Targeting HIV-1 Reverse Transcriptase Using a Fragment-Based Approach.

Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.

Discovery, synthesis and exploration of N-benzylsulfonyl-2-phenylazepanes as inhibitors of Bim expression in a mouse embryonic fibroblast model.

Chronic activation of beta-adrenergic receptors by the sympathetic nervous system results in the apoptosis of cardiomyocytes. Due to the inability of cardiomyocytes to regenerate, this can result in heart failure. Upregulation of the pro-apoptotic protein Bim has been implicated as the cause of cardiomyocyte apoptosis. Beta blockers are the frontline drug used to negate this apoptotic pathway, as no direct inhibitors of Bim expression currently exist. Unfortunately, treatment of heart failure using beta blockers is not optimal. Therefore, direct inhibition of Bim expression is an attractive strategy to provide protection against stress-induced apoptosis of cardiomyocytes. Herein we explore a class of N-benzylsulfonyl-2-phenylazepanes to obtain anti-apoptotic compounds capable of reducing Bim expression levels to 7% of the control at 10 µM in cardiomyocytes under conditions of chronic beta-adrenergic receptor activation with little inhibitory effect upon protein kinase A activity and minimal toxicity.

LMP2 immunoproteasome promotes lymphocyte survival by degrading apoptotic BH3-only proteins.

The role of the immunoproteasome is perceived as confined to adaptive immune responses given its ability to produce peptides ideal for MHC Class-I binding. Here, we demonstrate that the immunoproteasome subunit, LMP2, has functions beyond its immunomodulatory role. Using LMP2-deficient mice, we demonstrate that LMP2 is crucial for lymphocyte development and survival in the periphery. Moreover, LMP2-deficient lymphocytes show impaired degradation of key BH3-only proteins, resulting in elevated levels of pro-apoptotic BIM and increased cell death. Interestingly, LMP2 is the sole immunoproteasome subunit required for BIM degradation. Together, our results suggest LMP2 has important housekeeping functions and represents a viable therapeutic target for cancer.

Differential prevalence of transporter polymorphisms in symptomatic and asymptomatic falciparum malaria infections in Uganda.

We explored associations between Plasmodium falciparum drug resistance-mediating polymorphisms and clinical presentations in parasitemic children enrolled in a cross-sectional survey in Tororo, Uganda, using a retrospective case-control design. All 243 febrile children (cases) and 243 randomly selected asymptomatic children (controls) were included. In a multivariate analysis adjusting for age, complexity of infection, and parasite density, the prevalence of wild-type genotypes was significantly higher in febrile children compared to asymptomatic children (pfcrt K76T: odds ratio [OR] 4.41 [95% confidence interval {CI}, 1.28-15.1]; pfmdr1 N86Y: OR 4.08 [95% CI, 2.01-8.31], and pfmdr1 D1246Y: OR 4.90 [95% CI, 1.52-15.8]), suggesting greater virulence for wild-type parasites.

Validation of the ligase detection reaction fluorescent microsphere assay for the detection of Plasmodium falciparum resistance mediating polymorphisms in Uganda.

BACKGROUND: Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting. METHODS: For 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared. RESULTS: SNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K=0.77) for pfcrt K76T, 79% (K=0.55) for pfmdr1 N86Y, 83% (K=0.65) for pfmdr1 Y184F, and 91% (K=0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes. CONCLUSION: The LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.

Inflammatory and immunometabolic consequences of gut dysfunction in HIV: Parallels with IBD and implications for reservoir persistence and non-AIDS comorbidities.

The gastrointestinal mucosa is critical for maintaining the integrity and functions of the gut. Disruption of this barrier is a hallmark and a risk factor for many intestinal and chronic inflammatory diseases. Inflammatory bowel disease (IBD) and HIV infection are characterized by microbial translocation and systemic inflammation. Despite the clinical overlaps between HIV and IBD, significant differences exist such as the severity of gut damage and mechanisms of immune cell homeostasis. Studies have supported the role of metabolic activation of immune cells in promoting chronic inflammation in HIV and IBD. This inflammatory response persists in HIV+ persons even after long-term virologic suppression by antiretroviral therapy (ART). Here, we review gut dysfunction and microbiota changes during HIV infection and IBD, and discuss how this may induce metabolic reprogramming of monocytes, macrophages and T cells to impact disease outcomes. Drawing from parallels with IBD, we highlight how factors such as lipopolysaccharides, residual viral replication, and extracellular vesicles activate biochemical pathways that regulate immunometabolic processes essential for HIV persistence and non-AIDS metabolic comorbidities. This review highlights new mechanisms and support for the use of immunometabolic-based therapeutics towards HIV remission/cure, and treatment of metabolic diseases.

DR5 and caspase-8 are dispensable in ER stress-induced apoptosis.

The endoplasmic reticulum (ER) stress response constitutes cellular reactions triggered by a wide variety of stimuli that disturb folding of proteins, often leading to apoptosis. ER stress-induced apoptotic cell death is thought to be an important contributor to many human pathological conditions. The molecular mechanism of this apoptosis process has been highly controversial with both the receptor and the mitochondrial pathways being implicated. Using knockout mouse models and RNAi-mediated gene silencing in cell lines, our group and others had demonstrated the importance of the mitochondrial apoptotic pathway in ER stress-induced cell death, particularly the role of the pro-apoptotic BH3-only BCL-2 family members, BIM and PUMA. However, a recent report suggested a central role for the death receptor, DR5, activated in a ligand-independent manner, and the initiator caspase, caspase-8, in ER stress-induced cell death. This prompted us to re-visit our previous observations and attempt to reproduce the newly published findings. Here we report that the mitochondrial apoptotic pathway, activated by BH3-only proteins, is essential for ER stress-induced cell death and that, in contrast to the previous report, DR5 as well as caspase-8 are not required for this process.

Temporal changes in prevalence of molecular markers mediating antimalarial drug resistance in a high malaria transmission setting in Uganda.

Standard therapy for malaria in Uganda changed from chloroquine to chloroquine + sulfadoxine-pyrimethamine in 2000, and artemether-lumefantrine in 2004, although implementation of each change was slow. Plasmodium falciparum genetic polymorphisms are associated with alterations in drug sensitivity. We followed the prevalence of drug resistance-mediating P. falciparum polymorphisms in 982 samples from Tororo, a region of high transmission intensity, collected from three successive treatment trials conducted during 2003-2012, excluding samples with known recent prior treatment. Considering transporter mutations, prevalence of the mutant pfcrt 76T, pfmdr1 86Y, and pfmdr1 1246Y alleles decreased over time. Considering antifolate mutations, the prevalence of pfdhfr 51I, 59R, and 108N, and pfdhps 437G and 540E were consistently high; pfdhfr 164L and pfdhps 581G were uncommon, but most prevalent during 2008-2010. Our data suggest sequential selective pressures as different treatments were implemented, and they highlight the importance of genetic surveillance as treatment policies change over time.