Cleavage Of Polypeptide Research Articles

Molecular docking and molecular dynamic simulation-based phytoconstituents against SARS-CoV-2 with dual inhibition of the primary protease targets

A novel coronavirus has caused major health problems and is spreading globally. The main protease enzyme plays a significant role in the number of copies of ss-RNA produced during the proteolytic cleavage of polypeptides. This work aims to find possible dual inhibitors of the 3-Chymotrypsin-like proteases PDB-6W63 and 6LU7 which increase efficiency and faster inhibition activity. By using an in-silico technique, polyphenols are molecularly docked against these targets to inhibit protease enzymes. Some polyphenols, such as pelargonidin and naringin, have significant dual inhibition characteristics with remarkable binding affinities with active scaffolds of both proteins, which have important ADMET parameters. These organic molecules are strongly bonded with amino acids of protein via mostly hydrogen bonding. These polyphenols also have outstanding docking scores and MMGBSA energies. The validity of the docking score was evaluated using a molecular dynamics simulation that assessed the stability of the complex. With the aid of computer-aided drug design, we hypothesise that the dual inhibition of compounds pelargonidin and naringin could effectively and potentially oppose SARS-CoV-2.

Drug development targeting SARS-CoV-2 main protease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic with more than 6.5 million deaths since 2019. Although a number of vaccines significantly reduced the mortality rate, a large number of the world population is yet being infected with highly contagious omicron variants/subvarints. Additional therapeutic interventions are needed to reduce hospitalization and curb the ongoing pandemic. The activity of the SARS-CoV-2 enzyme; chymotrypsin-like main protease (Mpro) is essential for the cleavage of viral nonstructural polypeptides into individual functional proteins and therefore Mpro is an attractive drug target. The aim of this review is to summarize recent progress toward the development of therapeutic drugs against Mpro protease.

Novel Protease-Mediated Double Antigen Recognizing Chimeric Antigen Receptor (CAR) Enhances Directionality of CAR-T Cell Activity and Improves Target Cell Specificity

Recently, Chimeric Antigen Receptor (CAR)-T cell therapies show excellent therapeutic effect against various hematologic malignancies, including B-cell leukemia, B-cell lymphoma, and multiple myeloma. Expansion of target diseases of this attractive immunotherapy is one of the most important issues. To identify the novel specific tumor antigen as CAR-T cell target is the standard way to accomplish this goal. However, recent CAR-T cells recognize single tumor antigen, so normal tissues expressing the target antigen are possible to be attacked by CAR-T cells. For example, anti-CD19 CAR-T cells against B-cell malignancies kill not only tumor cells but also normal B-cells, so patients receiving the CAR-T cells showed severe immune deficiency. Improvement of target cell recognition system of CAR-T cells minimizes this adverse effect and contributes to expand target diseases. To improve target-cell-specificity of CAR-T cell, we established a protease-mediated regulatory CAR, named "Scissors-CAR" (panel A). This regulatory CAR is constituted with extracellular antigen recognition portion and cytoplasmic Human Immunodeficiency Virus protease (HIVPR) catalytic domain. When CAR-T cells expressing both anti-A Scissors-CAR and anti-B Counterpart-CAR that has HIVPR recognition polypeptide contact with target tumor expressing both antigen-A and B, these two CARs interact on cell membrane. This interaction induces Scissors-CAR-mediated cleavage of target polypeptide and structural change of Counterpart-CAR. Previously, we reported double antigen recognition system using Scissors-CAR. Previous Counterpart-CAR named "Signal-CAR" had HIVPR recognition polypeptide upstream of CD3-zeta, so Scissors-CAR-mediated cleavage broke the CAR protein structure and suppressed CAR-T cell activity (panel B, named "Negative system"). This system allowed CAR-T cells to directional attack against target cells expressing only antigen-A and protect the cells expressing both antigen-A and B. Here, we established another double-antigen recognition system using Scissors-CAR. In many cases including both hematologic malignancies and solid tumors, additional ectopic antigens are expressed on malignant cells. If CAR-T cells empowered to directional attack against target cells expressing both antigen-A and B, much more tumors can be treated with this tumor immunotherapy (panel C, named "Positive system"). To establish this directional CAR-T cells, we constructed novel Counterpart-CAR named as "Activating-CAR" constituted with target recognition portion, co-stimulatory domain, CD3-zeta, and protein destabilization motif known as degron. Between CD3-zeta and degron motif, HIVPR recognition polypeptide was inserted. In vitro analysis revealed that this degron motif destabilized Activating-CAR and HIVPR-mediated cleavage and that release of degron motif increased Activating-CAR stability. Furthermore, cell proliferation of Jurkat cells expressing both anti-CD19 Activating-CAR and anti-HER2 Scissors-CAR was enhanced co-cultivated with target cells expressing both CD19 and HER2. From these results, combined use of Activating-CAR and Scissors-CAR allowed CAR-T cells to directionally activate against target cells expressing two antigens recognized by each CAR. By choosing the Counterpart-CAR from "Signal-CAR" or "Activating-CAR", this protease-mediated double antigen recognition system could optimize target-cell-specificity of CAR-T cells for the individual tumor and contribute to expand the target tumors of CAR-T cell therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Easy access to α-ketoamides as SARS-CoV-2 and MERS Mpro inhibitors via the PADAM oxidation route

SARS-CoV-2 caused worldwide the current outbreak called COVID-19. Despite multiple countermeasures implemented, there is an urgent global need for new potent and efficient antiviral drugs against this pathogen. In this context, the main protease (Mpro) of SARS-CoV-2 is an essential viral enzyme and plays a pivotal role in viral replication and transcription. Its specific cleavage of polypeptides after a glutamine residue has been considered as a key element to design novel antiviral drugs. Herein, we reported the design, synthesis and structure-activity relationships of novel α-ketoamides as covalent reversible inhibitors of Mpro, exploiting the PADAM oxidation route. The reported compounds showed μM to nM activities in enzymatic and in the antiviral cell-based assays against SARS-CoV-2 Mpro.In order to assess inhibitors’ binding mode, two co-crystal structures of SARS-CoV-2 Mpro in complex with our inhibitors were solved, which confirmed the covalent binding of the keto amide moiety to the catalytic Cys145 residue of Mpro. Finally, in order to interrogate potential broad-spectrum properties, we assessed a selection of compounds against MERS Mpro where they showed nM inhibitory potency, thus highlighting their potential as broad-spectrum coronavirus inhibitors.

Parthenolide reveals an allosteric mode to inhibit the deISGylation activity of SARS-CoV‑2 papain-like protease.

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target. Here, we show that parthenolide, a germacrane sesquiterpene lactone, has SARS-CoV-2 PLpro inhibitory activity. Parthenolide covalently binds to Cys-191 or Cys-194 of the PLpro protein, but not the Cys-111 at the PLpro catalytic site. Mutation of Cys-191 or Cys-194 reduces the activity of PLpro. Molecular docking studies show that parthenolide may also form hydrogen bonds with Lys-192, Thr-193, and Gln-231. Furthermore, parthenolide inhibits the deISGylation but not the deubiquitinating activity of PLpro in vitro. These results reveal that parthenolide inhibits PLpro activity by allosteric regulation.

A human coronavirus OC43-derived polypeptide causes neuropathic pain.

Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV‐OC43 p65‐like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP‐derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV‐OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception‐, immune‐, and cellular energy‐related activities. Affinity capture shows the association of OC43p with an Na+/K+‐transporting ATPase, providing a potential direct target and mechanistic insight into virus‐induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV‐OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus‐induced neuropathic pain.

In Silico and In Vitro Identification of Pan-Coronaviral Main Protease Inhibitors from a Large Natural Product Library.

The main protease (Mpro or 3CLpro) in coronaviruses represents a promising specific drug target as it is essential for the cleavage of the virus polypeptide and has a unique cleavage site that does not exist in human host proteases. In this study, we explored potential natural pan-coronavirus drugs using in vitro and in silico approaches and three coronavirus main proteases as treatment targets. The PyRx program was used to screen 39,442 natural-product-like compounds from the ZINC database and 121 preselected phytochemicals from medicinal plants with known antiviral activity. After assessment with Lipinski’s rule of five, molecular docking was performed for the top 33 compounds of both libraries. Enzymatic assays were applied for the top candidates from both in silico approaches to test their ability to inhibit SARS-CoV-2 Mpro. The four compounds (hypericin, rosmarinic acid, isorhamnetin, and luteolin) that most efficiently inhibited SARS-CoV-2 Mpro in vitro were further tested for their efficacy in inhibiting Mpro of SARS-CoV-1 and MERS-CoV. Microscale thermophoresis was performed to determine dissociation constant (Kd) values to validate the binding of these active compounds to recombinant Mpro proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. The cytotoxicity of hypericin, rosmarinic acid, isorhamnetin, and luteolin was assessed in human diploid MRC-5 lung fibroblasts using the resazurin cell viability assay to determine their therapeutic indices. Sequence alignment of Mpro of SARS-CoV-2 demonstrated 96.08%, 50.83%, 49.17%, 48.51%, 44.04%, and 41.06% similarity to Mpro of other human-pathogenic coronaviruses (SARS-CoV-1, MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKU1, and HCoV-229E, respectively). Molecular docking showed that 12 out of 121 compounds were bound to SARS-CoV-2 Mpro at the same binding site as the control inhibitor, GC376. Enzyme inhibition assays revealed that hypericin, rosmarinic acid, isorhamnetin, and luteolin inhibited Mpro of SARS-CoV-2, while hypericin and isorhamnetin inhibited Mpro of SARS-CoV-1; hypericin showed inhibitory effects toward Mpro of MERS-CoV. Microscale thermophoresis confirmed the binding of these compounds to Mpro with high affinity. Resazurin assays showed that rosmarinic acid and luteolin were not cytotoxic toward MRC-5 cells, whereas hypericin and isorhamnetin were slightly cytotoxic. We demonstrated that hypericin represents a potential novel pan-anti-coronaviral agent by binding to and inhibiting Mpro of several human-pathogenic coronaviruses. Moreover, isorhamnetin showed inhibitory effects toward SARS-CoV-2 and SARS-CoV-1 Mpro, indicating that this compound may have some pan-coronaviral potential. Luteolin had inhibitory effects against SARS-CoV-2 Mpro.

Support Vector Machine as a Supervised Learning for the Prioritization of Novel Potential SARS-CoV-2 Main Protease Inhibitors.

In the last year, the COVID-19 pandemic has highly affected the lifestyle of the world population, encouraging the scientific community towards a great effort on studying the infection molecular mechanisms. Several vaccine formulations are nowadays available and helping to reach immunity. Nevertheless, there is a growing interest towards the development of novel anti-covid drugs. In this scenario, the main protease (Mpro) represents an appealing target, being the enzyme responsible for the cleavage of polypeptides during the viral genome transcription. With the aim of sharing new insights for the design of novel Mpro inhibitors, our research group developed a machine learning approach using the support vector machine (SVM) classification. Starting from a dataset of two million commercially available compounds, the model was able to classify two hundred novel chemo-types as potentially active against the viral protease. The compounds labelled as actives by SVM were next evaluated through consensus docking studies on two PDB structures and their binding mode was compared to well-known protease inhibitors. The best five compounds selected by consensus docking were then submitted to molecular dynamics to deepen binding interactions stability. Of note, the compounds selected via SVM retrieved all the most important interactions known in the literature.

In silico structure-based discovery of a SARS-CoV-2 main protease inhibitor.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel lineage B betacoroanvirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant mortality, morbidity, and socioeconomic disruptions worldwide. Effective antivirals are urgently needed for COVID-19. The main protease (Mpro) of SARS-CoV-2 is an attractive antiviral target because of its essential role in the cleavage of the viral polypeptide. In this study, we performed an in silico structure-based screening of a large chemical library to identify potential SARS-CoV-2 Mpro inhibitors. Among 8,820 compounds in the library, our screening identified trichostatin A, a histone deacetylase inhibitor and an antifungal compound, as an inhibitor of SARS-CoV-2 Mpro activity and replication. The half maximal effective concentration of trichostatin A against SARS-CoV-2 replication was 1.5 to 2.7µM, which was markedly below its 50% effective cytotoxic concentration (75.7µM) and peak serum concentration (132µM). Further drug compound optimization to develop more stable analogues with longer half-lives should be performed. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of SARS-CoV-2.

Conserved interactions required for inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C‐like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50 µM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.

Computational investigation of binding of chloroquinone and hydroxychloroquinone against PLPro of SARS-CoV-2

Novel coronavirus SARS-CoV-2 has infected 18 million people with 700,000+ mortalities worldwide and this deadly numeric figure is rapidly rising. With very few success stories, the therapeutic targeting of this epidemic has been mainly attributed to main protease (Mpro), whilst Papain-like proteases (PLpro) also plays a vital role in the processing of replicase polyprotein. Multifunctional roles of PLpro such as viral polypeptide cleavage, de-ISGlyation and immune suppression have made it a promising drug target for therapeutic interventions. Whilst there have been a number of studies and others are on-going on repurposing and new-small molecule screening, albeit previously FDA approved drugs viz. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have only been found effective against this pandemic. Inspired by this fact, we have carried out molecular docking and dynamics simulation studies of FDA approved CQ and HCQ against SARS-CoV-2 PLpro. The end aim is to characterise the binding mode of CQ and HCQ and identify the key amino acid residues involved in the mechanism of action. Further, molecular dynamics simulations (MDS) were carried out with the docked complex to search for the conformational space and for understanding the integrity of binding mode. We showed that the CQ and HCQ can bind with better binding affinity with PLpro as compared to reference known PLpro inhibitor. Based on the presented findings, it can be anticipated that the SARS-CoV-2 PLpro may act as molecular target of CQ and HCQ, and can be projected for further exploration to design potent inhibitors of SARS-CoV-2 PLpro in the near future.

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

2039-P: Plasmin-Mediated Cleavage of Human Islet Amyloid Polypeptide (hIAPP) Protects ß-Cells from Amyloid-Induced Toxicity

Islet amyloid deposition and reduced ß-cell mass are pathologic hallmarks of type 2 diabetes. Amyloid deposits contain the 37 amino acid hIAPP, aggregation of which is toxic to ß cells. The 20-29 amino acid region of the peptide is known to confer its amyloidogenic potential. We previously reported that plasmin, a fibrinolytic protease, cleaves hIAPP thereby inhibiting fibril formation. We now sought to determine whether hIAPP fragments arising from plasmin-mediated cleavage retain the potential to aggregate and induce cytotoxicity. The ß-cell line INS-1 was treated for 24 h with full-length hIAPP (20 µM) alone or in the presence of plasmin (0.4 µM); plasmin alone or buffer were used as controls. Exposure to hIAPP alone decreased cell viability (53±1.4% of buffer, p<0.05, n=5). Addition of plasmin to hIAPP restored cell viability (90±4.3% of buffer, p=0.3 vs. buffer and p<0.05 vs. hIAPP alone, n=5), while treatment with plasmin alone had no effect (102±2.9% of buffer, p=0.7, n=5). LC/MS analysis of hIAPP cleavage products showed that plasmin induced a rapid decrease in the abundance of full-length hIAPP and appearance of the hIAPP 1-11 and 12-37 fragments. Since the hIAPP 12-37 contains the amyloidogenic region, we compared the amyloidogenic and cytotoxic properties of this fragment to full-length hIAPP. In a thioflavin T assay, hIAPP 12-37 was less amyloidogenic than full-length hIAPP. As expected, treatment of INS-1 cells for 24 h with full-length hIAPP (0-60 µM) reduced cell viability in a dose-dependent manner (67±1.3%, 30±0.4% and 16±0.7% of buffer, at 20, 40 and 60 µM respectively; p<0.05 vs. buffer for all, n=2), while hIAPP 12-37 was not toxic at any dose tested (90±3.2%, 90±3.3% and 108±3.5% of buffer, at 20, 40 and 60 µM respectively; n=2). In summary, plasmin protects ß cells from hIAPP-induced toxicity by cleaving hIAPP between amino acids 11 and 12. Thus, increasing islet plasmin activity might be a strategy to limit ß-cell loss in type 2 diabetes. Disclosure N. Esser: None. M.F. Hogan: None. A.T. Templin: None. J.J. Castillo: None. D. Raleigh: None. J.S. Edgar: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. R.L. Hull: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc. Funding U.S. Department of Veterans Affairs (I01BX001060)

Differential cleavage of viral polypeptides by allotypic variants of granzyme B skews immunity to mouse cytomegalovirus

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

Recombinant Production of Opiorphin Pentapeptide as Tandem Multimers Through Rational Design of Primers

Opiorphin secreted into the human saliva is an endogenous regulator pentapeptide (QRFSR) showing a pain suppressant effect similar to morphine. We report here a strategy of producing tandemly repeated multimers of opiorphin peptide efficiently by expressing in Esherichia coli cells. For improving the expression level and increasing the yield of pentapeptide, 32-mer tandem sequence of opiorphin was generated by using partially complementary primers inserted into pGEX-2T expression vector, following fused with glutathione transferase (GST) gene. Recombinant tandem multimer was easily captured by GST column with a yield of 9.2 mg/L, and then 32-mer opiorphin polypeptide of 28.49 kDa size was cleaved into monomers by CNBr and endoproteinase Asp-N. Opiorphin polypeptides having a molecular weight of about 693 Da and containing 5 amino acids in length were then generated. The amount of cleaved and purified recombinant opiorphin monomers was calculated as 6.2 mg/L by the Bradford assay. Protein expression and cleavage of recombinant polypeptide were confirmed by SDS-PAGE and TLC. Results from this study clearly open a door to the mass production of the opiorphin peptide to use for laboratory research and industrial purposes. The overall gene construction and the direct cloning to an expression vector strategy using partially complementary primers with cleavable linker peptides could be applicable to many small peptides and their analogs for pharmaceutical and clinical application.

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.

Influenza A virus-induced host caspase and viral PA-X antagonize the antiviral host factor, histone deacetylase 4

Influenza A virus (IAV) effectively manipulates host machinery to replicate. There is a growing evidence that an optimal acetylation environment in the host cell is favorable to IAV proliferation and vice versa. The histone deacetylases (HDACs), a family of 18 host enzymes classified into four classes, are central to negatively regulating the acetylation level, hence the HDACs would not be favorable to IAV. Indeed, by using the RNAi and overexpression strategies, we found that human HDAC4, a class II member, possesses anti-IAV properties and is a component of host innate antiviral response. We discovered that IAV multiplication was augmented in HDAC4-depleted cells and abated in HDAC4-supplemented cells. Likewise, the expression of IFITM3, ISG15, and viperin, some of the critical markers of host anti-IAV response was abated in HDAC4-depleted cells and augmented in HDAC4-supplemented cells. In turn, IAV strongly antagonizes the HDAC4, by down-regulating its expression both at the mRNA level via viral RNA endonuclease PA-X and at the polypeptide level by inducing its cleavage via host caspase 3 in infected cells. Such HDAC4 polypeptide cleavage resulted in a ∼30 kDa fragment that is also observed in some heterologous systems and may have a significant role in IAV replication.

Mexican HIV-1 Protease Sequence Diversity.

Protease is one of three enzymes encoded within HIV's pol gene, responsible for the cleavage of viral Gag-Pol polypeptide into mature viral proteins and a target of current anti-retroviral therapy. Protease diversity analysis in Latin America has been lacking in spite of extensive studies of protease-inhibitor resistance mutations. We studied the diversity of 777 Mexican protease sequences and found that all were subtype B except one (CRF02_AG). Phylogenetic analysis suggested the existence of six different clades with geospecific contributions. Thirty-three percent of sites were conserved, 25% had conservative substitutions, and 41% exhibited physicochemical changes. The most conserved regions surrounded the active site, most of the flap domain, and a region between the 60's loop and C-terminal triad. A single sequence exhibited an active site mutation (T26S). Variable sites were mapped to a crystallographic structure, providing further insight into the distribution and functional relevance of variable sites among Mexican isolates.

Method to identify efficiently cleaved, membrane-bound, functional HIV-1 (Human Immunodeficiency Virus-1) envelopes

An ideal vaccine against HIV-1 will specifically elicit bNAbs (broadly neutralizing antibodies) which can cross-neutralize a wide spectrum of circulating viral strains belonging to different clades. The current paradigm for developing such a vaccine is to generate HIV-1 envelope (Env)-based immunogens which can specifically elicit bNAbs. For this purpose, it is necessary to identify Envs, belonging to different clades, suitable for immunogen design. Efficient cleavage of the HIV-1 Env precursor gp160 polypeptide into its constituent subunits determines its ability to selectively bind to bNAbs and poorly to non-NAbs (non-neutralizing antibodies), properties desirable in Env-based immunogens. Thus, efficiently cleaved HIV-1 Envs with desirable antigenic properties can be good candidates for developing immunogens. Here we describe in detail a six step method we have used in our laboratory to identify such efficiently cleaved Envs. Some of these protocols are optimizations of previously reported assays such as FACS-based cell surface antibody binding assay, pseudovirus neutralization assay and gp120 shedding assay. Other protocols like biotinylation-neutravidin-agarose pull-down assay and plasma membrane protein immunoprecipitation assay have been developed by taking inputs from reagent/kit manufacturer’s protocols and previous studies. These protocols will help the field in identifying more such Envs which can be used for immunogen development.•Six step process to identify efficiently cleaved, membrane-bound, functional HIV-1 Envs with high degree of repeatability.•Method applicable for characterizing any HIV-1 envelope protein.•New method of immunoprecipitation of plasma membrane fraction to validate efficiently cleaved HIV-1 envelopes.

OPTIMIZATION OF METHODS FOR PREPARATION OF RECOMBINANT TOBACCO ETCH VIRUS PROTEASE FROM ESCHERICHIA COLI CELLS

The catalytic domain of the nuclear inclusion protein of the tobacco etch virus protease, TEVp, is used for the cleavage of artificial fusion polypeptides. However, the production of a recombinant enzyme has certain difficulties, such as a low yield of the product and its low solubility in physiological solutions. The aim of the study was to optimize the methods of producing a recombinant enzyme TEVp from E. coli producing cells. Material and methods. The studies were carried out on E. coli cells st. BL21 (DE3). The enzyme was synthesized by cells in the form of a fusion polypeptide with maltose-binding protein (MBP), followed by self-cleavage. Biomass production was carried out under various conditions: a change in the temperature regime, the time of incubation of cells with an inducer, the concentration of the inducer and the growth phase of the culture with the addition of an inducer. The enzyme was isolated under native conditions and with an increased concentration of sodium chloride by affinity chromatography. The enzyme activity was tested on chimeric recombinant human apolipoprotein A-I (~33.4 kDa). Results and discussion. The results of the study showed that a significant influence on the final yield of the enzyme was provided by the growth phase of the culture when the inducer was added. Optimal conditions for obtaining biomass were found as follows: incubation temperature with an inducer 30 °C; incubation time 4 hours; inducer concentration 200 μM; optical density with inducer addition 2.0-2.5 optical units per ml. Sodium chloride concentration in the buffer solution during isolation of the protein was 150 mM. The yield of the enzyme under these conditions reached 50 mg from a liter of cell culture. A similar yield of the enzyme was obtained using the method of auto-induction of cell culture. In all cases, enzymes retained their activity. Conclusion. It was shown that the greatest influence on the yield of the recombinant enzyme from E. coli producing cells strain BL21 (DE3) in the expression vector pD441-MBP under the regulation of the bacteriophage promoter gene «T5» was exerted by the growth phase of the cell culture at the time of gene expression launch.