Effective estimation of the inhibitor affinity of HIV-1 protease via a modified LIE approach

The inhibition of the Human Immunodeficiency Virus Type 1 Protease (HIV-1 PR) can prevent the synthesis of new viruses. Computer-aided drug design (CADD) would enhance the discovery of new therapies, through which the estimation of ligand-binding affinity is critical to predict the most efficient inhibitor. A time-consuming binding free energy method would reduce the usefulness of CADD. The modified linear interaction energy (LIE) approach emerges as an appropriate protocol that performs this task. In particular, the polar interaction free energy, which is obtained via numerically resolving the linear Poisson–Boltzmann equation, plays as an important role in driving the binding mechanism of the HIV-1 PR + inhibitor complex. The electrostatic interaction energy contributes to the attraction between two molecules, but the vdW interaction acts as a repulsive factor between the ligand and the HIV-1 PR. Moreover, the ligands were found to adopt a very strong hydrophobic interaction with the HIV-1 PR. Furthermore, the results obtained corroborate the high accuracy and precision of computational studies with a large correlation coefficient value R = 0.83 and a small RMSE δ_RMSE = 1.25 kcal mol⁻¹. This method is less time-consuming than the other end-point methods, such as the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) and free energy perturbation (FEP) approaches. Overall, the modified LIE approach would provide ligand-binding affinity with HIV-1 PR accurately, precisely, and rapidly, resulting in a more efficient design of new inhibitors.

The inhibition of the Human Immunodeficiency Virus Type 1 Protease (HIV-1 PR) can prevent the synthesis of new viruses.     

Directed HIV-1 evolution of protease inhibitor resistance by second-generation short hairpin RNAs

Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches.     

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K_i = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4⁺ T cells (50% effective concentration [EC₅₀] = 3.4 to 11.5 nM), and macrophages (EC₅₀ = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC₅₀s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.Fifteen years ago, HIV protease (PR) inhibitors (PIs) were introduced into the clinic as a second class of antiretrovirals, after nucleosides, and launched the era of combination antiretroviral therapy (ART) that brought along a dramatic reduction of the morbidity and mortality among HIV-infected patients (7, 25, 29, 46). PIs evolved to be an important class of agents that are being widely used in combination with other antiretrovirals in both treatment-naïve and -experienced patients (48). On the basis of recent revisions of HIV treatment guidelines, one of several ritonavir-boosted PIs is recommended for use as a third agent of choice in combination with tenofovir and emtricitabine for first-line ART (8, 53). The choice of PIs over other antiretroviral agents is primarily driven by their clinical potency and a higher genetic barrier for resistance development (48). In addition, the clinical use of more recently developed PIs with improved resistance profiles, e.g., darunavir, in combination with new antiretrovirals may represent a promising nucleoside-sparing option for highly treatment-experienced patients (13, 50).Although a total of nine PIs is currently available for the treatment of HIV infection, only a few are widely used. In general, the long-term clinical benefit of PIs across all patient populations can be limited by various factors, including long-term safety and tolerability (3, 27, 38), resistance (36), and drug-drug interactions (18). Among these limitations, the development of viral resistance has been shown to be a major cause of therapy failure (1, 43). Several studies revealed that a significant proportion of patients with detectable viral loads harbor HIV strains resistant to at least one PI (36, 44). Furthermore, transmission of resistant viruses, including strains with reduced susceptibility to approved PIs, has been documented and may limit the choices for the first-line therapy (17, 47). The structural similarity among the multiple PIs used in the clinic increases the possibility of cross-resistance within this therapeutic class (52). Consequently, the mutations conferring resistance are frequently common to multiple PIs (14, 45). Therefore, the design of novel PIs with more favorable resistance profiles and improved pharmacological properties remains an area of high interest.Over more than 2 decades of intense development, HIV therapy became a complex and quickly evolving field of medical research. Novel therapeutic concepts and regimens using both the established and new antiretroviral drug classes are being explored with a primary goal to address limitations of ART in various patient populations. Increasing age of HIV-infected patients brings about additional challenges, such as long-term effects of HIV infection and tolerability of ART (15). In addition, ongoing health care reforms generate pressure to reduce treatment costs, providing an incentive for exploring novel simplified treatment regimens. These evolving aspects of anti-HIV therapy create additional need for potent, durable, and well-tolerated antiretrovirals, including novel PIs.Being aware of both the unique qualities and limitations of PIs, we explored the design of novel compounds through the application of novel modifications to established PI chemotypes. Recently, we reported on inhibitors containing a phosphonate moiety and showed that in comparison with the parent scaffolds, the phosphonate-modified compounds exhibit improved activity against a limited panel of PI-resistant viruses (4). Based on these initial data, we further explored a range of di(alkyl)phosphonate substituents in the structural context of TMC-126, a previously described bis-tetrahydrofuran (bis-THF) peptidomimetic PI (9, 10). Here and in a parallel report (12) we describe the profiling of GS-8374 (Fig. ​(Fig.1),1), a novel diethylphosphonate derivative of TMC-126 that exhibits favorable pharmacological properties, including a resistance profile superior to the profiles of all clinically approved PIs.Open in a separate windowFIG. 1.Structure of GS-8374.     

Effect of natural polymorphisms in the HIV-1 CRF02_AG protease on protease inhibitor hypersusceptibility

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.     

Evolution of primary protease inhibitor resistance mutations during protease inhibitor salvage therapy

In order to track the evolution of primary protease inhibitor (PI) resistance mutations in human immunodeficiency virus type 1 (HIV-1) isolates, baseline and follow-up protease sequences were obtained from patients undergoing salvage PI therapy who presented initially with isolates containing a single primary PI resistance mutation. Among 78 patients meeting study selection criteria, baseline primary PI resistance mutations included L90M (42% of patients), V82A/F/T (27%), D30N (21%), G48V (6%), and I84V (4%). Despite the switching of treatment to a new PI, primary PI resistance mutations present at the baseline persisted in 66 of 78 (85%) patients. D30N persisted less frequently than L90M (50% versus 100%, respectively; P < 0.001) and V82A/F/T (50% versus 81%, respectively; P = 0.05). HIV-1 isolates from 38 (49%) patients failing PI salvage therapy developed new primary PI resistance mutations including L90M, I84V, V82A, and G48V. Common combinations of primary and secondary PI resistance mutations after salvage therapy included mutations at amino acid positions 10, 82, and 46 and/or 54 in 16 patients; 10, 90, and 71 and/or 73 in 14 patients; 10, 73, 84, 90, and 46 and/or 54 in 5 patients; 10, 48, and 82 in 5 patients; and 30, 88 and 90 in 5 patients. In summary, during salvage PI therapy, most HIV-1 isolates with a single primary PI resistance mutation maintained their original mutations, and 49% developed additional primary PI resistance mutations. The persistence of L90M, V82A/F/T, G48V, and I84V during salvage therapy suggests that these mutations play a role in clinical resistance to multiple PIs.     

未接受蛋白酶抑制剂治疗者的HIV-1基因的多态性及蛋白酶抑制剂耐药相关性变异

目的:研究未接受蛋白酶抑制剂(PIs)治疗的人类获得性免疫缺陷病毒1(HIV-1)感染者HIV-1多态性以及PIs耐药相关性变异的分布情况.方法:取自中国4个省市(上海、安徽、河南和云南)81例未接受PIs治疗的HIV-1感染者中分离的病毒株,经反转录聚合酶链反应(RT-PCR)扩增HIV-1病毒的pol基因,并进行测序及进化分析.结果:HIV-1病毒株基因最多的为B'亚型(82.7%),其他分别为CRF01-AE亚型(11%)、CRF07_BC或CRF08_BC亚型(3.7%)、CRF02-AG亚型(1.2%)以及B亚型(1.2%).B'亚型主要存在于有偿助血员以及经输血感染的患者中.R41K、K70Q/R/E和L89M变异在非B亚型中比在B'和B亚型中更为常见.次要PIs耐药相关性变异I15M/V、K20M/R/I、M361和H69K在非B亚型中比在B和B'亚型中更为常见.相对于非B亚型,次要PIs耐药相关性变异I62V、L63P、A71V/T、V77I和193L则在B和B'亚型中更为常见.在3例(3.7%)感染B亚型的患者中发现了主要PIs耐药相关性变异M46I.结论:在未接受PIs治疗的患者中,HIV-1病毒基因多态性及次要PIs耐药相关性变异的出现率较高.而主要PIs耐药相关性变异的流行率则较低.     

Ahmpatinin iBu,a new HIV-1 protease inhibitor,from Streptomyces sp. CPCC 202950

Ahmpatinin ⁱBu (1) and statinin ⁱBu (2), two new linear peptides, a novel pyrrolidine derivative, (−)-(S)-2-[3-(6-methylheptanamido)-2-oxopyrrolidin-1-yl] acetic acid (3), and three known pepstatin derivatives (4–6) along with their corresponding methanolysis artifacts (7–9) were isolated from Streptomyces sp. CPCC 202950. Their structures were elucidated on the basis of extensive spectroscopic data using Marfey''s analysis, chiral-phase HPLC, and ECD and OR calculation to determine the absolute configurations. Compound 1 contains an unusual amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)pentanoic acid (Ahmppa), and 3 is the first natural product with a 2-(3-amino-2-oxopyrrolidin-1-yl)acetic acid system. Compounds 1, 2, and 4–9 are HIV-1 protease inhibitors. In particular, ahmpatinin ⁱBu (1) exhibits significant inhibitory activity against HIV-1 protease with an IC₅₀ value of 1.79 nM. A preliminary structure–activity relationship is discussed.

Ahmpatinin ⁱBu and statinin ⁱBu, two new linear peptides, were isolated from Streptomyces sp. CPCC 202950. Ahmpatinin ⁱBu exhibited significant inhibitory activity against HIV-1 protease with an IC₅₀ value of 1.79 nM.     

Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection

The distribution of secretory leukocyte protease inhibitor (SLPI) at entry portals indicates its involvement in defending the host from pathogens, consistent with the ability of SLPI to inhibit human immunodeficiency virus (HIV)-1 infection by an unknown mechanism. We now demonstrate that SLPI binds to the membrane of human macrophages through the phospholipid-binding protein, annexin II. Based on the recent identification of human cell membrane phosphatidylserine (PS) in the outer coat of HIV-1, we define a novel role for annexin II, a PS-binding moiety, as a cellular cofactor supporting macrophage HIV-1 infection. Moreover, this HIV-1 PS interaction with annexin II can be disrupted by SLPI or other annexin II-specific inhibitors. The PS-annexin II connection may represent a new target to prevent HIV-1 infection.     

Prevalence of darunavir resistance mutations in HIV-1-infected patients failing other protease inhibitors

BACKGROUND: To estimate to what extent darunavir might be effective in patients failing distinct protease inhibitors (PIs), the genotypic resistance scores recently reported for the drug were examined in a large clinical HIV-1 drug resistance database. METHODS: All clinical specimens from HIV-infected patients failing PI-based regimens referred for drug resistance testing between 1999 and 2007 to a reference centre in Madrid were analysed. Darunavir-specific resistance mutations listed by the September 2006 IAS-USA panel update were considered. RESULTS: A total of 1021 genotypes from patients failing lopinavir (39.2%), nelfinavir (28.1%), saquinavir (14.5%), indinavir (13.7%), atazanavir (6.6%), fosamprenavir (5.3%) and tipranavir (1.1%) were identified. The prevalence of major darunavir resistance mutations was I50V 2.1%, I54M 1.3%, L76V 2.7% and I84V 14.5%. For minor darunavir resistance mutations, the rates were V11I 3.3%, V32I 3.9%, L33F 11%, I47V 2.1%, I54L 2.3%, G73S 12.8% and L89V 2.4%. Overall, 6.7% (n = 68) of the genotypes had three or more darunavir resistance mutations, which corresponded to a mean total number of PI resistance mutations of 12.3 +/- 1.9. In the multivariate analysis, prior fosamprenavir failure, prior saquinavir failure, the total number of PI resistance mutations and the number of prior PIs used were all independently associated with having more darunavir resistance mutations. CONCLUSIONS: The prevalence of darunavir resistance mutations is low in patients failing other PI-based regimens, although prior failure to amprenavir and saquinavir might produce more cross-resistance to darunavir. Thus, darunavir may be a good option for patients who have failed other PI-based regimens.