Design of novel and highly selective SARS-CoV-2 main protease inhibitors.

We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro, including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy.

Therapeutic Potential of Cannabis: A Comprehensive Review of Current and Future Applications.

Historically, cannabis has been valued for its pain-relieving, anti-inflammatory, and calming properties. Ancient civilizations like the Egyptians, Greeks, and Chinese medicines recognized their therapeutic potential. The discovery of the endocannabinoid system, which interacts with cannabis phytoconstituents, has scientifically explained how cannabis affects the human immune system, including the central nervous system (CNS). This review explores the evolving world of cannabis-based treatments, spotlighting its diverse applications. By researching current research and clinical studies, we probe into how cannabinoids like Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) help to manage conditions ranging from chronic pain, persistent inflammation, cancer, inflammatory bowel disease, and neurological disorders to even viral diseases such as Human Immunodeficiency virus (HIV), SARS-CoV-2. and the emerging monkeypox. The long-term recreational use of cannabis can develop into cannabis use disorder (CUD), and therefore, understanding the factors contributing to the development and maintenance of cannabis addiction, including genetic predisposition, neurobiological mechanisms, and environmental influences, will be timely. Shedding light on the adverse impacts of CUD underscores the importance of early intervention, effective treatment approaches, and public health initiatives to address this complex issue in an evolving landscape of cannabis policies and perceptions.

Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection.

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.

Defining the Innate Immune Responses for SARS-CoV-2-Human Macrophage Interactions.

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end-organ malfunctions. All follow the presence of persistent viral components and virions without evidence of viral replication. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation, it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways, specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding end-organ tissue damage.

Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC50 values in the micromolar range (MU-UNMC-1: IC50 = 0.67 µM and MU-UNMC-2: IC50 = 1.72 µM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC50 = 9.27 and 3.00 µM) and Scotland variant B.1.222 (IC50 = 2.64 and 1.39 µM), respectively. Following these assays, we conducted "induced-fit (flexible) docking" to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir, the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2-specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 Spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir (RDV), suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19 patients.

Biomarkers of Activation and Inflammation to Track Disparity in Chronological and Physiological Age of People Living With HIV on Combination Antiretroviral Therapy.

With advancement, prompt use, and increasing accessibility of antiretroviral therapy, people with HIV are living longer and have comparable lifespans to those negative for HIV. However, people living with HIV experience tradeoffs with quality of life often developing age-associated co-morbid conditions such as cancers, cardiovascular diseases, or neurodegeneration due to chronic immune activation and inflammation. This creates a discrepancy in chronological and physiological age, with HIV-infected individuals appearing older than they are, and in some contexts ART-associated toxicity exacerbates this gap. The complexity of the accelerated aging process in the context of HIV-infection highlights the need for greater understanding of biomarkers involved. In this review, we discuss markers identified in different anatomical sites of the body including periphery, brain, and gut, as well as markers related to DNA that may serve as reliable predictors of accelerated aging in HIV infected individuals as it relates to inflammatory state and immune activation.

Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells.

The accurate estimation and eradication of Human Immunodeficiency Virus (HIV) viral reservoirs is limited by the incomplete reactivation of cells harboring the latent replication-competent virus. We investigated whether the in vitro and in vivo addition of retinoic acid (RA) enhances virus replication and improves the detection of latent virus. Peripheral blood mononuclear cells (PBMCs) from naive and anti-retroviral therapy (ART)-treated SIV-infected rhesus macaques (RMs) were cultured in vitro with anti-CD3/CD28 + IL-2 in the presence/absence of RA. Viral RNA and p27 levels were quantified using RT-qPCR and ELISA, respectively. Viral reservoirs were estimated using the Tat/Rev-Induced Limited Dilution Assay (TILDA) and Quantitative Viral Outgrowth Assay (QVOA). In vitro and in vivo measures revealed that there was also an increase in viral replication in RA-treated versus without RA conditions. In parallel, the addition of RA to either CD3/CD28 or phorbol myristate acetate (PMA)/ionomycin during QVOA and TILDA, respectively, was shown to augment reactivation of the replication-competent viral reservoir in anti-retroviral therapy (ART)-suppressed RMs as shown by a greater than 2.3-fold increase for QVOA and 1 to 2-fold increments for multi-spliced RNA per million CD4+ T cells. The use of RA can be a useful approach to enhance the efficiency of current protocols used for in vitro and potentially in vivo estimates of CD4+ T cell latent reservoirs. In addition, flow cytometry analysis revealed that RA improved estimates of various viral reservoir assays by eliciting broad CD4 T-cell activation as demonstrated by elevated CD25 and CD38 but reduced CD69 and PD-1 expressing cells.

Variability in HIV-1 Integrase Gene and 3'-Polypurine Tract Sequences in Cameroon Clinical Isolates, and Implications for Integrase Inhibitors Efficacy.

: Integrase strand-transfer inhibitors (INSTIs) are now included in preferred first-line antiretroviral therapy (ART) for HIV-infected adults. Studies of Western clade-B HIV-1 show increased resistance to INSTIs following mutations in integrase and nef 3'polypurine tract (3'-PPT). With anticipated shifts in Africa (where 25.6-million HIV-infected people resides) to INSTIs-based ART, it is critical to monitor patients in African countries for resistance-associated mutations (RAMs) affecting INSTIs efficacy. We analyzed HIV-1 integrase and 3'-PPT sequences in 345 clinical samples from INSTIs-naïve HIV-infected Cameroonians for polymorphisms and RAMs that affect INSTIs. Phylogeny showed high genetic diversity, with the predominance of HIV-1 CRF02_AG. Major INSTIs RAMs T66A and N155K were found in two (0.6%) samples. Integrase polymorphic and accessory RAMs found included T97A, E157Q, A128T, M50I, S119R, L74M, L74I, S230N, and E138D (0.3%-23.5% of samples). Ten (3.2%) samples had both I72V+L74M, L74M+T97A, or I72V+T97A mutations; thirty-one (9.8%) had 3'-PPT mutations. The low frequency of major INSTIs RAMs shows that INSTIs-based ART can be successfully used in Cameroon. Several samples had 1 INSTIs accessory RAMs known to reduce INSTIs efficacy; thus, INSTIs-based ART would require genetic surveillance. The 3'-PPT mutations could also affect INSTIs. For patients failing INSTIs-based ART with no INSTIs RAMs, monitoring 3'-PPT sequences could reveal treatment failure etiology.

HIV-1 Productively Infects and Integrates in Bronchial Epithelial Cells.

Background: The role of lung epithelial cells in HIV-1-related lung comorbidities remains unclear, and the major hurdle in curing HIV is the persistence of latent HIV reservoirs in people living with HIV (PLWH). The advent of combined antiretroviral therapy has considerably increased the life span; however, the incidence of chronic lung diseases is significantly higher among PLWH. Lung epithelial cells orchestrate the respiratory immune responses and whether these cells are productively infected by HIV-1 is debatable. Methods: Normal human bronchial epithelial cells (NHBEs) grown on air-liquid interface were infected with X4-tropic HIV-1LAV and examined for latency using latency-reversing agents (LRAs). The role of CD4 and CXCR4 HIV coreceptors in NHBEs were tested, and DNA sequencing analysis was used to analyze the genomic integration of HIV proviral genes, Alu-HIVgag-pol, HIV-nef, and HIV-LTR. Lung epithelial sections from HIV-infected humans and SHIV-infected macaques were analyzed by FISH for HIV-gag-pol RNA and epithelial cell-specific immunostaining. Results and Discussion: NHBEs express CD4 and CXCR4 at higher levels than A549 cells. NHBEs are infected with HIV-1 basolaterally, but not apically, by X4-tropic HIV-1LAV in a CXCR4/CD4-dependent manner leading to HIV-p24 antigen production; however, NHBEs are induced to express CCR5 by IL-13 treatment. In the presence of cART, HIV-1 induces latency and integration of HIV provirus in the cellular DNA, which is rescued by the LRAs (endotoxin/vorinostat). Furthermore, lung epithelial cells from HIV-infected humans and SHIV-infected macaques contain HIV-specific RNA transcripts. Thus, lung epithelial cells are targeted by HIV-1 and could serve as potential HIV reservoirs that may contribute to the respiratory comorbidities in PLWH.

A Tat/Rev Induced Limiting Dilution Assay to Measure Viral Reservoirs in Non-Human Primate Models of HIV Infection.

The establishment of latent infection and poorly characterized viral reservoirs in tissues represent major obstacles to a definitive cure for HIV. Non-human primate (NHP) models of HIV infection are critical to elucidate pathogenic processes and an essential tool to test novel therapeutic strategies. Thus, the availability of novel assays to measure residual viral replication and reservoirs in NHP models may increase their utility in the search for an HIV cure. We developed a tat/rev induced limiting dilution assay to measure the frequency of CD4+ T cells that express multiply-spliced(ms)_SIV RNA in presence and absence of stimulation. We validated the assay using cell lines and cells from blood and lymph nodes of SIV infected macaques. In vitro, SIV/SHIV TILDA detects only cells expressing viral proteins. In SIV/SHIV-infected macaques, CD4+ T cells that express msSIV/SHIV RNA (TILDA data) were detected also in the setting of very low/undetectable viremia. TILDA data were significantly higher after stimulation and correlated with plasma viral load (pVL). Interestingly, TILDA data from early cART initiation correlated with peak and AUC pVL post-cART interruption. In summary, we developed an assay that may be useful in characterizing viral reservoirs and determining the effect of HIV interventions in NHP models.

Pattern of variation in the mono- and dinucleotide repeat microsatellites associated with lynch syndrome in an Indian population.

BACKGROUND: Microsatellite instability (MSI) is associated with Lynch syndrome and hence is a surrogate marker for defective mismatch repair genes. The aim of this study was to investigate the degree of instability associated with each of the 5 microsatellite (MS) loci recommended by the National Cancer Institute (NCI), USA within an Indian population of mixed ethnicity and suffering from Lynch syndrome. METHODS: DNA from clinical samples originating from the study population (n = 130) were subjected to automated fragment analysis for all 5 MS loci, and data generated were analyzed to determine the frequency of variation of each of the MS in the resource population. RESULTS: Out of 130, 116 samples responded to polymerase chain reaction (PCR) for all 5 MS loci. 21 (16.15%) were MSI-high (MSI-H) while 27 (20.76%) and 68 (52.30%) were MSI-low (MSI-L) and microsatellite stable (MSS), respectively. D5S346 exhibited the highest instability (27 out of a total of 82 cases of instability recorded for all 5 MS in all 116 patients tested) followed by D2S123 (23/82), BAT26 (14/82), BAT25 (11/82), and D17S250 (7/82). CONCLUSION: MS D17S250 and BAT25 of the 5 MS panel recommended by the NCI are not informative enough and hence should be avoided for diagnosing Lynch syndrome in the Indian population.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Rare e14a3 (b3a3) BCR-ABL fusion in chronic myeloid leukemia in India: the threats and challenges in monitoring minimal residual disease (MRD).

OBJECTIVE: The primary objective of this work was to confirm the occurrence of rare BCR ABL fusion variant involving the a3 region of the ABL gene in a patient positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions and the challenges and threats that it poses in a routine laboratory setting which use commercial kits for monitoring the minimal residual disease. METHODS: A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. PCR amplicon generated from in the process was sequenced and analyzed. RESULTS: The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus of affected cells and presented a '1O1G2F' signal pattern. RT-PCR with probes from commercial kit designed to detect common breakpoints within the M- and m-BCR regions involving e13a2, e14a2 and e1a2 fusion variants respectively failed to generate any signal. Further investigation revealed presence of the rare e14a3 (b3a3) fusion. DISCUSSION: This is the first report of rare e14a3 fusion in the BCR ABL gene in a CML patient from India. The observation indicates the need for interrogating rare BCR ABL fusions when common breakpoint cluster regions are absent such that minimal residual disease (MRD), critical for disease monitoring, can be performed and false positive remission cases can be avoided. It also emphasizes the utility and significance of cytogenetics and FISH techniques in primary diagnosis of CML and use of RT-PCR based assays only for generating secondary information within special reference to MRD. CONCLUSION: The rare e14a3 (b3a3) fusion of the BCR ABL gene is present in Indian population as demonstrated from this first report and clinical laboratories using commercial kit that do not cover such rare fusions are likely to generate false result thereby declaring complete molecular remission in CML patients under therapy while conducting MRD assay using RT-PCR technology.

Distinct geographical clustering of HIV-1 and a signature amino acid at position 41 of the p24 unveiled by gag variability in India.

A portion of the gag gene cDNA for p24 protein from 30 Indian HIV-1 proviral DNA was amplified by PCR and sequenced. Phylogenetic analysis with reference samples of A1, A2, B, C, D, F1, F2, G, H, J, K, N and O subtypes revealed that 29 test samples aligned with subtype C reference strain while 1 matched with HIV-1 subtype A. Multiple alignment of predicted amino acid sequence of the Indian test samples and reference C subtype of HIV-1 samples from other countries indicated a molecular signature by way of rigid conservation of the amino acid 'S' at position 41 of the gag p24 protein in all Indian HIV-1 samples analyzed in this study as opposed to 'T' in the same position in C subtype sequences from other parts of the world. A phylogenetic analysis and visualization of the resulting tree in radial position showed distinct clubbing of all Indian C subtypes and formation of a cluster when compared to C subtype sequences from other countries with a single Chinese sample as an exception which was found in the Indian cluster. The use of a portion of p24 gene sequence as tool for subtyping as well as phylogenetic grouping with special reference to its geographical location is discussed.