FDA-approved Protease Inhibitors Research Articles

Examination of two different proteasome inhibitors in reactivating mutant human cystathionine β-synthase in mice.

Classic homocystinuria is an inborn error of metabolism caused mainly by missense mutations leading to misfolded and/or unstable human cystathionine β-synthase (CBS) protein, causing the accumulation of excess total homocysteine (tHcy) in tissues. Previously, it has been shown that certain missense containing human CBS proteins can be functionally rescued in mouse models of CBS deficiency by treatment with proteasome inhibitors. The rescue by proteasome inhibitors is thought to work both by inhibiting the degradation of misfolded CBS protein and by inducing the levels of heat-shock chaperone proteins in the liver. Here we examine the effectiveness of two FDA approved protease inhibitors, carfilzomib and bortezomib, on various transgenic mouse models of human CBS deficiency. Our results show that although both drugs are effective in inducing the liver chaperone proteins Hsp70 and Hsp27, and are effective in inhibiting proteasome function, bortezomib was somewhat more robust in restoring the mutant CBS function. Moreover, there was no significant correlation between proteasome inhibition and CBS activity, suggesting that some of bortezomib's effects are via other mechanisms. We also test the use of low-doses of bortezomib and carfilzomib on various mouse models for extended periods of time and find that while low-doses are less toxic, they are also less effective at restoring CBS function. Overall, these results show that while restoration of mutant CBS function is possible with proteasome inhibitors, the exact mechanism is complicated and it will likely be too toxic for long-term patient treatment.

629. High Efficacy of Bictegravir/Emtricitabine/Tenofovir Alafenamide (B/F/TAF) in African American Adults with HIV Including Those with Preexisting Resistance, Viral Blips, and Suboptimal Adherence

BackgroundBRAAVE 2020 demonstrated the efficacy of switching to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) among African American adults with suppressed HIV through Week (W) 48 (Figure 1). We present resistance, viral blips, adherence, and virologic outcomes through W72.Figure 1. BRAAVE 2020 study design (phase 3, randomized, open-label, multicenter [USA], active-controlled study) and virologic suppression at weeks 24 and 48 *Allowed 3rd agents: any FDA-approved protease inhibitor, nonnucleoside reverse transcriptase inhibitor (except etravirine), integrase strand transfer inhibitor (except bictegravir), or maraviroc.MethodsEnrollment criteria permitted NNRTI resistance (-R), PI-R, and certain NRTI-R (M184V/I allowed; K65R/E/N, ≥3 thymidine analog mutations [TAMs], or T69-insertions excluded) and excluded known primary INSTI-R. Preexisting drug resistance was assessed with historical genotypes and retrospective baseline proviral DNA genotyping. Adherence was calculated by pill count. Viral blips (transient HIV-1 RNA ≥50 copies/mL) and outcomes based on last available on-treatment HIV-1 RNA were assessed.Results489 participants received B/F/TAF and had ≥1 post-switch HIV-1 RNA measurement. Baseline genotypic data from cumulative historical and/or proviral genotypes were available for 96% (468/489) in protease/reverse transcriptase and 93% (453/489) in integrase. Preexisting NRTI-R, M184V/I, ≥1 TAMs, NNRTI-R, and PI-R were observed in 15% (68/468), 11% (50/468), 8% (36/468), 22% (101/468), and 13% (61/468), respectively. Primary INSTI-R was detected post-randomization in 2% (11/453); all remained in the study and were included in efficacy analyses. Through W72, 99% (486/489) of participants had HIV-1 RNA < 50 copies/mL at their last study visit, including all with baseline NRTI-R or INSTI-R (Figure 2). Mean frequency of viral blips was 1% per timepoint, and blips were not associated with virologic failure. 112 participants (23%) had < 95% adherence by pill count, 98% (110/112) of whom had HIV-1 RNA < 50 copies/mL at last visit, including 14 of 14 (100%) with < 80% adherence. No participant discontinued due to lack of efficacy or had treatment emergent resistance to study drugs.Figure 2. Virologic suppression by preexisting resistance, viral blips, and adherence ConclusionVirologic suppression was maintained through W72 of B/F/TAF treatment, including those with preexisting resistance, viral blips, and suboptimal adherence. Continued HIV suppression and absence of treatment-emergent resistance demonstrate the efficacy of B/F/TAF in African Americans regardless of adherence or preexisting resistance to NNRTIs, PIs, or non-tenofovir NRTIs.Disclosures Kristen Andreatta, MSc, Gilead Sciences, Inc (Employee, Shareholder) Michelle L. D'Antoni, PhD, Gilead Sciences (Employee, Shareholder)Gilead Sciences, Inc (Employee, Shareholder) Silvia Chang, Masters, Gilead Sciences, Inc (Employee, Shareholder) Aiyappa Parvangada, MS Computational Biology, Gilead Sciences, Inc (Employee, Shareholder) Ross Martin, PhD, Gilead Sciences, Inc (Employee, Shareholder) Christiana Blair, MS, Gilead Sciences, Inc (Employee, Shareholder) Sean E. Collins, MD, MS, Gilead Sciences, Inc (Employee, Shareholder) Kirsten L. White, PhD, Gilead Sciences, Inc (Employee, Shareholder)

Arjunetin as a promising drug candidate against SARS-CoV-2: molecular dynamics simulation studies

Stem and bark of the tree Terminalia arjuna Wight & Arn. (Combretaceae) has been documented to exhibit therapeutic properties like cardiotonic, anticancer, antiviral, antibacterial, antifungal, hypercholesterolemia, hypolipidemic, and anti-coagulant. Our previous studies have shown that, ethanolic extract of T. arjuna bark exhibits radical scavenging anti-oxidant activity and also effectively inhibited catalase activity. In this study, oleanane triterpenoids type compounds viz., oleanolic acid, arjunolic acid, arjunolitin, arjunetin were isolated from ethanolic bark extract as bio-active compound and their structures were elucidated using 1H, 13C NMR, HR-ESIMS, IR. Of the various compounds, Arjunetin showed significant inhibition of catalase activity as compared to the other compounds. Based on the structural similarity between arjunetin and current antiviral drugs, we propose that arjunetin might exhibit antiviral activity. Molecular docking and molecular dynamics studies showed that arjunetin binds to the binds to key targets of SARS-CoV-2 namely, 3CLpro, PLpro, and RdRp) with a higher binding energy values (3CLpro, −8.4 kcal/mol; PLpro, −7.6 kcal/mol and RdRp, −8.1 kcal/mol) as compared with FDA approved protease inhibitor drugs to Lopinavir (3CLpro, −7.2 kcal/mole and PLpro −7.7 kcal/mole) and Remdesivir (RdRp −7.6 kcal/mole). To further investigate this, we performed 200–500 ns molecular dynamics simulation studies. The results transpired that the binding affinity of Arjunetin is higher than Remdesivir in the RNA binding cavity of RdRp. Based on structural similarity between arjunetin and Saikosaponin (a known antiviral agents) and based on our molecular docking and molecular dynamic simulation studies, we propose that arjunetin can be a promising drug candidate against Covid-19. Communicated by Ramaswamy H. Sarma

Abstract 1164: Human endogenous retrovirus type K promotes proliferation of Merlin negative schwannoma and meningioma which can be inhibited by anti-retroviral and anti-TEAD drugs

Abstract Deficiency of the tumor suppressor Merlin causes the development of schwannoma, meningioma and ependymoma tumors. These can occur spontaneously or in the hereditary disease Neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Currently available treatments are surgery or radiosurgery which are only partially effective, with tumors frequently reoccurring and in case of meningioma often as a higher grade. There is an urgent need for effective drug treatments.In this study, we investigated the role of Human Endogenous Retrovirus Type K (HERV-K) and its targetability in Merlin negative schwannoma and meningioma tumors by using tissues and primary cells isolated form patients employing immunohistochemistry, immunocytochemistry, western blotting and proliferation assays. We demonstrate that HERV-K proteins are overexpressed in Merlin negative schwannoma and all meningioma grades. Furthermore, we implicate CRL4DCAF1 and YAPT/EAD Hippo pathway in this overexpression and suggest that exosome transport of the HERV-K Envelope (Env) protein might contribute to schwannoma development. We also show that: (i) Ectopic overexpression of HERV-K Env in normal Schwann cells increased proliferation and upregulated the transcription factor c-Jun. Env overexpression also increased activity of the extracellular signal-regulated kinase 1/2 (pERK1/2), a known mitogenic pathway in schwannoma. (ii) An anti-Env monoclonal antibody decreased pERK1/2 and reduced proliferation of schwannoma cells. iii) FDA-approved retroviral protease inhibitors Ritonavir, Atazanavir and Lopinavir decreased pERK1/2 and cyclin D1 and reduced proliferation of schwannoma and grade I meningioma cells. We provide evidence for HERV-K Env contributing to the development of NF2-associated schwannomas and meningiomas. Considering the urgent need of drugs to treat NF2, we suggest the trialling of antiretroviral protease inhibitors, and consideration of anti-HERV-K Env immunotherapy, as well as TEAD-specific inhibitors in these patients. The above treatments would also be potentially beneficial for patients with other tumors caused by Merlin deficiency. Citation Format: Sylwia Ammoun, Emmanuel A. Maze, Bora Agit, Robert Belshaw, C Oliver Hanemann. Human endogenous retrovirus type K promotes proliferation of Merlin negative schwannoma and meningioma which can be inhibited by anti-retroviral and anti-TEAD drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1164.

Single-Agent and Fixed-Dose Combination HIV-1 Protease Inhibitor Drugs in Fission Yeast (Schizosaccharomyces pombe).

Successful combination antiretroviral therapies (cART) eliminate active replicating HIV-1, slow down disease progression, and prolong lives. However, cART effectiveness could be compromised by the emergence of viral multidrug resistance, suggesting the need for new drug discoveries. The objective of this study was to further demonstrate the utility of the fission yeast cell-based systems that we developed previously for the discovery and testing of HIV protease (PR) inhibitors (PIs) against wild-type or multi-PI drug resistant M11PR that we isolated from an infected individual. All thirteen FDA-approved single-agent and fixed-dose combination HIV PI drugs were tested. The effect of these drugs on HIV PR activities was tested in pure compounds or formulation drugs. All FDA-approved PI drugs, except for a prodrug FPV, were able to suppress the wild-type PR-induced cellular and enzymatic activities. Relative drug potencies measured by EC50 in fission yeast were discussed in comparison with those measured in human cells. In contrast, none of the FDA-approved drugs suppressed the multi-PI drug resistant M11PR activities. Results of this study show that fission yeast is a reliable cell-based system for the discovery and testing of HIV PIs and further demonstrate the need for new PI drugs against viral multi-PI resistance.

Potential protease inhibitors and their combinations to block SARS-CoV-2

COVID-19, which has emerged recently as a pandemic viral infection caused by SARS-coronavirus 2 has spread rapidly around the world, creating a public health emergency. The current situation demands an effective therapeutic strategy to control the disease using drugs that are approved, or by inventing new ones. The present study examines the possible repurposing of existing anti-viral protease inhibitor drugs. For this, the structural features of the viral spike protein, the substrate for host cell protease and main protease of the available SARS CoV-2 isolates were established by comparing with related viruses for which antiviral drugs are effective. The results showed 97% sequence similarity among SARS and SARS-CoV-2 main protease and has same cleavage site positions and ACE2 receptor binding region as in the SARS–CoV spike protein. Though both are N-glycosylated, unlike SARS-CoV, human SARS-CoV-2 S-protein was O-glycosylated as well. Molecular docking studies were done to explore the role of FDA approved protease inhibitors to control SARS-CoV-2 replication. The results indicated that, Ritonavir has the highest potency to block SARS-CoV-2 main protease and human TMPRSS2, a host cell factor that aids viral infection. Other drugs such as Indinavir and Atazanavir also showed favourable binding with Cathepsin B/L that helped viral fusion with the host cell membrane. Further molecular dynamics simulation and MM-PBSA binding free energy calculations confirmed the stability of protein-drug complexes. These results suggest that protease inhibitors particularly Ritonavir, either alone or in combination with other drugs such as Atazanavir, have the potential to treat COVID 19. Communicated by Ramaswamy H. Sarma

Pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and FDA-approved inhibitors of SARS-CoV-2 main protease 3CLpro

The SARS-CoV-2 was confirmed to cause the global pandemic of coronavirus disease 2019 (COVID-19). The 3-chymotrypsin-like protease (3CLpro), an essential enzyme for viral replication, is a valid target to combat SARS-CoV and MERS-CoV. In this work, we present a structure-based study to identify potential covalent inhibitors containing a variety of chemical warheads. The targeted Asinex Focused Covalent (AFCL) library was screened based on different reaction types and potential covalent inhibitors were identified. In addition, we screened FDA-approved protease inhibitors to find candidates to be repurposed against SARS-CoV-2 3CLpro. A number of compounds with significant covalent docking scores were identified. These compounds were able to establish a covalent bond (C–S) with the reactive thiol group of Cys145 and to form favorable interactions with residues lining the substrate-binding site. Moreover, paritaprevir and simeprevir from FDA-approved protease inhibitors were identified as potential inhibitors of SARS-CoV-2 3CLpro. The mechanism and dynamic stability of binding between the identified compounds and SARS-CoV-2 3CLpro were characterized by molecular dynamics (MD) simulations. The identified compounds are potential inhibitors worthy of further development as COVID-19 drugs. Importantly, the identified FDA-approved anti-hepatitis-C virus (HCV) drugs paritaprevir and simeprevir could be ready for clinical trials to treat infected patients and help curb COVID-19. Communicated by Ramaswamy H. Sarma

Diversity of Mexican HIV-1 Protease Sequences.

Protease is one of three enzymes encoded within HIV's pol gene and a retroviral aspartyl-protease which unlike other aspartyl-proteases form a 22kDa homodimer to exert its activity. Protease is responsible for the cleavage of viral Gag-Pol polypeptide into mature viral proteins and a target of current anti-retroviral therapy (ART). Saquinavir was the first FDA-approved protease inhibitor. This image maps Shannon entropy levels of variable sites as derived from the study of 777 Mexican protease nucleotide sequences. Shannon entropy was calculated employing amino acid equivalents representing increases in entropy with regards to HXB2 reference. Entropy levels were arbitrarily classified as high (> 0.6), mid (between 0.6 and 0.2) and low (below 0.2). Protease sites having high and mid entropy levels were mapped into an X-ray diffraction crystal of HIV-1 protease dimer coupled to the anti-retroviral drug saquinavir. All sites exhibiting high and mid entropy affected the surface of the protein and were confined to an area located between the elbow and the 60's loop. High levels of entropy were observed in residues surrounding the 10's loop, the elbow region, the 60's loop and in the first half of the C-terminal domain. Protease sequence diversity is mostly a consequence of ART-driven selection for mutations that lower affinity for a drug, enabling viral replication in spite of continued ART use. Genetic variation continues to represent a major challenge for HIV prevention and treatment.

Targeting eukaryotic proteases for natural products-based drug development.

Covering: up to April 2020 Proteases are involved in the regulation of many physiological processes. Their overexpression and dysregulated activity are linked to diseases such as hypertension, diabetes, viral infections, blood clotting disorders, respiratory diseases, and cancer. Therefore, they represent an important class of therapeutic targets. Several protease inhibitors have reached the market and >60% of them are directly related to natural products, even when excluding synthetic natural product mimics. Historically, natural products have been a valuable and validated source of therapeutic agents, as over half of the marketed drugs across targets and diseases are inspired by natural product structures. In the past two decades the number of new protease inhibitors discovered from nature has sharply increased. Additionally, the availability of 3D structural information for proteases has permitted structure-based design and accelerated the synthesis of optimized lead structures with improved potency and selectivity profiles, resulting in some of the most-potent-in-class inhibitors. These discoveries were oftentimes maximized by in-depth biological assessments of lead inhibitors, linking them to a relevant disease state. This review will discuss some of the current and emerging drug targets and their involvement in various disease processes, highlighting selected success stories behind several FDA-approved protease inhibitors that have natural products scaffolds as well as recent selected pharmacologically well-characterized inhibitors derived from marine or terrestrial sources.

Molecular alteration in drug susceptibility against subtype B and C-SA HIV-1 proteases: MD study

HIV-1 protease (HIV PR) is one of the most promising targets for anti-HIV drug discovery. Extensive anti-HIV drug discovery research was focused on HIV-1 subtype B protease though FDA-approved protease inhibitors (PIs) generally exhibit altered biological activities against other protease subtypes such as South African subtype C (C-SA) protease that is prevalent in sub-Saharan Africa. Recent experimental reports highlight the differences in dynamic behaviors between these two protease subtypes. Herein, the dynamics of three FDA approved PIs, namely atazanavir (ATV), darunavir (DRV), and ritonavir (RTV), were thoroughly analyzed for both subtypes B and C protease complexes using molecular dynamic (MD) simulation in explicit solvent. The comparative MD post-analyses through flap dynamics, cross-correlation analyses, principal component analyses (PCA), per residue decomposition analyses, and MM-GBSA binding free energy analyses, revealed the altered responses observed in the complexes of these PIs in these two protease subtypes. The calculated theoretical binding energy analyses are consistent with the experiment to a considerable extent. Theoretical binding energies combined with flap dynamics appear to correspond to altered drug susceptibility against subtype B and C-SA HIV-1 proteases. The current analyses provide useful insight on the mechanism of action of PIs against these two different HIV-1 protease subtypes which will be helpful in developing new anti-protease compounds.

Kinetic and thermodynamic characterisation of HIV-protease inhibitors against E35D↑G↑S mutant in the South African HIV-1 subtype C protease

Herein, we report the effect of nine FDA approved protease inhibitor drugs against a new HIV-1 subtype C mutant protease, E35D↑G↑S. The mutant has five mutations, E35D, two insertions, position 36 (G and S), and D60E. Kinetics, inhibition constants, vitality, Gibbs free binding energies are reported. The variant showed a decreased affinity for substrate and low catalytic efficiency compared to the wild type. There was a significant decrease in the binding of seven FDA approved protease inhibitors against the mutant (p < .0001). Amprenavir and ritonavir showed the least decrease, but still significant reduced activity in comparison to the wildtype (4 and 5 folds, respectively, p = .0021 and .003, respectively). Nelfinavir and atazanavir were the worst inhibitors against the variant as seen from the IC50, with values of 1401 ± 3.0 and 685 ± 3.0 nM, respectively. Thermodynamics data showed less favourable Gibbs free binding energies for the protease inhibitors to the mutant.

Structural Analysis of Darunavir Analogs Against Primary Resistance Mutations in HIV Protease

Antiretroviral therapies (ARTs) involve combinations of small molecule inhibitors that suppress the viral load of Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Symptom (AIDS). ARTs, currently the only defense against HIV, target critical proteins necessary for viral replication, such as HIV‐1 protease. The most potent FDA approved protease inhibitor, darunavir (DRV), is still susceptible to resistance if the protease acquires mutations in and around the active site. One method to avoid resistance is to optimize the inhibitor to fit the consensus volume that the protease's natural substrates occupy. Using this strategy, DRV analogs with larger moieties at the P1’ position were designed and synthesized to leverage unexploited space and create additional protease‐inhibitor interactions, resulting in inhibitors that are more potent and less susceptible to resistance. To elucidate the molecular mechanisms of active site resistance mutations against these inhibitors, we used a multidisciplinary approach, including biochemical assays and x‐ray crystallography. Results suggest that modifications to the DRV scaffold result in inhibitors with potency comparable to DRV, but structural analysis highlights differences in intermolecular and intramolecular interactions, which will inform future drug design.Support or Funding InformationThis work was supported in part by a grant from the National Institute of General Medical Sciences, National Institutes of Health (P01‐GM109767).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors.

Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2' pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine FDA-approved HIV-1 protease inhibitors (PI) are active against HIV-2. The underlying reasons for intrinsic PI resistance in HIV-2 are not known. We examined the contributions of four amino acids in the ligand-binding pocket of the enzyme that differ between HIV-1 and HIV-2 by constructing HIV-2 clones encoding the corresponding HIV-1 amino acids and testing the PI susceptibilities of the resulting viruses. We found that the HIV-2 clone containing all four changes (PRΔ4) was as susceptible as HIV-1 to all nine PI. We also modeled the PRΔ4 enzyme structure and compared it to existing crystallographic structures of HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir. Our findings demonstrate that four positions in the ligand-binding cleft of protease are the primary cause of HIV-2 PI resistance.

Simeprevir and sofosbuvir: Two new drugs for chronic HCV infection

Simeprevir and sofosbuvir: Two new drugs for chronic HCV infection

Why is Substrate Peptide Binding Unsusceptible to Multidrug-Resistant Mutations in HIV-1 Protease? A Structural and Energetic Analysis

Understanding of the molecular mechanism and biological implication underlying the difference in binding of substrate peptides and small-molecule inhibitors to multidrug-resistant mutants of HIV-1 protease would help to develop new anti-HIV agents combating drug resistance. Here, an integration of rigorous quantum mechanics/molecular mechanics (QM/MM) analysis and empirical Poisson–Boltzmann/surface area (PB/SA) model is described to investigate the structural basis and energetic property of wild-type HIV-1 protease and its mutants in recognizing and binding with a wide variety of ligands, including the peptides derived from its cognate cleavage sites and the cleavage site variants as well as a number of FDA-approved protease inhibitors, attempting to explain why is substrate binding unsusceptible to most observed HIV-1 protease mutations. A preliminary test study demonstrates that the combined QM/MM–PB/SA scheme is able to effectively reproduce the relative ligand binding energy changes upon protease single- and double-mutations, albeit the absolute values appear to be different significantly between the calculated and experimental results. With the QM/MM–PB/SA calculations a complete mutation energy map of HIV-1 protease–ligand interactions is created, which unravels distinct affinity pictures of wild-type substrates, substrate variants and, particularly, the protease inhibitors bound to HIV-1 protease mutants, suggesting that, on the one hand, the evaluation pressure under anti-HIV chemotherapies addresses site-directed protease mutations that impair and undermine the intermolecular interactions specific to inhibitors but not substrates; on the other hand, co-evaluation of protease and its substrate peptides provides a more effective mechanism to avoid therapeutic surveillance. Further, nonbonded interaction analysis and computational alanine scanning reveal 12 key residues that is critical for substrate binding, from which the Asn25, Gly27, Ala28, Asp29 and Pro81 are identified that have not yet been found to cause drug resistance and hence would be the promising sites targeted by new protease inhibitors.

Comparative analysis of ER stress response into HIV protease inhibitors: Lopinavir but not darunavir induces potent ER stress response via ROS/JNK pathway

HIV protease inhibitor (PI)-induced ER stress has been associated with adverse effects. Although it is a serious clinical problem for HIV/AIDS patients, comparative analyses of ER stress induction by clinically used PIs have rarely been done. Especially, there is no report on the differential ER stress response between lopinavir (LPV) and darunavir (DRV), although these PIs are the most clinically used PIs. We show here that LPV induces the most potent CHOP expression, ER stress marker, among the 9 Food and Drug Administration (FDA)-approved PIs in human peripheral blood mononuclear cells, several human epithelial cells, and mouse embryonic fibroblasts. LPV induced the most potent ROS production and JNK activation in 9 PIs. A comparison among the most clinically used PIs, ritonavir (RTV), LPV, and DRV, revealed that LPV potently and RTV moderately but not DRV induced ER stress via ROS-dependent JNK activation rather than proteasome inhibition. Finally, we analyzed ER stress induction in tissues of mice intraperitoneally injected with RTV, LPV, and DRV. RTV and LPV but not DRV showed ER stress induction in several mice tissues. In conclusion, we first identify LPV as the most potent ER stress inducing PI among 9 FDA-approved PIs in human cells, and although clinical verification is necessary, we show here that DRV has the advantage of less ROS and ER stress induction potential compared with LPV in vitro and in vivo.

Inhibitor-induced conformational shifts and ligand-exchange dynamics for HIV-1 protease measured by pulsed EPR and NMR spectroscopy.

Double electron-electron resonance (DEER) spectroscopy was utilized to investigate shifts in conformational sampling induced by nine FDA-approved protease inhibitors (PIs) and a nonhydrolyzable substrate mimic for human immunodeficiency virus type 1 protease (HIV-1 PR) subtype B, subtype C, and CRF_01 A/E. The ligand-bound subtype C protease has broader DEER distance profiles, but trends for inhibitor-induced conformational shifts are comparable to those previously reported for subtype B. Ritonavir, one of the strong-binding inhibitors for subtypes B and C, induces less of the closed conformation in CRF_01 A/E. (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR (1)H-(15)N HSQC titration data show that inhibitor residence time in the protein binding pocket, inferred from resonance exchange broadening, shifting or splitting correlates with the degree of ligand-induced flap closure measured by DEER spectroscopy. These parallel results show that the ligand-induced conformational shifts resulting from protein-ligand interactions characterized by DEER spectroscopy of HIV-1 PR obtained at the cryogenic temperature are consistent with more physiological solution protein-ligand interactions observed by solution NMR spectroscopy.

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

Site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

Targeting the PI3K/AKT/Survivin Pathway with Ritonavir In Therapy-Resistant Mantle Cell Lymphoma

AbstractAbstract 2855Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma accounting for about 6% of non-Hodgkin's lymphoma cases in the US. While multiple therapy regimens are available to treat MCL, patients ultimately relapse within 3–4 years from therapy-resistant MCL, making MCL carry the worst prognosis of all non-Hodgkin's B cell lymphomas. To improve therapies for patients with MCL, we must understand the biological causes for relapse and therapy-resistance in MCL and develop mechanisms to target the unique properties in relapsing MCL. Recently, we have isolated and produced therapy-resistant MCL cell lines by inoculating NOD-SCID mice with a human MCL cell line, Granta-519 (GP), and subsequently treating with CHOP chemotherapy regimen plus bortezomib. When mice relapsed following therapy, tumor cells were isolated from the kidneys and livers, and cultured to produce stable cell lines, named GRK and GRL, respectively. GRK and GRL cells exhibited increased proliferative and therapy-resistant properties in vitro, and GRL showed increased aggressiveness in vivo also, as compared with GP. Using quantitative PCR (qRT-PCR) to assess the activation of pathways relating to lymphoma progression, the PI3K/Akt/survivin pathway was found to be differentially activated in GRL and GRK compared to GP, including a 6.5 fold increase in survivin in GRL. Therefore, we inhibited survivin in GRL using the FDA-approved protease inhibitor ritonavir (Abbott Laboratories), as recent studies suggest this compound is able to downregulate survivin in lymphoblastoid B cells (Dewan, et al. Int J Cancer 2009; 124[3]: 622–629). When GRL cells were incubated with ritonavir plus vincristine or doxorubicin, MTT assays showed significant inhibition of cell proliferation/survival (p < 0.05) compared to the effects of ritonavir alone. Also, flow cytometric analysis of Annexin V demonstrated dose-dependent induction of apoptosis upon ritonavir treatment in GRL. In addition, qRT-PCR analysis demonstrated a decrease in mRNA expression of the pro-survival genes cyclin D2, Rel A, survivin, and BCL2 in GP and GRL cells after ritonavir treatment. Together, these studies demonstrate the potentials of utilizing ritonavir in a combined treatment regimen designed to target therapy-resistant MCL. As ritonavir is already an FDA-approved drug, these studies hold promise for expediting future in vivo and clinical studies that may eliminate therapy-resistant cells responsible for relapse in MCL patients.(This research was supported by the Lymphoma Research Foundation, New York, NY). Disclosures:No relevant conflicts of interest to declare.

Molecular Basis for Drug Resistance in HIV-1 Protease

HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.