86-531-88382005 b Department of Pharmacology, Key laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012, Jinan, Shandong, P. R. China

have been regarded as highly validated but still underexploited targets of antiviral therapies. During the past two decades, significant advances have been made in the discovery of novel antiviral drugs, nearly 30 potent inhibitors have been licensed as anti-HIV drugs, and they have already gained definitive places in the treatment of AIDS patients. As well known, in clinical use, there is a high probability for the development of (cross) resistance among the compounds with the same mechanism of action. Given this, there is still a significant need to identify new, safe, and effective antiviral molecules targeting newly emerging targets, alternative mechanisms or new binding sites on traditional targets.1,2 Advances in structural biology reveal the in-depth understanding of the structures and functions of these three highly

Dongwei Kang was born in 1990 in Liaocheng, Shandong province, China. In 2012, he graduated from the School of Hebei University of Technology and obtained his BS degree. He is currently studying for master’s degree in the Department of Medicinal Chemistry of the School of Pharmaceutical Sciences in Shandong University. Dongwei Kang

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Peng Zhan

Dr Peng Zhan was born in 1983 in Ji’nan, Shandong province, China. He obtained his BS degree from Shandong University, China, in 2005. Then he earned his MS degree and PhD in medicinal chemistry from Shandong University in 2008 and 2010, respectively. He is now working as an associate professor in Shandong University. In 2012, he was appointed as a JSPS Postdoctoral Fellow in Japan. So far he has published more than 50 SCI academic peer review papers.

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Fig. 1 Schematic diagram showing the licensed anti-HIV drugs and the antiviral arsenal with novel agents aiming at three highly validated but currently underexploited viral targets, which were highlighted in blue and pink, respectively.

validated HIV targets, which have provided novel insights into inhibition, such as via targeting alternative binding sites, or via inhibiting multimerization or interaction with cellular cofactors. Consequently, recently, there has been renewed interest in

Prof. Dr Xinyong Liu was born in 1963 in Qingdao, Shandong province, China. He received his BS and MS degrees from School of Pharmaceutical Sciences, Shandong University, 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 PhD from Shandong University in 2004. He is currently the Director Xinyong Liu of the Institute of Medicinal Chemistry, Shandong University. His research area involves discovery of bioactive molecules based on rational drug design approaches. He has contributed about 200 scientific publications and patents as well as many monographs.

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the discovery of antiviral arsenal with novel agents aiming at alternative mechanisms and binding sites on traditional targets (Fig. 1), which will be summarized in this article. Besides, the exploitation of newly emerging methodologies in medicinal chemistry that contribute to the identification of novel antiviral drugs remains an active area, which will also be illustrated by representative examples.

2. Targeting the traditional drug targets with alternative mechanisms 2.1

HIV-1 RT inhibitors with innovative mechanisms of action

HIV-1 RT is a multifunctional protein catalyzing the synthesis of proviral DNA from the genomic viral RNA by RNA-/DNAdependent DNA polymerase activities (namely, RDDP/DDDP) and ribonuclease H (RNase H) activity concerning the digestion of only RNA of the RNA–DNA hybrid.3,4 Up to now, RT has been regarded as an indispensable target for anti-HIV therapy since most approved antiretroviral drugs target the RT. The 11 existing RT inhibitors (RTIs) approved by FDA are divided into two categories: the nucleos(t)ide-RT inhibitors (NRTIs) compete with the natural nucleoside substrate and act as chain terminators of DNA synthesis after incorporation into the primer strand, while the non-nucleoside RT


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Review

Fig. 2

Structures of nucleoside derivatives as RT-directed mutagenic inducers.

Fig. 3

Structures of NNRTIs with not-conventionally-binding modes and/or alternative mechanisms.

inhibitors (NNRTIs) bind to an allosteric and hydrophobic pocket (non-nucleoside inhibitor binding pocket, NNIBP) and inhibit polymerization noncompetitively. All of the licensed RT drugs inhibit only the RT-associated polymerase activity.3,4 The intrinsic conformational flexibility of

RT can provide novel allosteric sites and/or alternative mechanisms that can be exploited to develop new therapeutic classes of RTIs.5 As a consequence, other RTIs with distinct mechanisms have been exploited or are currently under development, including RT-directed mutagenic inducers, nucleotide-competing RT

Fig. 4 The exclusive hydrophobicity (a) and the particular shape (b) of 7-bound NNIBP (PDB code: 3IRX, resolution: 2.8 Å). These figures were generated using PyMol (www.pymol.org).


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Fig. 5 Crystal structure of HIV-1 RT complexed with ‘‘dragon’’ shaped (Body, Head, Wings, Tail) TSAO-T 8 (PDB code: 3QO9, resolution: 2.6 Å). This figure was generated using PyMol (www.pymol.org).

Fig. 6 CP-94,707 (in pink) bound in HIV-1 RT (PDB code: 1TV6, resolution: 2.8 Å).

inhibitors (NcRTIs), RT-associated RNase H function inhibitors, primer/template-competing RT inhibitors and dual inhibitors of the HIV-1 RT-associated polymerase and RNase H activities. In this section, we will review current progress in the understanding of innovative mechanisms of action related to these compounds and make comments on opportunities and challenges in anti-HIV drug research. 2.1.1 RT-directed mutagenic inducers. The RT-directed mutagenic inducers can incorporate into the viral DNA thereby increasing the mutation rate of HIV by mispairing, and resulting in defective viruses leading to lethal mutagenesis, but they do not block viral DNA synthesis.6,7 These inhibitors include several nucleoside derivatives (Fig. 2), such as 5-OH-dC (1),8 5-AZC (2),9 KP-1212 (3), KP-1461 (4),10 5-hydroxymethyl-2 0 -dU (5) and 5-hydroxymethyl-2 0 -dC (6).11 Their antiviral profiles

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along with the innovative mechanism of action make these mutagenic inducers a very promising class of molecules to be exploited for more effective antiviral arsenal. 2.1.2 NNRTIs with not-conventionally-binding modes and/or alternative mechanisms. In contrast to the established NNRTIs class, several NNRTIs with novel inhibitory mechanisms for RT or unorthodox binding modes have been proposed (Fig. 3), such as alkenyldiarylmethane (ADAM) 7,12,13 ‘‘dragon’’ shaped-tertbutyldimethylsilyl-spiroaminooxathioledioxide (TSAO) 8,14 and CP-94,707 (9).15 These NNRTIs present distinct resistance profiles, rendering attractive modes of RT inhibition. The X-ray structures (Fig. 4–6) can help illuminate the structural information of RT bound to these compounds and thereby facilitate the design of more potent second-generation NNRTIs. 2.1.3 Nucleotide-competing RT inhibitors (NcRTIs). A few years ago, a novel family of RT inhibitors, termed nucleotidecompeting RT inhibitors (NcRTIs), have been reported.16 For instance, 2-methylsulfonyl-4-dimethylamino-6-vinylpyrimidine (DAVP-1, 10),17 indolopyridinones CBL-4.0 (11a) and CBL-4.1 (INDOPY-1) (11b),18–21 and benzofurano[3,2-d]pyrimidine-2-ones 12a–e (Fig. 7).22 Like the NNRTIs class, NcRTIs are structurally distinct from the deoxyribonucleotide triphosphates (dNTPs) and the established NRTIs. They do not act as chain terminators, but reversibly inhibit binding of the incoming nucleotide to the RT catalytic site.16 The NcRTIs could circumvent some of the limitations associated with current RTIs drugs caused by drugresistant mutations and might provide a promising opportunity for antiviral therapy. Besides, the X-ray structure of HIV-1 RT bound to DAVP-1 shows a relatively untapped binding site close to the polymerase active site (Fig. 8).17 2.1.4 Primer/template-competing RT inhibitors. Recently, compounds KM-1 (13),23,24 SY-3E4 (14),25 and N-{2-[4-(aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide (NAPETA, 15)26 were reported as primer/template-competing RT inhibitors (Fig. 9), which inhibited RT via a distinct mechanism compared with that of the classic approved NNRTIs drugs. For instance, KM-1 could distort RT conformation and misalign DNA at the active site.23,24 SY-3E4 could displace the aptamer from the RT, selectively inhibit DDDP, but not RDDP activity, and can inhibit the replication of both the wild-type (WT) and resistant HIV strains.25 NAPETA inhibited both RDDP and DDDP activities, with IC50 values of 1.2 and 2.1 mM, respectively. It also interfered with the formation of the RT-DNA complex.26 There is no denying the fact that these molecules provided previously unexplored opportunities for discovery of novel anti-HIV agents. 2.1.5 RT-associated RNase H function inhibitors. A perusal of the current literature revealed that extensive efforts are on-going to develop inhibitors against the second enzymatic activity of HIV-1 RT, namely, RNase H function – an exciting yet little explored target.27,28 The HIV-1 RNase H inhibitors can be divided into active site inhibitors and allosteric inhibitors. The active site inhibitors are structurally diversified divalent metal ion chelators (Fig. 10), which bind to the RNase H active site and inhibit the enzyme by chelating the Mg2+/Mn2+ ions. Representative compounds include a-hydroxytropolone manicol (16) and b-thujaplicinol (17),29


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Fig. 7 Structures of NcRTIs.

N-hydroxyimides 18a and 18b,30 3-hydroxyquinolin-2(1H)-one 19,31 2-hydroxyisoquinoline-1,3(2H,4H)-dione 20,32 pyrimidinol carboxylic acids 21a and 21b,33a,b N-hydroxy quinazolinedione 22,33b

dioxobutanoic acid derivatives RDS1643 (23),34 BTDBA (24),35 1-hydroxy-1,8-naphthyridin-2(1H)-ones 25–27,36 1-hydroxy-1,8naphthyridin-2(1H)-one 28,37,38 and 5-nitro-furan-2-carboxylic

Fig. 8 Crystal structure of DAVP-1 bound to the unligated RT (PDB code: 3ISN). The sulfone forms multiple H-bonds with the amino acid residues around it. Key H-bond interactions are shown as yellow dashed lines.


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Fig. 9


Structures of primer/template-competing RT inhibitors.

Fig. 10 Structures of divalent metal ion chelators as RNase H active site inhibitors.

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Fig. 11 Co-crystallized RNAse H inhibitors and relative PDB codes. The inhibitors in a–h can bind with the two metal ions in the RNase H catalytic center, which is involved in the inhibition of RNase H function. (a) Manicol complexed with the RNase H active site of liganded HIV-1 RT (Rilpivirine) (PDB code: 3QLH); (b) HIV-1 RT with b-thujaplicinol bound at the RNase H active site (PDB code: 3IG1); (c) HIV-1 RNase H p15 with the engineered E. coli loop and active site inhibitor 21a (PDB code: 3HYF); (d) HIV-1 RNase H p15 with engineered E. coli loop and 21b (PDB code: 3QIN); (e) RNase H with the engineered E. coli loop and N-hydroxy quinazolinedione inhibitor 22 (PDB code: 3QIO); (f) MK1 with liganded RT (NVP) (PDB code: 3LP0); (g) MK2 with liganded RT (NVP) (PDB code: 3LP1); (h) MK3 binding mode in the catalytic pocket (PDB code: 3LP3); (i) MK3 binding mode in the NNIBP (PDB code: 3LP2).

Fig. 12

The allosteric RNase H inhibitors.

acid carbamoylmethyl ester (NACME) derivatives 29 and 30.39 These compounds hopefully signify a renewed courage to pursue non-traditional classes of RTIs and to rapidly expand


the antiviral therapeutic arsenal. It should be noted that the widespread use of the RNase H active site inhibitors will ultimately depend upon a better understanding of how to

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Fig. 13 HIV-1 RT bound with DHBNH (PDB code: 2I5J). It shows the following mode of RT inhibition: DHBNH not only interacts with conserved residues (Trp229, Asp186) but forms substantial interactions with the main-chains of some less well-conserved residues.42

improve the selectivity of these agents and to low the cytotoxicity. Moreover, the obtained high resolution co-crystal structures illustrated that the two metal ions in the RNase H catalytic center and the conserved His539 residue are involved in the inhibition of RNase H function, providing potential implications for the design of new inhibitors effective against multidrugresistant HIV strains (Fig. 11). Unlike divalent metal-chelating active site inhibitors described above, vinylogous urea NSC727447 (31),40 thienopyrimidinones 32–3441 and dihydroxy benzoyl naphthyl hydrazone (DHBNH, 35),42 were identified as RT RNase H antagonists with an allosteric mechanism of action (Fig. 12). Although DHBNH primarily inhibits the RNase H activity, based on the crystal structure

Fig. 14

of RT complexed with DHBNH, it binds at a site which is 450 Å away from the RNase H active site and partially overlaps the NNIBP (Fig. 13). Binding of DHBNH directly affects the conformation of the polymerase primer grip and the thumb, which is responsible for its anti-HIV activity.42 Moreover, more potential sites for inhibiting the HIV RNase H have been disclosed recently, which provided an attractive venue for developing additional allosteric RNase H inhibitors.43,44 2.1.6 Dual inhibitors of the HIV-1 RT-associated polymerase and RNase H activities. As described in the above section, the DHBNH binding site gives opportunities to design novel derivatives with multiple RT inhibitory activities. Consequently, through chemical modifications of DHBNH, compounds 36 and 37 were developed as the inhibitors of RT-associated polymerase and the RNase H activities (Fig. 14). In addition, alizarine analogues K-49 (38) and KNA-53 (39) were identified as dual inhibitors of the DNA polymerase and RNase H activities.45 Besides, hydrazonoindolin-2-one derivative 40 was found to be active for RNase H (IC50: B2 mM) and RDDP activity (IC50: B1.4 mM).46 The dual properties of these compounds provided a very good basis for the consequent analoging-based optimization of discovery novel multitarget-directed ligands with improved druggability. 2.1.7 Others. Biochemical studies have found that the compounds CP-94,707 (9) and acylhydrazone DHBNH (35) also specifically inhibited the initiation of reverse transcription.47,48 Still, there are additional sites on RT that can be exploited as novel druggable target sites, such as the Knuckles site, the NNRTI adjacent site, and the incoming nucleotide binding site, which were identified using X-ray crystallography-based fragment screening (Fig. 15).49 The proper filling of these newly identified binding sites beyond the main NNIBP is beneficial for improving binding affinity and selectivity of NNRTIs hits and leads. On the other hand, fragment screening against HIV1 RT has been employed as a useful tool to better understand the mechanism of a flexible target binding with the ligand.

Dual inhibitors of the RT-associated polymerase and RNase H (abbreviated RNH) activities.

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Fig. 15 Novel HIV-1 RT binding sites with bound fragments: (a) near Knuckles site (4IG3); (b) at the incoming dNTP binding site (4ICL); (c,d) at NNRTI adjacent site (4I7G or 4KFB). Because of its flexibility and plasticity, the RT is like a ‘‘coat’’ with many ‘‘pockets’’. The NNRTI adjacent site is apparently able to make additional favorable binding contacts with RT by introducing suitable groups, which can be regarded as a tolerant region for the structural modifications of NNRTIs.

2.2

HIV protease inhibitors with novel binding modes

At present, most of the protease inhibitors (PIs) are pseudopeptides with limited bioavailability and low metabolic stability,

and their usage is greatly compromised by side effects, high costs, and emerging of resistant strains.50 Consequently, discovery of novel HIV PIs with the unprecedented mechanism of

Fig. 16 (a) The X-ray structure of cobalt bis(1,2-dicarbollide) complexed with PR (PDB code: 1ZTZ); Two molecules of the parent compound can bind with the hydrophobic pockets in the flap-proximal domain of the S3 and S3 0 subsites in PR.51 (b) The X-ray structure of polyhedral metallacarboranes complexed with PR (PDB code: 3I8W).52 It suggested that these compounds can block flap closure besides filling the corresponding binding sites as conventional PIs.


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Fig. 17 Structures of compounds 41 and 42, and the X-ray structure of dibenzo[b,e][1,4]diazepinone derivative 41 (duplicate, blue and red) complexed with HIV PR (PDB code: 3T11). Key H-bond interactions are shown as yellow dashed lines.

action has emerged as an interesting trend in the current antiretroviral drug research field. Recently, substituted metallacarboranes were identified as potent, specific (weakly inhibited human homologues) and competitive nonpeptidic inhibitors against WT and drug-resistant HIV PR mutants.51–53 The inhibitory profile of metallacarboranes can be explained by their unorthodox binding mode in the PR binding pocket and their ability to adjust their position within the PR substrate binding cleft (Fig. 16).52,53 Besides, X-ray structure (PDB code: 3T11) of a dibenzo[b,e][1,4]diazepinone 41 complexed with HIV PR (Fig. 17) suggested that duplicate compounds also bind to the PR active site. Molecular modeling demonstrated that the dimeric inhibitor 42 formed two key H-bonds with the catalytic aspartates which is the cause of their improved potency compared to the monomeric structure.54 These results will accelerate the rational design of future generations of non-peptide PIs that inhibit multi-mutated strains. An increasing effort in HIV-1 PR drug discovery is looking beyond the active site, towards allosteric inhibitors, which is an alternative method to limit the development of drug-resistance. Fragment screening has successfully detected binding of novel fragments to undescribed sites on HIV-1 PR. It was shown that indole-carboxylate derivatives (indole-6-carboxylic acid (1F1, 43), 3-indolepropionic acid (1F1-N, 44)) occupy an external site (the flap site) on the PR (Fig. 18).55 Although the identified compounds by this approach display relatively weak affinity, they are highly efficient ligands with respect to their size. These data strengthen the case of allosteric inhibition of HIV PR,

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Fig. 18 The X-ray structures of indole-carboxylate small molecules complexed with HIV PR. (a,b) compound 1F1 (PDB code: 4EJ8); (c,d) compound 1F1-N1 (PDB code: 4EJL). Key H-bond interactions are shown as yellow dashed lines.


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Fig. 19 The structure of carbonylhydrazide-based derivative 45.

Fig. 20 The structure of benzoic acid derivative D77.

providing a good paradigm for designing improved inhibitors for use in combination antiviral therapy. Importantly, the inhibition of HIV-1 PR dimerization represents a unique and practicable approach for antiviral drug design. The related inhibitors can dissociate intermolecular b-sheets involved in protein–protein interactions.56 The carbonylhydrazide-based derivative 45 (Fig. 19) was a recently reported non-peptide inhibitor of PR dimerization with a Kid of 50 nM for PR, 80 nM for the multimutated PR strain ANAM-11, and no inhibition against the aspartic proteases renin and pepsin.57 Besides, alkyl hydroxybenzoic acid derivatives (structures were not shown) can also inhibit HIV-1 PR dimerization.58 These findings are informative for designing potent inhibitors of HIV-1 PR dimerization. 2.3

Non-catalytic site (allosteric) IN inhibitors

Several IN inhibitors in preclinical development act via the identical mechanism as the approved drug raltegravir, namely IN strand transfer inhibitors (INSTIs). It was suggested that HIV-1 clinical isolates that are resistant to raltegravir are also resistant to some of the advanced clinical candidates. Therefore, there is a continuous effort to discover creative inhibitors targeting distinct steps of the IN-mediated integration process, which was exemplified by recent reports on the discovery of allosteric IN inhibitors (ALLINIs), including compounds inhibiting the interaction of IN with cellular cofactors (especially, lens epithelium-derived growth factor (LEDGF/p75)) and IN dimerization inhibitors.59,60 2.3.1 Small-molecules targeting LEDGF/p75-IN interaction. ALLINIs can disturb the LEDGF/p75-IN interaction, disrupt IN assembly with viral DNA and inhibit enzyme function allosterically. ALLINIs display a synergistic effect with INSTIs, highlighting the clinical potential of this novel inhibitor family.61–63


Review

The benzoic acid derivative D77 (46, Fig. 20) was the firstly discovered small molecular allosteric IN inhibitor that blocks HIV-1 IN interaction with LEDGF/p75 and cooperatively inhibits HIV-1(IIIB) replication in the MT-4 cell with an EC50 of 23.8 mg mL1 (5.03 mg mL1 for C8166 cells). Besides, cytotoxicities in mockinfected MT-4 and C8166 cells were observed with a CC50 of 76.82, 26.36 mg mL1, respectively.64 In recent years, 2-(quinolin-3-yl)acetic acid derivatives have attracted tremendous attention (Fig. 21).65–72 Several compounds are being evaluated as drug candidates at various stages of development for the treatment of HIV infection, which is a reflection of the growing maturity and acceptance of ALLINIs. Most notably, BI 224436 (51) is an investigational new drug candidate in phase Ia clinical trials, which can inhibit the replication of WT and raltegravir-resistant strains of HIV-1.68 BI 224436 exhibited high bioavailability with good tolerability (even at single doses ranging up to 200 mg) and good dosedependent pharmacokinetics (single dose: 100 mg), as well as favorable plasma levels.69 Moreover, the crystal structures of IN complexed with 2-(quinolin-3-yl)acetic acids have been solved (Fig. 22), providing detailed insights for designing prospective chemical structures inhibiting IN-LEDGF/p75 interaction. What needs highlighting is that tert-butoxy-(4-phenyl-quinolin3-yl)-acetic acids (tBPQA, compounds GS-A, GS-B, and GS-C) displayed not only inhibition of IN-LEDGF interaction but also inhibition of IN-viral DNA assembly.71 In view of this, more and more groups have focused their efforts on the search of structurally distinct inhibitors targeting HIV-1 IN-LEDGF/p75 interaction. Meanwhile, different types of novel medicinal chemistry strategies, such as the pharmacophorebased approach (Fig. 23),73 the scaffold hopping approach by combining the privileged fragments of lead compounds (Fig. 24),74 structure-based virtual screening,75 substructure and similarity searching using the structure-based pharmacophore model,76 as well as the ‘‘privileged’’ fragment-based discovery,77 have been carried out (Fig. 25), which resulted in the identification of structurally diverse compounds 60–64. These results further indicate the feasibility of inhibiting the HIV-1 IN-LEDGF/ p75 interaction by small molecules, providing an alternative way to exploration of HIV inhibitors with the unexploited mechanism of action. What cannot be neglected is that peptide mimicks of the IN binding domain of LEDGF/p75, cyclic peptides and the stapled peptides also exhibited potent inhibition of IN.78–80 2.3.2 Small molecule HIV-1 IN dimerization inhibitors. HIV IN requires a dynamic and precise equilibrium between several oligomeric units (at least a dimer) for its catalytic functions, as a consequence, the development of inhibitors targeting IN dimerization constitutes an interesting antiviral strategy. As illustrated in Fig. 26, several structurally diverse compounds were reported as allosteric IN dimerization inhibitors. For instance, compounds 65 and 66 from AlphaScreen-based assay for high-throughput screening (HTS) displayed significant activity with percentages of IN inhibition at 100 mM of 50% and 83%, respectively.81a,b Coumarin 67 identified by photoaffinity

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Fig. 21

2-(Quinolin-3-yl)acetic acid derivatives as HIV-1 IN allosteric inhibitors.

labeling and mass spectrometric analysis could bind to a site that may interfere with formation of active IN multimers.82 Compound 68 strongly modulated dynamic interactions between IN subunits.83 Moreover, crystal structure of the IN core domain

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complexed with sucrose (69) (Fig. 27) revealed that the sucrose binding site is in a region of the dimer interface, which represents a potential target for the development of allosteric inhibitors of IN dimerization.84


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Fig. 22 The structures of the HIV-1 IN catalytic core domain complexed with allosteric inhibitors deposited in the PDB with the following accession codes: (a) Compound LEDGIN-6 (47) (PDB code: 3LPU);65 (b) Compound 48 (PDB code: 3LPT);65 (c) Compound BI-1001 (49) (PDB code: 4DMN);66 (d) Compound 50 (PDB code: 4ID1).67 Key H-bond interactions are shown as yellow dashed lines. In the binding conformation, the substituted phenyl was perpendicular to the quinoline ring.

2.3.3 HIV IN inhibitors targeting other undescribed binding sites. The combined NMR and X-ray-based fragment-based screening approach and/or surface plasmon resonance were employed to identify and characterize the previously undescribed IN binding sites.85–88 For instance, through a fragmentbased screening method, a novel binding site and related ligand 3-(7-bromobenzo[d][1,3]dioxol-5-yl)-1-methyl-1H-pyrazol-5-amine (70) was identified. This compound displayed high affinity in the micromolar range and good ligand efficiency, providing

an excellent starting point for further hit-to-lead development (Fig. 28).88

3. Exploiting new drug design strategies for highly validated HIV targets In this section, we have selected a few successful case studies to illustrate the current state of the art in drug design exploiting

Fig. 23 Discovery of (4-hydroxy-1H-indol-3-yl)-4-oxobut-2-enoic acids as novel inhibitors of LEDGF/p75-IN interaction employing a pharmacophorebased computational approach.73


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Fig. 24 Scaffold hopping strategy for discovery of LEDGF/p75-IN interaction inhibitors by combining the privileged fragments of catechol and salicylate.74

novel medicinal chemistry strategies against these three highly validated HIV targets. 3.1

Rationally designed multitarget-directed ligands (MTDLs)

Design of multitarget-directed ligands (MTDLs) has proved to be an emerging and appealing anti-HIV drug discovery strategy.89a,b The key to the rational design of MTDLs via a knowledge-based pharmacophore combination approach would be to identify a

Fig. 25

Fig. 27 Crystal structure of the IN core domain complexed with sucrose (69) (PDB code: 3L3V).

tolerant domain in the drug target. Recently, the persistent work in a group of Minnesota University reported exciting results involving the rational construction of fused and highly merged MTDLs via a minimal structural variation of the HEPT platform to introduce inhibitory activity against IN (Fig. 29 and 30). Crystallographic studies have suggested that certain NNRTI scaffolds had structural moieties that were situated in the

Discovery of LEDGF/p75-IN interaction inhibitors via other medicinal chemistry approaches.

Fig. 26 Structures of small molecule HIV-1 IN dimerization inhibitors.

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Fig. 28

New fragment targeting a novel site on HIV-1 IN (PDB code: 3AO2).

solvent-exposed region controlled by the P236 loop of the NNRTI binding pocket (NNIBP) and were not directly involved

Review

in RT binding, where some limited structural changes could be tolerated. For instance, the phenyl group in the N-1 substituent of the HEPTs was very amenable to chemical modifications. In addition, IN binding requires minimally a hydrophobic benzyl group and a two metal ion chelating functionality. Therefore, this tolerant region in NNRTIs provided a prerequisite for incorporating a relatively hydrophilic IN chelating functionality to construct the bifunctional platform.90 Based on this general knowledge of pharmacophoric characteristics and SARs of the existing inhibitors, several classes of RT/IN dual inhibition scaffolds were established by incorporating a pharmacophore element for IN inhibitors to this tolerant region of potent NNRTIs (Fig. 29). Especially, the identified dual compounds 73a and 73b demonstrated excellent anti-HIV potencies at the nanomolar level, which effectively highlighted the MTDLs strategy. These compounds could be considered as good candidates with a potential impact for further pharmacological development in AIDS therapy.91a,b Moreover, as an extension to this work, a new molecular scaffold 3-hydroxypyrimidine-2,4-dione (exemplified by compounds 74a–c) featuring an N-OH moiety and capable of inhibiting both RT and IN was rationally driven from the HEPTs scaffold. The structural evolution includes a minimal 3-N hydroxylation in the pyrimidine ring of HEPTs to provide a chelating triad motif along with the existing benzyl group, which appeared to meet

Fig. 29 Discovery of fused RT/IN dual inhibitors combining RT inhibitor 71 with IN inhibitor 72.

Fig. 30 Design of highly merged RT/IN dual inhibitors combining RT inhibitor 71 with IN inhibitor 72.


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analoging-based optimization guided by computational modeling were often employed for developing MTDLs. For instance, the HIV-1 IN and the RNase H domain in RT shared structural and functional similarities. And several divalent metal ion chelators, such as diketo acids, hydroxytropolones, N-hydroxyimides, 2-hydroxyisoquinoline-1,3(2H,4H)-diones were identified as dual inhibitors targeting both at HIV-1 IN and the RT RNase H domain, which lays a solid foundation for the following analoging-based optimization strategy.91 3.2

Fig. 31

Bisubstrate-type inhibitor of HIV-1 RT.

the key structural requirements for IN inhibition. Meanwhile, the newly introduced OH could potentially form H-bonds with the consecutive lysine residues motif (Lys101–Lys104) in the NNIBP, and globally, based on the assay results, the bioactive conformation of the ligand should be kept, thus this chemical alteration did not severely impair the compound’s ability to bind with RT.91c–e The preliminary SAR studies allowed for a thorough understanding of the optimum for potent inhibition of RT and IN and paved the way for developing a novel family of anti-HIV therapeutics. Finally we also have to say that structural information on viral targets provides invaluable guidance in rational designing MTDLs. Besides, other strategies including HTS and consequent

Fig. 32

Multivalent ligands (mixed site inhibitor)

Multivalent ligands were composed of multiple conjugated fragments, each targeted to an additional binding site of a multisubstrate enzyme simultaneously. The principle advantage of multivalent inhibitors was their ability to generate more interactions with the binding pocket that could yield significantly improved affinity and selectivity, when compared with singlesite ligands, providing a more attractive strategy to design novel HIV therapeutic agents.92 The close proximity (10–15 Å) of the NRTI and NNRTI binding sites afforded a favorable platform and inspired several research teams to create bisubstrate-type NRTI-NNRTI (chimeric) divalent inhibitors with the potential to simultaneously occupy both binding sites. In 2013, the preparation and biochemical assay of a novel bifunctional RT inhibitor d4T-4PEG-TMC (76) utilizing d4T (NRTI) and a TMC-derivative 75 linked via a poly(ethylene glycol) (PEG) linker was reported (Fig. 31). It was illustrated that HIV-1 RT could smoothly incorporate the triphosphate of 76 in a base-specific manner. In addition, this compound displayed low nanomolar potency with 4300-fold and 4.3-fold increase of polymerization inhibition in vitro relative to the parent d4T and 75, respectively, which provided a proof-of-concept for the

Discovery of a potent HIV-1 PR inhibitor by using in situ click chemistry.

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Fig. 33 Discovery of NNRTI candidate Lersivirine via scaffold refining and LE/LLE-guided optimization. HLM = human liver microsome; ligand efficiency (LE) is the binding energy per heavy atom (kcal mol1) = 1.4 log (RT IC50)/number of heavy atoms; Ligand-lipophilicity efficiency (LLE) = log (RT IC50)clog P.

application of bifunctional RT inhibitors as a promising strategy of anti-HIV drug research.93 In theory, this strategy is applicable to all the protein with multiple binding sites, thus it could be used to rapidly develop potent and uniquely selective inhibitors. 3.3

Dynamic ligation screening (DLS)-based drug discovery

Combinatorial chemistry is a powerful tool to rapidly afford a large number of potentially bioactive molecules. In the last decade, dynamic ligation screening (DLS) has been introduced as a creative methodology in fragment-based drug discovery (FBDD) that combines dynamic, target-guided synthesis (TGS) of inhibitory ligands from a pool of smaller reactive fragments. It is not uncommon to notice that in situ click chemistry employing Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) has been widely applied in DLS for efficient assembly of diverse compound collections.94 Recently, this methodology has been successfully explored for the rapid synthesis and discovery of nonpeptidic HIV-1 PR inhibitors (Fig. 32).95 Concretely, azide (77, IC50 = 4.2 mM) was incubated with alkynes (78a–e, IC50s of all of these alkynes 4100 mM) in the presence of HIV PR. Compound 79 (IC50 = 6 nM, Ki = 1.7 nM) was selectively picked

Fig. 34

up by the PR, which was active against the WT and drug resistant mutants in nanomolar concentrations. This procedure significantly saved the load of chemical synthesis and separation and was considered to be an effective method for drug discovery. In addition, diversity-oriented rapid synthesis of potent HIV-1 PR inhibitors by in situ click chemistry and aminocarbonylation reaction was also reported, which provided the possibility to generate other bioavailable nonpeptidic inhibitors of HIV-1 PR.96,97 3.4 Ligand efficiency (LE)/ligand-lipophilicity efficiency (LLE)-guided drug discovery Ligand efficiency (LE) is an important indicator of ‘‘druglikeness’’, which is dependent on the molecule’s potency and physicochemical properties. The introduction of LE concept promoted the normalization of the free energy of binding of a ligand. Henceforth, optimizing compounds will be based on their effective binding and pharmacokinetic related properties, and the size effects will be removed.98 In addition, it was well recognized that the improvement of ligand-lipophilicity efficiency (LLE) was beneficial to increasing the potency and metabolic stability.

Novel indazole NNRTIs (83a,b) using molecular hybridization based on crystallographic overlays.


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Fig. 35 X-ray cocrystal structure determination of 83a (red) with the NNRTI binding site (PDB code: 2JLE). Compound 83a forms the pivotal edge-to-face p-interactions with the highly conserved residue Trp229, and the indazole NH forms a key hydrogen bond with the backbone of the Lys101 residue.


Current NNRTIs optimization was not purely affinity-driven, instead LE/LLE were used to guide the elaboration of the core motif and branching moieties. For instance, scaffold refining of the disused NNRTI Capravirine (80) led to the identification of pyrazole heterocycle as a suitable template for obtaining novel NNRTIs. Further, structure and medicinal chemistry knowledgebased optimization with emphasis on LE resulted in identification of several novel inhibitor chemotypes, which displayed very impressive anti-HIV activities (Fig. 33).99a,b Especially, Lersivirine (UK-453061, 81b) was selected for further clinical evaluation due to its excellent broad spectrum anti-HIV potency against a broad panel of key HIV-1 mutants, safety, and pharmacokinetic profiles.99c–e Therefore, LE/LLE were regarded as important variables in silico in NNRTIs hit identification against WT and drug-resistant RTs.100 Taken together, these case analyses revealed the robustness and efficiency of the creative medicinal chemistry strategies in lead discovery and structural optimization, which implicates a broad application of these approaches to many newly emerging anti-HIV targets, beyond these highly validated targets.

Fig. 36 Schematic diagram showing medicinal chemistry methodologies and the clinical medication of these highly validated viral targets as well as implications for innovative HIV targets.

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3.5 Crystallographic overlay-based (structure-guided) molecular hybridization Among the medicinal chemistry tricks of scaffold evolution, molecular hybridization is a very robust tactic in the discovery of bioactive molecules on the basis of combining basic pharmacophore elements from different bioactive substances to construct a new hybrid entity endowed with improved affinity and potency, when compared to the prototypes.90c The structural biology of primitive proteins in HIV-l replication uncovers the important binding mode of the inhibitor indicating the basic requirements determining their binding affinity and provides new clues about opportunities for evolving the scaffold and improving drug efficacy.90 In 2009, a novel series of indazole derivatives with potent inhibition against both WT and clinically relevant RT mutations was efficiently created by using crystallographic overlay-based molecular hybridization of existing NNRTIs Capravirine (80) and Efavirenz (82) (Fig. 34). Especially, compounds 83a and 83b possess potent antiviral activity and retain excellent metabolic stability.101 Besides, exploitation of the crystallographically determined binding mode of 83a for enhancement of antiviral potency was also described (Fig. 35). This case provided a proof-of-concept for the utility of crystallographic overlay-based molecular hybridization in the generation of novel derivatives from sub-structures of preexisting ligands bound to a common target. Admittedly, in an era of increasingly high-resolution crystallographic structures, the success rate of this approach should be superior to more traditional de novo design methods. Such a structure-guided molecular hybridization method by taking full advantage of structural information will become increasingly valuable.102–105

4. Conclusion and perspective In spite of HIV-1 RT, IN and PR being most widely exploited enzymatic targets for anti-HIV drugs, there is still great scope for further development of novel inhibitors with the unprecedented mechanism of action targeting these enzymes. The availability of vast quantities of structural biology information revealing distinctive binding modes for inhibitors and mechanisms of resistance at the molecular and atomic levels will assist in structure-based rational drug design to overcome drug resistance. Consequently, the application of medicinal chemistry methodologies, such as fragment-based drug discovery (fragment linking and elaboration), multitarget-directed ligands, multivalent ligands, dynamic ligation screening, ligand efficiency, crystallographic overlays-based molecular hybridization, and ‘‘privileged structure’’-guided scaffold refining106 will provide new tools to accelerate drug discovery (Fig. 36). The undeniable fact is that the experimental screening (or HTS) of in-house chemical libraries to find ‘‘original hits’’ with potency continues to be a common avenue for the discovery of additional antiviral agents with the unexpected mechanism of action, although the outcome still remains unpredictable. Fragment screening by biophysical technologies (such as X-ray


Review

crystallography, NMR spectroscopy, or surface plasmon resonance) has proven to be an effective supplement to traditional screening methods for drug discovery.107 A perusal of the literature revealed that fragment screening has been utilized for a variety of targets, including these three HIV-1 proteins. In the future research, the combination of fragment screening together with the visualization of the binding properties by X-ray crystallography will offer great benefits.108 From the clinical medication in the treatment of HIV infection point of view, an allosteric inhibitor together with an active site inhibitor would present a good combination to lessen the evolution of resistance, as an allosteric inhibitor could restore the potency of an active site inhibitor against multidrug-resistant mutants. There is also no denying that as the rapidly increasing availability of high-resolution structures of viral particles, it is equally important to exploit new agents inhibiting innovative HIV targets. As Confucius said: ‘‘consider the past (old), and you shall know the future (new)’’, it can be anticipated that valuable insights will be obtained through reviewing the stories of ‘‘Old Dogs with New Tricks’’, namely, medicinal chemistry methodologies dealing with these clinically validated antiviral targets. All the figures of binding modes were generated using PyMol (www.pymol.org).

Conflicts of interest The authors declare no conflict of interest.

Acknowledgements The financial support from the National Natural Science Foundation of China (NSFC No. 81102320, No. 81273354), Key Project of NSFC for International Cooperation (No. 30910103908), Research Fund for the Doctoral Program of Higher Education of China (No. 20110131130005, 20110131120037), and China Postdoctoral Science Foundation funded project (No. 20100481282, 2012T50584) is gratefully acknowledged.

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Mol. BioSyst.

New dogs, old tricks.

Pristinamycin: old drug, new tricks?

On Teaching Old Dogs New Tricks.

tRNAs: new tricks from old dogs.

A medical homily: old dogs do teach new tricks.

Old Dogs Learning New Tricks: Neuroplasticity Beyond the Juvenile Period.

Tumor-related interleukins: old validated targets for new anti-cancer drug development.

Myosin light chains: Teaching old dogs new tricks.

Evidence-based maternity care: can new dogs learn old tricks?

New (or not-so-new) tricks for old dogs: ultrasound imaging in anatomy laboratories.

Arginase: an old enzyme with new tricks.

Pinaverium in Irritable Bowel Syndrome: Old Drug, New Tricks?

New tricks for old dogs: countering antibiotic resistance in tuberculosis with host-directed therapeutics.

Antimicrobials: New tricks for old drugs.

New tricks for an old workhorse.

Programming T cell Killers for an HIV Cure: Teach the New Dogs New Tricks and Let the Sleeping Dogs Lie.

You can teach an old dog new tricks. How AIDS trials are pioneering new strategies.

New targets for drug action: is high selectivity always beneficial?

Old dogs with new tricks: Detecting accelerated long-term forgetting by extending traditional measures.

Water-pipe smoking and albuminuria: new dog with old tricks.

NSAIDs: learning new tricks from old drugs.

Baculovirus expression: old dog, new tricks.

DNA methylation: old dog, new tricks?

Old dog, new tricks: novel cardiac targets and stress regulation by protein kinase G.