NNRTI Binding Pocket Research Articles

Discovery of Novel Amino Acids (Analogues)-Substituted Thiophene[3,2-d]pyrimidine Derivatives as Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: Design, Synthesis, and Biological Evaluation.

Inspired by our previous work on the modification of diarylpyrimidine-typed non-nucleoside reverse transcriptase inhibitors (NNRTIs) and the reported crystallographic studies, a series of novel amino acids (analogues)-substituted thiophene[3,2-d]pyrimidine derivatives were designed and synthesized by targeting the solvent-exposed region of the NNRTI-binding pocket. The biological evaluation results showed that compound 5k was the most active inhibitor, exhibiting moderate-to-excellent potency against HIV-1 wild-type (WT) and a panel of NNRTI-resistant strains, with EC50 values ranging from 0.042 μM to 7.530 μM. Of special note, 5k exhibited the most potent activity against single-mutant strains (K103N and E138K), with EC50 values of 0.031 μM and 0.094 μM, being about 4.3-fold superior to EFV (EC50 = 0.132 μM) and 1.9-fold superior to NVP (EC50 = 0.181 μM), respectively. In addition, 5k demonstrated lower cytotoxicity (CC50 = 27.9 μM) and higher selectivity index values. The HIV-1 reverse transcriptase (RT) inhibition assay was further performed to confirm their binding target. Moreover, preliminary structure-activity relationships (SARs) and molecular docking studies were also discussed in order to provide valuable insights for further structural optimizations. In summary, 5k turned out to be a promising NNRTI lead compound for further investigations of treatments for HIV-1 infections.

Design and synthesis of Fsp3-enriched spirocyclic-substituted diarylpyrimidine derivatives as novel HIV-1 NNRTIs.

In this study, a novel series of diarylpyrimidine derivatives with Fsp3-enriched spirocycles were designed and synthesized to further explore the chemical space of the hydrophobic channel of the NNRTI-binding pocket. The biological evaluation results showed that most of the compounds displayed effective inhibitory potency against the HIV-1 wild-type strain, with EC50 values ranging from micromolar to submicromolar levels. Among them, TT6 turned out to be the most effective inhibitor with an EC50 value of 0.17 μM, demonstrating up to 47 times more active than that of reference drug 3TC (EC50 = 8.01 μM). More encouragingly, TT6 was found to potently inhibit the HIV-1 mutant strain K103N with an EC50 value of 0.69 μM, being about 6-fold more potent than 3TC (EC50 = 3.68 μM) and NVP (EC50 = 4.62 μM). Furthermore, TT6 exhibited the most potent inhibitory activity toward HIV-1 reverse transcriptase with an IC50 value of 0.33 μM. Additionally, molecular simulation studies were conducted to investigate the binding modes between TT6 and NNRTI-binding pocket, which may provide valuable clues for the follow-up structural optimizations.

Structure-based design and optimization lead to the identification of novel dihydrothiopyrano[3,2-d]pyrimidine derivatives as potent HIV-1 inhibitors against drug-resistant variants

With our continuous endeavors in seeking potent anti-HIV-1 agents, we reported here the discovery, biological characterization, and druggability evaluation of a class of nonnucleoside reverse transcriptase inhibitors. To fully explore the chemical space of the NNRTI-binding pocket, novel series of dihydrothiopyrano [3,2-d]pyrimidines were developed by employing the structure-based design strategy. Most of the derivatives were endowed with prominent antiviral activities against HIV-1 wild-type and resistant strains at nanomolar levels. Among them, compound 23h featuring the aminopiperidine moiety was identified as the most potent inhibitor, with EC50 values ranging from 3.43 to 21.4 nmol/L. Especially, for the challenging double-mutants F227L + V106A and K103N + Y181C, 23h exhibited 2.3- to 14.5-fold more potent activity than the first-line drugs efavirenz and etravirine. Besides, the resistance profiles of 23h achieved remarkable improvement compared to efavirenz and etravirine. The binding target of 23h was further confirmed to be HIV-1 reverse transcriptase. Molecular modeling studies were also performed to elucidate the biological evaluation results and give guidance for the optimization campaign. Furthermore, no apparent inhibition of the major CYP450 enzymes and hERG channel was observed for 23h. Most importantly, 23h was characterized by good pharmacokinetic properties and excellent safety in vivo. Collectively, 23h holds great promise as a potential candidate for its effective antiviral efficacy and favorable drug-like profiles.

Targeting HIV-1 Reverse Transcriptase Using a Fragment-Based Approach.

Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.

Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.

In silico studies on new oxathiadiazoles as potential anti-HIV agents

The first generation NNRTIs - tivirapine, a TIBO derivative, and loviride, an α-APA derivative, though effective against wild type HIV-1, showed lower potency when tested against common NNRTI-resistant mutants. A systematic structure based molecular modeling study played a key role in understanding the three dimensional structure activity relationships (3D SAR) in these two chemically distinct series. These derivatives were docked into allosteric pocket of HIV-RT to check their ability to inhibit polymerization by HIV-1 RT. The Lipinski's rule of five was also used to determine the biological activity or drug-likeness of the designed inhibitors. The TOPKAT protocol available in DS 3.0 was used to predict the activity and carcinogenicity.Docking experiments have shown good protein-ligand interactions. For the most of the NNRTIs, π-stacking and van der Waals interactions between the inhibitors and the NNRTI binding pocket amino acid residues were observed. All derivatives showed the formation of hydrogen bonds with lysine 101 and lysine 103 and π-interactions with aromatic residues Tyr181, Tyr188, and Trp229. Better optimum oral bioavailability, absorption and permeation have been predicted for these molecules on the basis of ADMET protocol available within DS 3.0.

Identification of Adjacent NNRTI Binding Pocket in Multi-mutated HIV1- RT Enzyme Model: An in silico Study.

A possible strategy to combat mutant strains is to have a thorough structural evaluation before and after mutations to identify the diversity in the non-nucleoside inhibitor binding pocket and their effects on enzyme-ligand interactions to generate novel NNRTI's accordingly. The primary objective of this study was to find effects of multiple point mutations on NNRTI binding pocket. This study included the contribution of each individual mutation in NNIBP that propose an adjacent binding pocket which can be used to discover novel NNRTI derivatives. An in Silico model of HIV-1 RT enzyme with multiple mutations K103N, Y181C and Y188L was developed and evaluated. Two designed NNRTI pyridinone derivatives were selected as ligands for docking studies with the homology model through alignment based docking and residue based docking approaches. Binding pockets of wild type HIV-1 RT and multi-mutated homology model were compared thoroughly. K103N mutation narrowed the entrance of NNRTI binding pocket and forbade electrostatic interaction with α amino group of LYS103. Mutations Y181C and Y188L prevented NNRTI binding by eliminating aromatic π interactions offered by tyrosine rings. Docking study against new homology model suggested an adjacent binding pocket with combination of residues in palm and connection domains. This pocket is approximately 14.46Å away from conventional NNRTI binding site. Increased rigidity, steric hindrance and losses of important interactions cumulatively prompt ligands to adapt adjacent NNRTI binding pocket. The proposed new and adjacent binding pocket is identified by this study which can further be evaluated to generate novel derivatives.

The structure of FIV reverse transcriptase and its implications for non-nucleoside inhibitor resistance.

Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the “closed” pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more “closed” conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.

Discovery of novel diarylpyrimidines as potent HIV-1 NNRTIs by investigating the chemical space of a less explored "hydrophobic channel".

A new series of diarylpyrimidines (DAPYs) were designed, synthesized and evaluated as novel HIV-1 NNRTIs to further explore the chemical space surrounding the "hydrophobic channel" of the NNRTI binding pocket (NNIBP), guided by the comprehensive analysis of X-ray structural biology data of HIV-1 RT/NNRTI complexes and molecular modeling. Encouragingly, most of the synthesized DAPYs were found to be active against the HIV-1 wild-type (WT) strain with EC50 values ranging from 3 nM to 63 nM, and displayed significantly reduced cytotoxicity compared with etravirine (ETV) and rilpivirine (RPV). Among them, two most promising compounds Z10 (EC50 = 3 nM) and Z13 (EC50 = 3 nM) showed equivalent potency against the HIV-1 WT strain to the reference drugs efavirenz (EFV, EC50 = 3 nM) and ETV (EC50 = 3 nM). Notably, Z13 also showed the most potent activity against HIV-1 mutant strains including K103N (EC50 = 10 nM), E138K (EC50 = 22 nM) and RES056 (EC50 = 0.935 μM). Against mutant strains Y181C, Y188L and F227L + V106A, Z17 showed double-digit nanomolar inhibitory activity with EC50 values 27 nM, 98 nM and 30 nM, respectively. The structure-activity relationships (SARs) and molecular docking studies provided important clues for further molecular elaboration. Collectively, this study provides useful information to guide lead optimization and drug discovery via the exploration of this seldom investigated region.

Identification of novel bifunctional HIV-1 reverse transcriptase inhibitors.

The increasing prevalence of mutations in HIV-1 reverse transcriptase (RT) that confer resistance to existing NRTIs and NNRTIs underscores the need to develop RT inhibitors with novel mode-of-inhibition and distinct resistance profiles. Biochemical assays were employed to identify inhibitors of RT activity and characterize their mode of inhibition. The antiviral activity of the inhibitors was assessed by cell-based assays using laboratory HIV-1 isolates and MT4 cells. RT variants were purified via avidin affinity columns. Compound A displayed equal or greater potency against many common NNRTI-resistant RTs (K103N and Y181C RTs) relative to WT RT. Despite possessing certain NNRTI-like properties, such as being unable to inhibit an engineered variant of RT lacking an NNRTI-binding pocket, we found that compound A was dependent on Mg2+ for binding to RT. Optimization of compound A led to more potent analogues, which retained similar activities against WT and K103N mutant viruses with submicromolar potency in a cell-based assay. One of the analogues, compound G, was crystallized in complex with RT and the structure was determined at 2.6 Å resolution. The structure indicated that compound G simultaneously interacts with the active site (Asp186), the highly conserved primer grip region (Leu234 and Trp229) and the NNRTI-binding pocket (Tyr188). These findings reveal a novel class of RT bifunctional inhibitors that are not sensitive to the most common RT mutations, which can be further developed to address the deficiency of current RT inhibitors.

The HIV-1 p66 homodimeric RT exhibits different conformations in the binding-competent and -incompetent NNRTI site.

Non-nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase (RT), NNRTIs, which bind to the p66/p51 heterodimeric RT, also interact with the p66/p66 homodimer, whose structure is unknown. 19 F nuclear magnetic resonance of a single 4-trifluoromethylphenylalanine (tfmF) residue, incorporated into the NNRTI binding pocket of the p66/p66 homodimer at position 181, was used to investigate NNRTI binding. In the NNRTI-bound homodimer complex, two different 19 F signals are observed, with the resonance frequencies matching those of the NNRTI-bound p66/p51 heterodimer spectra, in which the individual p66-subunit or p51-subunit were labeled with tfmF at positions 181. These data suggest that the NNRTI-bound p66/p66 homodimer conformation, particularly around residue 181, is very similar to that in the p66/p51 heterodimer, explaining why NNRTI binding to p66/p66 enhances dimer formation.

Natural product-inspired esters and amides of ferulic and caffeic acid as dual inhibitors of HIV-1 reverse transcriptase

Using an HIV-1 Reverse Transcriptase (RT)-associated RNase H inhibition assay as lead, bioguided fractionation of the dichloromethane extract of the Ocimum sanctum leaves led to the isolation of five triterpenes (1–5) along with three 3-methoxy-4-hydroxy phenyl derivatives (6–8). The structure of this isolates were determined by 1D and 2D NMR experiments as well as ESI-MS. Tetradecyl ferulate (8) showed an interesting RNase H IC50 value of 12.4 μM and due to the synthetic accessibility of this secondary metabolite, a structure-activity relationship study was carried out. A series of esters and amides of ferulic and caffeic acids were synthesized and, among all, the most active was N-oleylcaffeamide displaying a strong inhibitory activity towards both RT-associated functions, ribonuclease H and DNA polymerase. Molecular modeling studies together with Yonetani-Theorell analysis, demonstrated that N-oleylcaffeamide is able to bind both two allosteric site located one close to the NNRTI binding pocket and the other close to RNase H catalytic site.

Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study

HIV-1 reverse transcriptase (RT) is a major drug target in the treatment of HIV-1 infection. RT inhibitors currently in use include non-nucleoside, allosteric RT inhibitors (NNRTIs), which bind to a hydrophobic pocket, distinct from the enzyme's active site. We investigated RT-NNRTI interactions by solution (19)F nuclear magnetic resonance (NMR), using singly (19)F-labeled RT proteins. Comparison of (19)F chemical shifts of fluorinated RT and drug-resistant variants revealed that the fluorine resonance is a sensitive probe for identifying mutation-induced changes in the enzyme. Our data show that in the unliganded enzyme, the NNRTI-binding pocket is highly plastic and not locked into a single conformation. Upon inhibitor binding, the binding pocket becomes rigidified. In the inhibitor-bound state, the (19)F signal of RT is similar to that of drug-resistant mutant enzymes, distinct from what is observed for the free state. Our results demonstrate the power of (19)F NMR spectroscopy to characterize conformational properties using selectively (19)F-labeled protein.

Structural optimization of pyridine-type DAPY derivatives to exploit the tolerant regions of the NNRTI binding pocket

Based on the crystallographic studies of diarylpyrimidines (DAPYs), we embarked on incorporating the hydrophilic piperidyl or morpholinyl group into the known DAPY derivatives bearing the pyridine moiety as a core structure, with the double aim to exploit additional interactions with the HIV-1 NNRTI binding pocket (NNIBP), as well as to improve the compound solubility. The antiviral evaluation result show that the most potent compounds I-8b2, I-8b3, I-8b4 and I-8c3 exhibited anti-HIV-1 (IIIB) strain activity ranging from 7.4 nM to 9.4 nM (SI = 168–1283), superior to FDA-approved drugs of nevirapine (NVP), lamivudine (3TC) and delavirdine (DLV), and comparable to etravirine (ETV), zidovudine (AZT) and efavirenz (EFV). Additionally, compounds I-8c2 and I-8c3 showed moderate activity against NNRTI resistant strains baring mutations K103N and Y181C with EC50 values of 6.2 μM and 6.8 μM, respectively. Preliminary structure-activity relationships (SARs), reverse transcriptase inhibition efficacy and molecular modeling of selected compounds are also presented. These outcomes support our design hypothesis and demonstrate that the piperidyl group modified pyridine-typed DAPY derivatives are highly potent NNRTIs with improved water solubility.

Design, synthesis and evaluation of novel HIV-1 NNRTIs with dual structural conformations targeting the entrance channel of the NNRTI binding pocket

On the basis of structure-based bioisosteric replacement and molecular hybridization strategy, a series of novel dual structural-conformation inhibitors targeting the “entrance channel” of HIV-1 NNRTIs binding pocket (NNIBP) were designed and synthesized. All of the new compounds were evaluated for their anti-HIV activities in MT-4 cells using the MTT method. Five compounds exhibited moderate to excellent potencies inhibiting wild-type (wt) HIV-1 replication with EC50 values ranging from 31.36 μM to 0.11 μM. Among them, compound 15b was identified as the most potent inhibitor with EC50 values of 0.11 μM and 2.18 μM against wt and K103N/Y181C double mutant HIV-1 strain (RES056), respectively. In addition, preliminary structure–activity relationships (SARs) and molecular simulation studies were discussed, which may provide valuable insights for further optimization.

Design, synthesis and anti-HIV evaluation of novel diarylpyridine derivatives targeting the entrance channel of NNRTI binding pocket

The development of novel NNRTIs with activity against variants of HIV-1RT is crucial for overcoming treatment failure. In the present study, a series of novel 6-substituted diarylpyridine derivatives targeting the entrance channel of the NNIBP of RT were designed through a molecular hybridization strategy. Encouragingly, these new diarylpyridine derivatives were found to be active against wild-type (WT) HIV-1 with an EC50 values ranging from 0.035 μM to 1.99 μM. Nearly half of them exhibited more potent inhibitory activities in cellular assays than the control drug nevirapine (NVP). Notably, three most promising compounds If (EC50 = 35 nM), Ia (EC50 = 43 nM) and IIa (EC50 = 41 nM) showed high potency against WT and were comparable to the reference drug delavirdine (DLV) (EC50 = 33 nM). Moreover, compounds Ib, IIb and IIh displayed effective activity against the most common clinically observed single and double-mutated HIV-1 strains in micromolar concentrations. In particular, the inhibition of IIb against the K103N mutation (EC50 = 49 nM), which confers resistance to a wide variety of NNRTIs, was about 140 times more effective than NVP (EC50 = 6.78 μM), 50 times more than DLV (EC50 = 2.48 μM) and about 3 times more than EFV (EC50 = 0.12 μM), indicating that the newly designed compounds have great potential to be further developed as new anti-HIV-1 agents. Preliminary structure-activity relationships (SARs) and molecular modeling of the new diarylpyridine derivatives were discussed in detail.

Structure-enhanced methods in the development of non-nucleoside inhibitors targeting HIV reverse transcriptase variants.

Resistance continues to emerge as a leading cause for antiretroviral treatment failure. Several mutations in HIV reverse transcriptase (RT) confer resistance to non-nucleoside inhibitors (NNRTIs), vital components of antiretroviral combination therapies. Since the majority of mutations are located in the NNRTI binding pocket, crystal structures of RT variants in complex with NNRTIs have provided ideas for new drug design strategies. This article reviews the impact of RT crystal structures on the multidisciplinary design and development of new inhibitors with improved resistance profiles.

Factors influencing the efficacy of rilpivirine in HIV-1 subtype C in low- and middle-income countries.

The use of the NNRTI rilpivirine in low- and middle-income countries (LMICs) is under debate. The main objective of this study was to provide further clinical insights and biochemical evidence on the usefulness of rilpivirine in LMICs. Rilpivirine resistance was assessed in 5340 therapy-naive and 13,750 first-generation NNRTI-failed patients from Europe and therapy-naive HIV-1 subtype C (HIV-1C)-infected individuals from India (n = 617) and Ethiopia (n = 127). Rilpivirine inhibition and binding affinity assays were performed using patient-derived HIV-1C reverse transcriptases (RTs). Primary rilpivirine resistance was rare, but the proportion of patients with >100,000 HIV-1 RNA copies/mL pre-ART was high in patients from India and Ethiopia, limiting the usefulness of rilpivirine as a first-line drug in LMICs. In patients failing first-line NNRTI treatments, cross-resistance patterns suggested that 73% of the patients could benefit from switching to rilpivirine-based therapy. In vitro inhibition assays showed ∼ 2-fold higher rilpivirine IC50 for HIV-1C RT than HIV-1B RT. Pre-steady-state determination of rilpivirine-binding affinities revealed 3.7-fold lower rilpivirine binding to HIV-1C than HIV-1B RT. Structural analysis indicated that naturally occurring polymorphisms close to the NNRTI-binding pocket may reduce rilpivirine binding, leading to lower susceptibility of HIV-1C to rilpivirine. Our clinical and biochemical findings indicate that the usefulness of rilpivirine has limitations in HIV-1C-dominated epidemics in LMICs, but the drug could still be beneficial in patients failing first-line therapy if genotypic resistance testing is performed.

NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs.

Structure-based evaluation of non-nucleoside inhibitors with improved potency and solubility that target HIV reverse transcriptase variants.

The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series.