Resistant mechanism against nelfinavir of human immunodeficiency virus type 1 proteases

Inhibitors against human immunodeficiency virus type-1 (HIV-1) proteases are finely effective for anti-HIV-1 treatments. However, the therapeutic efficacy is reduced by the rapid emergence of inhibitor-resistant variants of the protease. Among patients who failed in the inhibitor nelfinavir (NFV) treatment, D30N, N88D, and L90M mutations of HIV-1 protease are often observed. Despite the serious clinical problem, it is not clear how these mutations, especially nonactive site mutations N88D and L90M, affect the affinity of NFV or why they cause the resistance to NFV. In this study, we executed molecular dynamics simulations of the NFV-bound proteases in the wild-type and D30N, N88D, D30N/N88D, and L90M mutants. Our simulations clarified the conformational change at the active site of the protease and the change of the affinity with NFV for all of these mutations, even though the 88th and 90th residues are not located in the NFV-bound cavity and not able to directly interact with NFV. D30N mutation causes the disappearance of the hydrogen bond between the m-phenol group of NFV and the 30th residue. N88D mutation alters the active site conformation slightly and induces a favorable hydrophobic contact. L90M mutation dramatically changes the conformation at the flap region and leads to an unfavorable distortion of the binding pocket of the protease, although 90M is largely far apart from the flap region. Furthermore, the changes of binding energies of the mutants from the wild-type protease are shown to be correlated with the mutant resistivity previously reported by the phenotypic experiments.     

分子动力学研究V82A和L90M变异对HIV PR-IDV复合物的影响

为了说明V82A和L90M变异对蛋白酶(PR)和茚地那韦(IDV)复合物的影响,进行了5.5ns的MD模拟.用MM-PBSA方法计算了体系的结合自由能,计算和实验结果一致.分解自由能为不同能量项说明,这两个变异引起熵的贡献变化大于焓的贡献变化.分解自由能到每个残基说明Wild,V82A和L90M具有相似的结合模式,结合能的贡献主要来源于A28/A28',I50/I50'和I84/I84'这六个残基组,详细分析了Wild和IDV的结合模式,对比分析了V82A和L90M变异引起结合模式的细小变化.V82A变异引起结合模式的变化是由于变异后位阻减小导致的.L90M变异引起D25和L90间的作用增强并引起结合模式的细小变化.研究结果有助于更好地理解变异对抑制剂和HIV-1PR结合模式的影响,并可以用来帮助设计更高效的PR抑制剂.     

An insight to the molecular interactions of the FDA approved HIV PR drugs against L38L↑N↑L PR mutant

The aspartate protease of the human immune deficiency type-1 virus (HIV-1) has become a crucial antiviral target in which many useful antiretroviral inhibitors have been developed. However, it seems the emergence of new HIV-1 PR mutations enhances drug resistance, hence, the available FDA approved drugs show less activity towards the protease. A mutation and insertion designated L38L↑N↑L PR was recently reported from subtype of C-SA HIV-1. An integrated two-layered ONIOM (QM:MM) method was employed in this study to examine the binding affinities of the nine HIV PR inhibitors against this mutant. The computed binding free energies as well as experimental data revealed a reduced inhibitory activity towards the L38L↑N↑L PR in comparison with subtype C-SA HIV-1 PR. This observation suggests that the insertion and mutations significantly affect the binding affinities or characteristics of the HIV PIs and/or parent PR. The same trend for the computational binding free energies was observed for eight of the nine inhibitors with respect to the experimental binding free energies. The outcome of this study shows that ONIOM method can be used as a reliable computational approach to rationalize lead compounds against specific targets. The nature of the intermolecular interactions in terms of the host–guest hydrogen bond interactions is discussed using the atoms in molecules (AIM) analysis. Natural bond orbital analysis was also used to determine the extent of charge transfer between the QM region of the L38L↑N↑L PR enzyme and FDA approved drugs. AIM analysis showed that the interaction between the QM region of the L38L↑N↑L PR and FDA approved drugs are electrostatic dominant, the bond stability computed from the NBO analysis supports the results from the AIM application. Future studies will focus on the improvement of the computational model by considering explicit water molecules in the active pocket. We believe that this approach has the potential to provide information that will aid in the design of much improved HIV-1 PR antiviral drugs.     

Structure, dynamics and solvation of HIV-1 protease/saquinavir complex in aqueous solution and their contributions to drug resistance: molecular dynamic simulations

As it is known that the understanding of the basic properties of the enzyme/inhibitor complex leads directly to enhancing the capability in drug designing and drug discovery. Molecular dynamics simulations have been performed to examine detailed information on the structure and dynamical properties of the HIV-1 PR complexed with saquinavir in the three protonated states, monoprotonates at Asp25 (Mono-25) and Asp25'(Mono-25') and diprotonate (Di-Pro) at both Asp25 and Asp25'. The obtained results support clinical data which reveal that Ile84 and Gly48 are two of the most frequent residues where mutation toward a protease inhibitor takes place. In contrast to the Ile84 mutation due to high displacement of Ile84 in the presence of saquinavir, source of the Gly48 mutation was observed to be due to the limited space in the HIV-1 PR pocket. The Gly48 was, on one side, found to form strong hydrogen bonds with saquinavir, while on the other side this residue was repelled by the hydrophobic Phe53 residue. In terms of inhibitor/enzyme binding, interactions between saquinavir and a catalytic triad of the HIV-1 PR were calculated using the ab initio method. The results show an order of the binding energy of Mono25相似文献   

Energetic basis for drug resistance of HIV-1 protease mutants against amprenavir

Amprenavir (APV) is a high affinity (0.15 nM) HIV-1 protease (PR) inhibitor. However, the affinities of the drug resistant protease variants V32I, I50V, I54V, I54M, I84V and L90M to amprenavir are decreased 3 to 30-fold compared to the wild-type. In this work, the popular molecular mechanics Poisson-Boltzmann surface area method has been used to investigate the effectiveness of amprenavir against the wild-type and these mutated protease variants. Our results reveal that the protonation state of Asp25/Asp25′ strongly affects the dynamics, the overall affinity and the interactions of the inhibitor with individual residues. We emphasize that, in contrast to what is often assumed, the protonation state may not be inferred from the affinities but requires pK_a calculations. At neutral pH, Asp25 and Asp25′ are ionized or protonated, respectively, as suggested from pK_a calculations. This protonation state was thus mainly considered in our study. Mutation induced changes in binding affinities are in agreement with the experimental findings. The decomposition of the binding free energy reveals the mechanisms underlying binding and drug resistance. Drug resistance arises from an increase in the energetic contribution from the van der Waals interactions between APV and PR (V32I, I50V, and I84V mutant) or a rise in the energetic contribution from the electrostatic interactions between the inhibitor and its target (I54M and I54V mutant). For the V32I mutant, also an increased free energy for the polar solvation contributes to the drug resistance. For the L90M mutant, a rise in the van der Waals energy for APV-PR interactions is compensated by a decrease in the polar solvation free energy such that the net binding affinity remains unchanged. Detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.     

Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79′s loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50′. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1′ subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2′ subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhancing protein backbone binding--a fruitful concept for combating drug-resistant HIV

The evolution of drug resistance is one of the most fundamental problems in medicine. In HIV/AIDS, the rapid emergence of drug-resistant HIV-1 variants is a major obstacle to current treatments. HIV-1 protease inhibitors are essential components of present antiretroviral therapies. However, with these protease inhibitors, resistance occurs through viral mutations that alter inhibitor binding, resulting in a loss of efficacy. This loss of potency has raised serious questions with regard to effective long-term antiretroviral therapy for HIV/AIDS. In this context, our research has focused on designing inhibitors that form extensive hydrogen-bonding interactions with the enzyme's backbone in the active site. In doing so, we limit the protease's ability to acquire drug resistance as the geometry of the catalytic site must be conserved to maintain functionality. In this Review, we examine the underlying principles of enzyme structure that support our backbone-binding concept as an effective means to combat drug resistance and highlight their application in our recent work on antiviral HIV-1 protease inhibitors.     

Design of dimerization inhibitors of HIV-1 aspartic proteinase: A computer-based combinatorial approach

Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.     

Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy‐Resistant L858R/T790M/C797S Mutant

The treatment of non‐small‐cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations. Third‐generation EGFR inhibitors have been designed to overcome resistance through covalent binding to the Cys 797 residue of the enzyme, and these inhibitors are effective against most clinically relevant EGFR mutants. However, the high dependence of these recent EGFR inhibitors on this particular interaction means that additional mutation of Cys 797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients. A new generation of irreversible and reversible mutant EGFR inhibitors was developed with strong noncovalent binding properties, and these compounds show high inhibitory activities against the cysteine‐mutated L858R/T790M/C797S EGFR.     

Combating susceptibility to drug resistance: lessons from HIV-1 protease

Drug resistance is a major obstacle in modern medicine. However, resistance is rarely considered in drug development and may inadvertently be facilitated, as many designed inhibitors contact residues that can mutate to confer resistance, without significantly impairing function. Contemporary drug design often ignores the detailed atomic basis for function and primarily focuses on disrupting the target's activity, which is necessary but not sufficient for developing a robust drug. In this study, we examine the impact of drug-resistant mutations in HIV-1 protease on substrate recognition and demonstrate that most primary active site mutations do not extensively contact substrates, but are critical to inhibitor binding. We propose a general, structure-based strategy to reduce the probability of drug resistance by designing inhibitors that interact only with those residues that are essential for function.     

Inhibitor docking screened by the modified SAFE_p scoring function: application to cyclic urea HIV-1 PR inhibitors

Our laboratory has in the past developed a method for the prediction of ligand binding free energies to proteins, referred to as SAFE_p (Solvent free energy predictor). Previously, we have applied this protocol for the prediction of the binding free energy of peptidic and cyclic urea HIV-1 PR inhibitors, whose X-ray structures bound to enzyme are known. In this work, we present the first account of a docking simulation, where the ligand conformations were screened and inhibitor ranking was predicted on the basis of a modified SAFE_p approach, for a set of cyclic urea-HIV-1 PR complexes whose structures are not known. We show that the optimal dielectric constant for docking is rather high, in line with the values needed to reproduce some protein residue properties, like pKa's. Our protocol is able to reproduce most of the observed binding ranking, even in the case that the components of the equation are not fitted to experimental data. Partition of the binding free energy into pocket and residue contributions sheds light into the importance of the inhibitor's fragments and on the prediction of "hot spots" for resistance mutations.     

Interpretation of protein/ligand crystal structure using QM/MM calculations: case of HIV-1 protease/metallacarborane complex

Deltahedral metallacarborane compounds have recently been discovered as potent, specific, stable, and nontoxic inhibitors of HIV-1 protease (PR), the major target for AIDS therapy. The 2.15 A-resolution X-ray structure has exhibited a nonsymmetrical binding of the parental compound [Co(3+)-(C2B9H11)2](-) (GB-18) into PR dimer and a symmetrical arrangement in the crystal of two PR dimer complexes into a tetramer. In order to explore structural and energetic details of the inhibitor binding, quantum mechanics coupled with molecular mechanics approach was utilized. Realizing the close positioning of anionic inhibitors in the active site cavity, the possibility of an exchange of structural water molecules Wat50 and Wat128 by Na+ counterions was studied. The energy profiles for the rotation of the GB-18 molecules along their longitudinal axes in complex with PR were calculated. The results show that two Na+ counterions are present in the active site cavity and provide energetically favorable and unfavorable positions for carbon atoms within the carborane cages. Eighty-one rotamer combinations of four molecules of GB-18 bound to PR out of 4 x 10(5) are predicted to be highly populated. These results lay ground for further calculations of interaction energies between GB-18 and amino acids of PR active site and will make it possible to interpret computationally the binding of similar metallacarborane molecules to PR as well as to resistant PR variants. Moreover, this computational tool will allow the design of new, more potent metallacarborane-based HIV-1 protease inhibitors.     

Resolution of discordant HIV-1 protease resistance rankings using molecular dynamics simulations

The emergence of drug resistance is a major challenge for the effective treatment of HIV. In this article, we explore the application of atomistic molecular dynamics simulations to quantify the level of resistance of a patient-derived HIV-1 protease sequence to the inhibitor lopinavir. A comparative drug ranking methodology was developed to compare drug resistance rankings produced by the Stanford HIVdb, ANRS, and RegaDB clinical decision support systems. The methodology was used to identify a patient sequence for which the three rival online tools produced differing resistance rankings. Mutations at only three positions ( L10I , A71IV, and L90M ) influenced the resistance level assigned to the sequence. We use ensemble molecular dynamics simulations to elucidate the origin of these discrepancies and the mechanism of resistance. By simulating not only the full patient sequences but also systems containing the constituent mutations, we gain insight into why resistance estimates vary and the interactions between the various mutations. In the same way, we also gain valuable knowledge of the mechanistic causes of resistance. In particular, we identify changes in the relative conformation of the two beta sheets that form the protease dimer interface which suggest an explanation of the relative frequency of different amino acids observed in patients at residue 71.     

Molecular basis of drug resistance in aurora kinases

Aurora kinases have emerged as potential targets in cancer therapy, and several drugs are currently undergoing preclinical and clinical validation. Whether clinical resistance to these drugs can arise is unclear. We exploited a hypermutagenic cancer cell line to select mutations conferring resistance to a well-studied Aurora inhibitor, ZM447439. All resistant clones contained dominant point mutations in Aurora B. Three mutations map to residues in the ATP-binding pocket that are distinct from the "gatekeeper" residue. The mutants retain wild-type catalytic activity and were resistant to all of the Aurora inhibitors tested. Our studies predict that drug-resistant Aurora B mutants are likely to arise during clinical treatment. Furthermore, because the plasticity of the ATP-binding pocket renders Aurora B insensitive to multiple inhibitors, our observations indicate that the drug-resistant Aurora B mutants should be exploited as novel drug targets.     

Molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza A virus M2

Influenza A virus M2 (A/M2) forms a homotetrameric proton selective channel in the viral membrane. It has been the drug target of antiviral drugs such as amantadine and rimantadine. However, most of the current virulent influenza A viruses carry drug-resistant mutations alongside the drug binding site, such as S31N, V27A, and L26F, etc., each of which might be dominant in a given flu season. Among these mutations, the V27A mutation was prevalent among transmissible viruses under drug selection pressure. Until now, V27A has not been successfully targeted by small molecule inhibitors, despite years of extensive medicinal chemistry research efforts and high throughput screening. Guided by molecular dynamics (MD) simulation of drug binding and the influence of drug binding on the dynamics of A/M2 from earlier experimental studies, we designed a series of potent spirane amine inhibitors targeting not only WT, but also both A/M2-27A and L26F mutants with IC(50)s similar to that seen for amantadine's inhibition of the WT channel. The potencies of these inhibitors were further demonstrated in experimental binding and plaque reduction assays. These results demonstrate the power of MD simulations to probe the mechanism of drug binding as well as the ability to guide design of inhibitors of targets that had previously appeared to be undruggable.     

Molecular insight into the T798M gatekeeper mutation-caused acquired resistance to tyrosine kinase inhibitors in ErbB2-positive breast cancer

Human epidermal growth factor receptor 2 (ErbB2) is an attractive therapeutic target for metastatic breast cancer. The kinase has been clinically observed to harbor a gatekeeper mutation T798M in its active site, which causes acquired resistance to the first-line targeted breast cancer therapy with small-molecule tyrosine kinase inhibitors. Previously, several theories have been proposed to explain the molecular mechanism of gatekeeper mutation-caused drug resistance, such as blocking of inhibitor binding and increasing of ATP affinity. In the current study, the direct binding of three wild type-selective inhibitors (Lapatinib, AEE788 and TAK-285) and two wild type-sparing inhibitors (Staurosporine and Bosutinib) to the wild-type ErbB2 and its T798M mutant are investigated in detail by using rigorous computational analysis and binding affinity assay. Substitution of the polar threonine with a bulky methionine at residue 798 can impair and improve the direct binding affinity of wild type-selective and wild type-sparing inhibitors, respectively. Hindrance effect is responsible for the affinity decrease of wild type-selective inhibitors, while additional nonbonded interactions contribute to the affinity increase of wild type-sparing inhibitors, thus conferring selectivity to the inhibitors for mutant over wild type. The binding affinity of Staurosporine and Bosutinib to ErbB2 kinase domain is improved by 11.9-fold and 2.1-fold upon T798M mutation, respectively. Structural analysis reveals that a nonbonded network of S–π contact interactions (for Staurosporine) or an S-involving halogen bond (for Bosutinib) forms with the sulfide group of mutant Met798 residue.     

HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants

The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.     

Prediction and analysis of binding affinities for chemically diverse HIV-1 PR inhibitors by the modified SAFE_p approach

One of the biggest challenges in the "in silico" screening of enzyme ligands is to have a protocol that could predict the ligand binding free energies. In our group we have developed a very simple screening function (referred to as solvent accessibility free energy of binding predictor, SAFE_p) which we have applied previously to the study of peptidic HIV-1 protease (HIV-1 PR) inhibitors and later to cyclic urea type HIV-1 PR inhibitors. In this work, we have extended the SAFE_p protocol to a chemically diverse set of HIV-1 PR inhibitors with binding constants that differ by several orders of magnitude. The resulting function is able to reproduce the ranking and in many cases the value of the inhibitor binding affinities for the HIV-1 PR, with accuracy comparable with that of costlier protocols. We also demonstrate that the binding pocket SAFE_p analysis can contribute to the understanding of the physical forces that participate in ligand binding. The analysis tools afforded by our protocol have allowed us to identify an induced fit phenomena mediated by the inhibitor and have demonstrated that larger fragments do not necessarily contribute the most to the binding free energy, an outcome partially brought about by the substantial role the desolvation penalty plays in the energetics of binding. Finally, we have revisited the effect of the Asp dyad protonation state on the predicted binding affinities.