Comparison of feline and human immunodeficiency virus reverse transcriptase enzymes through chemical screening and computational analysis.

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 µM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.

Structure-guided design of novel biphenyl-quinazoline derivatives as potent non-nucleoside reverse transcriptase inhibitors featuring improved anti-resistance, selectivity, and solubility.

In pursuit of enhancing the anti-resistance efficacy and solubility of our previously identified NNRTI 1, a series of biphenyl-quinazoline derivatives were synthesized employing a structure-based drug design strategy. Noteworthy advancements in anti-resistance efficacy were discerned among some of these analogs, prominently exemplified by compound 7ag, which exhibited a remarkable 1.37 to 602.41-fold increase in potency against mutant strains (Y181C, L100I, Y188L, F227L + V106A, and K103N + Y181C) in comparison to compound 1. Compound 7ag also demonstrated comparable anti-HIV activity against both WT HIV and K103N, albeit with a marginal reduction in activity against E138K. Of significance, this analog showed augmented selectivity index (SI > 5368) relative to compound 1 (SI > 37764), Nevirapine (SI > 158), Efavirenz (SI > 269), and Etravirine (SI > 1519). Moreover, it displayed a significant enhancement in water solubility, surpassing that of compound 1, Etravirine, and Rilpivirine. To elucidate the underlying molecular mechanisms, molecular docking studies were undertaken to probe the critical interactions between 7ag and both WT and mutant strains of HIV-1 RT. These findings furnish invaluable insights driving further advancements in the development of DAPYs for HIV therapy.

HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro.

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.

Role of I182, R187, and K188 Amino Acid Residues in the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration.

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.

HIV-1 reverse transcriptase stability correlates with Gag cleavage efficiency: reverse transcriptase interaction implications for modulating protease activation.

Proteolytic processing of human immunodeficiency virus type 1 particles mediated by viral protease (PR) is essential for acquiring virus infectivity. Activation of PR embedded in Gag-Pol is triggered by Gag-Pol dimerization during virus assembly. We previously reported that amino acid substitutions at the RT tryptophan repeat motif destabilize virus-associated RT and attenuate the ability of efavirenz (EFV, an RT dimerization enhancer) to increase PR-mediated Gag cleavage efficiency. Furthermore, a single amino acid change at RT significantly reduces virus yields due to enhanced Gag cleavage. These data raise the possibility of the RT domain contributing to PR activation by promoting Gag-Pol dimerization. To test this hypothesis, we investigated the putative involvement of a hydrophobic leucine repeat motif (LRM) spanning RT L282 to L310 in RT/RT interactions. We found that LRM amino acid substitutions led to RT instability and that RT is consequently susceptible to degradation by PR. The LRM mutants exhibited reduced Gag cleavage efficiencies while attenuating the EFV enhancement of Gag cleavage. In addition, an RT dimerization-defective mutant, W401A, reduced enhanced Gag cleavage via a leucine zipper (LZ) motif inserted at the deleted Gag-Pol region. Importantly, the presence of RT and integrase domains failed to counteract the LZ enhancement of Gag cleavage. A combination of the Gag cleavage enhancement factors EFV and W402A markedly impaired Gag cleavage, indicating a disruption of W402A Gag-Pol dimerization following EFV binding to W402A Gag-Pol. Our results support the idea that RT modulates PR activation by affecting Gag-Pol/Gag-Pol interaction. IMPORTANCE A stable reverse transcriptase (RT) p66/51 heterodimer is required for HIV-1 genome replication in host cells following virus entry. The activation of viral protease (PR) to mediate virus particle processing helps viruses acquire infectivity following cell release. RT and PR both appear to be major targets for inhibiting HIV-1 replication. We found a strong correlation between impaired p66/51RT stability and deficient PR-mediated Gag cleavage, suggesting that RT/RT interaction is critical for triggering PR activation via the promotion of adequate Gag-Pol dimerization. Accordingly, RT/RT interaction is a potentially advantageous method for anti-HIV/AIDS therapy if it is found to simultaneously block PR and RT enzymatic activity.

Prediction of HIV-1 protease cleavage site from octapeptide sequence information using selected classifiers and hybrid descriptors.

BACKGROUND: In most parts of the world, especially in underdeveloped countries, acquired immunodeficiency syndrome (AIDS) still remains a major cause of death, disability, and unfavorable economic outcomes. This has necessitated intensive research to develop effective therapeutic agents for the treatment of human immunodeficiency virus (HIV) infection, which is responsible for AIDS. Peptide cleavage by HIV-1 protease is an essential step in the replication of HIV-1. Thus, correct and timely prediction of the cleavage site of HIV-1 protease can significantly speed up and optimize the drug discovery process of novel HIV-1 protease inhibitors. In this work, we built and compared the performance of selected machine learning models for the prediction of HIV-1 protease cleavage site utilizing a hybrid of octapeptide sequence information comprising bond composition, amino acid binary profile (AABP), and physicochemical properties as numerical descriptors serving as input variables for some selected machine learning algorithms. Our work differs from antecedent studies exploring the same subject in the combination of octapeptide descriptors and method used. Instead of using various subsets of the dataset for training and testing the models, we combined the dataset, applied a 3-way data split, and then used a "stratified" 10-fold cross-validation technique alongside the testing set to evaluate the models. RESULTS: Among the 8 models evaluated in the "stratified" 10-fold CV experiment, logistic regression, multi-layer perceptron classifier, linear discriminant analysis, gradient boosting classifier, Naive Bayes classifier, and decision tree classifier with AUC, F-score, and B. Acc. scores in the ranges of 0.91-0.96, 0.81-0.88, and 80.1-86.4%, respectively, have the closest predictive performance to the state-of-the-art model (AUC 0.96, F-score 0.80 and B. Acc. ~ 80.0%). Whereas, the perceptron classifier and the K-nearest neighbors had statistically lower performance (AUC 0.77-0.82, F-score 0.53-0.69, and B. Acc. 60.0-68.5%) at p < 0.05. On the other hand, logistic regression, and multi-layer perceptron classifier (AUC of 0.97, F-score > 0.89, and B. Acc. > 90.0%) had the best performance on further evaluation on the testing set, though linear discriminant analysis, gradient boosting classifier, and Naive Bayes classifier equally performed well (AUC > 0.94, F-score > 0.87, and B. Acc. > 86.0%). CONCLUSIONS: Logistic regression and multi-layer perceptron classifiers have comparable predictive performances to the state-of-the-art model when octapeptide sequence descriptors consisting of AABP, bond composition and standard physicochemical properties are used as input variables. In our future work, we hope to develop a standalone software for HIV-1 protease cleavage site prediction utilizing the linear regression algorithm and the aforementioned octapeptide sequence descriptors.

Effectively predicting HIV-1 protease cleavage sites by using an ensemble learning approach.

BACKGROUND: The site information of substrates that can be cleaved by human immunodeficiency virus 1 proteases (HIV-1 PRs) is of great significance for designing effective inhibitors against HIV-1 viruses. A variety of machine learning-based algorithms have been developed to predict HIV-1 PR cleavage sites by extracting relevant features from substrate sequences. However, only relying on the sequence information is not sufficient to ensure a promising performance due to the uncertainty in the way of separating the datasets used for training and testing. Moreover, the existence of noisy data, i.e., false positive and false negative cleavage sites, could negatively influence the accuracy performance. RESULTS: In this work, an ensemble learning algorithm for predicting HIV-1 PR cleavage sites, namely EM-HIV, is proposed by training a set of weak learners, i.e., biased support vector machine classifiers, with the asymmetric bagging strategy. By doing so, the impact of data imbalance and noisy data can thus be alleviated. Besides, in order to make full use of substrate sequences, the features used by EM-HIV are collected from three different coding schemes, including amino acid identities, chemical properties and variable-length coevolutionary patterns, for the purpose of constructing more relevant feature vectors of octamers. Experiment results on three independent benchmark datasets demonstrate that EM-HIV outperforms state-of-the-art prediction algorithm in terms of several evaluation metrics. Hence, EM-HIV can be regarded as a useful tool to accurately predict HIV-1 PR cleavage sites.

Computational and Crystallographic Analysis of Binding Structures of Inhibitory Compounds for HIV-1 RNase H Activity.

Chemotherapy of human immunodeficiency virus type-1 (HIV-1) has significantly developed over the last three decades. The emergence of drug-resistant variants is, however, still a severe problem. The RNase H activity of HIV-1 reverse transcriptase is an attractive target for a new class of antiviral drugs because there is no approved inhibitor. The nitro-furan-carbonyl and nitro-thiophene-carbonyl groups are potent scaffolds for the HIV-1 RNase H inhibitor. In this work, the binding structures of six inhibitory compounds were obtained by X-ray crystal analysis in a complex with a recombinant protein of HIV-1 RNase H domain. Every inhibitory compound was found to be bound to the catalytic site with the furan- or thiophene-ring coordinated to two divalent metal ions at the binding pocket. All the atoms in nitro, furan, carbonyl, and two metals were aligned in the nitro-furan derivatives. The straight line connecting nitro and carboxyl groups was parallel to the plane made by two metal ions and a furan O atom. The binding modes of the nitro-thiophene derivatives were slightly different from those of the nitro-furan ones. The nitro and carbonyl groups deviated from the plane made by two metals and a thiophene S atom. Molecular dynamics simulations suggested that the furan O or thiophene S atom and carbonyl O atom were firmly coordinated to the metal ions. The simulations made the planar nitro-furan moiety well aligned to the line connecting the two metal ions. In contrast, the nitro-thiophene derivatives were displaced from the initial positions after the simulations. The computational findings will be a sound basis for developing potent inhibitors for HIV-1 RNase H activity.

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.

The HIV-1 Viral Protease Is Activated during Assembly and Budding Prior to Particle Release.

HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that prematurely activating, rather than inhibiting, protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation is challenging to induce efficiently. Here, monitoring viral protease activity with sensitive techniques, including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a precursor conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics is required to induce potent and specific elimination of HIV-infected cells. IMPORTANCE HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.

Prediction and molecular field view of drug resistance in HIV-1 protease mutants.

Conquering the mutational drug resistance is a great challenge in anti-HIV drug development and therapy. Quantitatively predicting the mutational drug resistance in molecular level and elucidating the three dimensional structure-resistance relationships for anti-HIV drug targets will help to improve the understanding of the drug resistance mechanism and aid the design of resistance evading inhibitors. Here the MB-QSAR (Mutation-dependent Biomacromolecular Quantitative Structure Activity Relationship) method was employed to predict the molecular drug resistance of HIV-1 protease mutants towards six drugs, and to depict the structure resistance relationships in HIV-1 protease mutants. MB-QSAR models were constructed based on a published data set of Ki values for HIV-1 protease mutants against drugs. Reliable MB-QSAR models were achieved and these models display both well internal and external prediction abilities. Interpreting the MB-QSAR models supplied structural information related to the drug resistance as well as the guidance for the design of resistance evading drugs. This work showed that MB-QSAR method can be employed to predict the resistance of HIV-1 protease caused by polymorphic mutations, which offer a fast and accurate method for the prediction of other drug target within the context of 3D structures.

Drug Resistance in the HIV-1 Subtype C Protease Enzyme: A High Throughput Virtual Screening Approach in Search of New Ligands with Activity.

BACKGROUND: HIV-1 subtype C protease is a strategic target for antiretroviral treatment. However, resistance to protease inhibitors appears after months of treatment. Chromones and 2- biscoumarin derivatives show potential for inhibition of the HIV- subtype C protease. OBJECTIVE: Different heterocyclic structures from the ZINC database were docked against Human Immunodeficiency Virus-1 (HIV) subtype C protease crystal structure 2R5Q and 2R5P. The 5 best molecules were selected to be docked against 62 homology models based on HIV-protease sequences from infants failing antiretroviral protease treatment. This experimentation was performed with two molecular docking programs: Autodock and Autodock Vina. These molecules were modified by substituting protons with different moieties, and the derivatives were docked against the same targets. Ligand-protein interactions, physical/chemical proprieties of the molecules, and dynamics simulations were analyzed. METHODS: Docking of all of the molecules was performed to find out the binding sites of HIV-1 subtype C proteases. An in-house script was made to substitute protons of molecules with different moieties. According to the Lipinski rule of five, physical and chemical properties were determined. Complexes of certain ligands-protease were compared to the protein alone in molecular dynamics simulations. RESULTS: From the first docking results, the 5 best (lowest energy) ligands (dibenz[a,h]acridine, dibenz[a, i]acridine, NSC114903, dibenz[c,h]acridine, benzo[a]acridine) were selected. The binding energy of the modified ligands increased, including the poorest-performing molecules. A correlation between nature, the position, and the resulting binding energy was observed. According to the Lipinski rules, the physico-chemical characteristics of the five best-modified ligands are ideal for oral bioavailability. Molecular dynamics simulations show that some lead-protease complexes were stable. CONCLUSION: Dibenz[a,h]acridine, dibenz[a, i]acridine, NSC114903, dibenz[c,h]acridine, benzo[ a]acridine and their derivatives might be considered as promising HIV-1 subtype C protease inhibitors. This could be confirmed through synthesis and subsequent in vitro assays.

Development of Novel Dihydrofuro[3,4-d]pyrimidine Derivatives as HIV-1 NNRTIs to Overcome the Highly Resistant Mutant Strains F227L/V106A and K103N/Y181C.

Here, we report the design, synthesis, structure-activity relationship studies, antiviral activity, enzyme inhibition, and druggability evaluation of dihydrofuro[3,4-d]pyrimidine derivatives as a potent class of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Compounds 14b (EC50 = 5.79-28.3 nM) and 16c (EC50 = 2.85-18.0 nM) exhibited superior potency against a panel of HIV-1-resistant strains. Especially, for the changeling mutations F227L/V106A and K103N/Y181C, both compounds exhibited remarkably improved activity compared to those of etravirine and rilpivirine. Moreover, 14b and 16c showed moderate RT enzyme inhibition (IC50 = 0.14-0.15 µM), which demonstrated that they acted as HIV-1 NNRTIs. Furthermore, 14b and 16c exhibited favorable pharmacokinetic and safety properties, making them excellent leads for further development.

Joint neutron/molecular dynamics vibrational spectroscopy reveals softening of HIV-1 protease upon binding of a tight inhibitor.

Biomacromolecules are inherently dynamic, and their dynamics are interwoven into function. The fast collective vibrational dynamics in proteins occurs in the low picosecond timescale corresponding to frequencies of ∼5-50 cm-1. This sub-to-low THz frequency regime covers the low-amplitude collective breathing motions of a whole protein and vibrations of the constituent secondary structure elements, such as α-helices, ß-sheets and loops. We have used inelastic neutron scattering experiments in combination with molecular dynamics simulations to demonstrate the vibrational dynamics softening of HIV-1 protease, a target of HIV/AIDS antivirals, upon binding of a tight clinical inhibitor darunavir. Changes in the vibrational density of states of matching structural elements in the two monomers of the homodimeric protein are not identical, indicating asymmetric effects of darunavir on the vibrational dynamics. Three of the 11 major secondary structure elements contribute over 40% to the overall changes in the vibrational density of states upon darunavir binding. Molecular dynamics simulations informed by experiments allowed us to estimate that the altered vibrational dynamics of the protease would contribute -3.6 kcal mol-1 at 300 K, or 25%, to the free energy of darunavir binding. As HIV-1 protease drug resistance remains a concern, our results open a new avenue to help establish a direct quantitative link between protein vibrational dynamics and drug resistance.

Antiviral Activity and Resistance Profile of the Novel HIV-1 Non-Catalytic Site Integrase Inhibitor JTP-0157602.

HIV-1 integrase (IN) is an essential enzyme for viral replication. Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 IN inhibitors and a potential new class of antiretrovirals. In this report, we identified a novel NCINI, JTP-0157602, with an original scaffold. JTP-0157602 exhibited potent antiviral activity against HIV-1 and showed a serum-shifted 90% effective concentration (EC90) of 138 nM, which is comparable to those of the FDA-approved IN strand transfer inhibitors (INSTIs). This compound was fully potent against a wide range of recombinant viruses with IN polymorphisms, including amino acids 124/125, a hot spot of IN polymorphisms. In addition, JTP-0157602 retained potent antiviral activity against a broad panel of recombinant viruses with INSTI-related resistance mutations, including multiple substitutions that emerged in clinical studies of INSTIs. Resistance selection experiments of JTP-0157602 led to the emergence of A128T and T174I mutations, which are located at the lens epithelium-derived growth factor/p75 binding pocket of IN. JTP-0157602 inhibited HIV-1 replication mainly during the late phase of the replication cycle, and HIV-1 virions produced by reactivation from HIV-1 latently infected Jurkat cells in the presence of JTP-0157602 were noninfectious. These results suggest that JTP-0157602 and analog compounds can be used to treat HIV-1 infectious diseases. IMPORTANCE Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 integrase (IN) inhibitors that bind to the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. NCINIs are expected to be a new class of anti-HIV-1 agents. In this study, we present a novel NCINI, JTP-0157602, which has potent activity against a broad range of HIV-1 strains with IN polymorphisms. Furthermore, JTP-0157602 shows strong antiviral activity against IN strand transfer inhibitor-resistant mutations, suggesting that JTP-0157602 and its analogs are potential agents for treating HIV-1 infections. Structural modeling indicated that JTP-0157602 binds to the LEDGF/p75 binding pocket of IN, and the results of in vitro resistance induction revealed the JTP-0157602 resistance mechanism of HIV-1. These data shed light on developing novel NCINIs that exhibit potent activity against HIV-1 with broad IN polymorphisms and multidrug-resistant HIV-1 variants.

Structure and function of retroviral integrase.

A hallmark of retroviral replication is establishment of the proviral state, wherein a DNA copy of the viral RNA genome is stably incorporated into a host cell chromosome. Integrase is the viral enzyme responsible for the catalytic steps involved in this process, and integrase strand transfer inhibitors are widely used to treat people living with HIV. Over the past decade, a series of X-ray crystallography and cryogenic electron microscopy studies have revealed the structural basis of retroviral DNA integration. A variable number of integrase molecules congregate on viral DNA ends to assemble a conserved intasome core machine that facilitates integration. The structures additionally informed on the modes of integrase inhibitor action and the means by which HIV acquires drug resistance. Recent years have witnessed the development of allosteric integrase inhibitors, a highly promising class of small molecules that antagonize viral morphogenesis. In this Review, we explore recent insights into the organization and mechanism of the retroviral integration machinery and highlight open questions as well as new directions in the field.

Indolylarylsulfones bearing phenylboronic acid and phenylboronate ester functionalities as potent HIV­1 non-nucleoside reverse transcriptase inhibitors.

To explore the chemical space around the entrance channel of the HIV-1 reverse transcriptase (RT) binding pocket, we innovatively designed and synthesized a series of novel indolylarylsulfones (IASs) bearing phenylboronic acid and phenylboronate ester functionalities at the indole-2-carboxamide as new HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) through structure-based drug design. All the newly synthesized compounds exhibited excellent to moderate potency against wild-type (WT) HIV-1 with EC50 values ranging from 6.7 to 42.6 nM. Among all, (3-ethylphenyl)boronic acid substituted indole-2-carboxamide and (4-ethylphenyl) boronate ester substituted indole-2-carboxamide were found to be the most potent inhibitors (EC50 = 8.5 nM, SI = 3310; EC50 = 6.7 nM, SI = 3549, respectively). Notably, (3-ethylphenyl)boronic acid substituted indole-2-carboxamide maintained excellent activities against the single HIV-1 mutants L100I (EC50 = 7.3 nM), K103N (EC50 = 9.2 nM), as well as the double mutant V106A/F227L (EC50 = 21.1 nM). Preliminary SARs and molecular modelling studies are also discussed in detail.

Towards the De Novo Design of HIV-1 Protease Inhibitors Based on Natural Products.

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) continues to be a public health problem. In 2020, 680,000 people died from HIV-related causes, and 1.5 million people were infected. Antiretrovirals are a way to control HIV infection but not to cure AIDS. As such, effective treatment must be developed to control AIDS. Developing a drug is not an easy task, and there is an enormous amount of work and economic resources invested. For this reason, it is highly convenient to employ computer-aided drug design methods, which can help generate and identify novel molecules. Using the de novo design, novel molecules can be developed using fragments as building blocks. In this work, we develop a virtual focused compound library of HIV-1 viral protease inhibitors from natural product fragments. Natural products are characterized by a large diversity of functional groups, many sp3 atoms, and chiral centers. Pseudo-natural products are a combination of natural products fragments that keep the desired structural characteristics from different natural products. An interactive version of chemical space visualization of virtual compounds focused on HIV-1 viral protease inhibitors from natural product fragments is freely available in the supplementary material.

HIV-1 gp120 Antagonists Also Inhibit HIV-1 Reverse Transcriptase by Bridging the NNRTI and NRTI Sites.

HIV-1 infection is typically treated using ≥2 drugs, including at least one HIV-1 reverse transcriptase (RT) inhibitor. Drugs targeting RT comprise nucleos(t)ide RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NRTI-triphosphates bind at the polymerase active site and, following incorporation, inhibit DNA elongation. NNRTIs bind at an allosteric pocket ∼10 Å away from the polymerase active site. This study focuses on compounds ("NBD derivatives") originally developed to bind to HIV-1 gp120, some of which inhibit RT. We have determined crystal structures of three NBD compounds in complex with HIV-1 RT, correlating with RT enzyme inhibition and antiviral activity, to develop structure-activity relationships. Intriguingly, these compounds bridge the dNTP and NNRTI-binding sites and inhibit the polymerase activity of RT in the enzymatic assays (IC50 < 5 µM). Two of the lead compounds, NBD-14189 and NBD-14270, show potent antiviral activity (EC50 < 200 nM), and NBD-14270 shows low cytotoxicity (CC50 > 100 µM).