Reciprocal Modulation of Antiretroviral Drug and Steroid Receptor Function In Vitro.

Millions of women are exposed simultaneously to antiretroviral drugs (ARVs) and progestin-based hormonal contraceptives. Yet the reciprocal modulation by ARVs and progestins of their intracellular functions is relatively unexplored. We investigated the effects of tenofovir disoproxil fumarate (TDF) and dapivirine (DPV), alone and in the presence of select steroids and progestins, on cell viability, steroid-regulated immunomodulatory gene expression, activation of steroid receptors, and anti-HIV-1 activity in vitro Both TDF and DPV modulated the transcriptional efficacy of a glucocorticoid agonist via the glucocorticoid receptor (GR) in the U2OS cell line. In TZM-bl cells, DPV induced the expression of the proinflammatory interleukin 8 (IL-8) gene while TDF significantly increased medroxyprogesterone acetate (MPA)-induced expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. However, peripheral blood mononuclear cell (PBMC) and ectocervical explant tissue viability and gene expression results, along with TZM-bl HIV-1 infection data, are reassuring and suggest that TDF and DPV, in combination with dexamethasone (DEX) or MPA, do not reciprocally modulate key biological effects in primary cells and tissue. We show for the first time that TDF induces progestogen-independent activation of the progesterone receptor (PR) in a cell line. The ability of TDF and DPV to influence GR and PR activity suggests that their use may be associated with steroid receptor-mediated off-target effects. This, together with cell line and individual donor gene expression responses in the primary models, raises concerns that reciprocal modulation may cause side effects in a cell- and donor-specific manner in vivo.

Shining new light on ancient drugs: preparation and subcellular localisation of novel fluorescent analogues of Cinchona alkaloids in intraerythrocytic Plasmodium falciparum.

Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, suitable for cellular imaging have been synthesised by attaching the small extrinsic fluorophore, NBD. Interactions of these derivatives with ferriprotoporphyrin IX were evaluated to verify that insights generated by live-cell imaging were relevant to the parent molecules. These analogues are shown by confocal and super-resolution microscopy to accumulate selectively in Plasmodium falciparum. Localisation to the region corresponding to the digestive vacuole supports the putative primary role of these alkaloids as haemozoin inhibitors. Quantitative analysis revealed minimal accumulation within the nucleus, rejecting the disruption of DNA replication as a possible mode of action. While extensive localisation to phospholipid structures and associated organelles was observed, the analogues did not show evidence of association with neutral lipid bodies.

Ongoing Implementation and Prospective Validation of Artificial Intelligence/Machine Learning Tools at an African Drug Discovery Center.

Artificial intelligence (AI) and machine learning (ML) are anticipated to accelerate drug discovery programs. Following our development of an end-to-end virtual screening cascade at the University of Cape Town (UCT) Holistic Drug Discovery and Development (H3D) Center, we report the ongoing implementation of open-source AI/ML tools for use in resource-constrained settings.

First fully-automated AI/ML virtual screening cascade implemented at a drug discovery centre in Africa.

Streamlined data-driven drug discovery remains challenging, especially in resource-limited settings. We present ZairaChem, an artificial intelligence (AI)- and machine learning (ML)-based tool for quantitative structure-activity/property relationship (QSAR/QSPR) modelling. ZairaChem is fully automated, requires low computational resources and works across a broad spectrum of datasets. We describe an end-to-end implementation at the H3D Centre, the leading integrated drug discovery unit in Africa, at which no prior AI/ML capabilities were available. By leveraging in-house data collected over a decade, we have developed a virtual screening cascade for malaria and tuberculosis drug discovery comprising 15 models for key decision-making assays ranging from whole-cell phenotypic screening and cytotoxicity to aqueous solubility, permeability, microsomal metabolic stability, cytochrome inhibition, and cardiotoxicity. We show how computational profiling of compounds, prior to synthesis and testing, can inform progression of frontrunner compounds at H3D. This project is a first-of-its-kind deployment at scale of AI/ML tools in a research centre operating in a low-resource setting.

Drug discovery in Africa tackles zoonotic and related infections.

Zoonotic and related infections pose an enormous health threat to the world's second-most populous continent. Despite the challenges faced by drug discovery scientists in Africa, recent progress toward identifying potential medicines across diverse disease areas is a cause for optimism and an indicator of progress in African-led scientific initiatives.

Thwarting protein synthesis leads to malaria parasite paralysis.

Inhibiting translation presents a tantalizing strategy to tackle the most virulent human malaria parasite. Xie et al. disclose a compound that binds selectively to Plasmodium falciparum tyrosine aminoacyl-tRNA synthetase, preventing the incorporation of tyrosine into nascent proteins and paving the way for a new generation of safe, effective antimalarials.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Differential metabolism of clinically-relevant progestogens in cell lines and tissue: Implications for biological mechanisms.

Steroid hormones regulate a variety of physiological processes, including reproductive function, and are widely used in hormonal therapy. Synthetic progestogens, or progestins, were designed to mimic progesterone (P4) for use in contraception and hormonal replacement therapy in women. Medroxyprogesterone acetate (MPA) and norethisterone (NET) are the most widely used injectable contraceptives in the developing world, while other progestins such as levonorgestrel (LNG), etonogestrel (ETG) and nestorone (NES) are used in or being developed for other forms of contraception. As concerns remain about the most appropriate choice of progestin and dosage, and the associated side-effects, the mechanisms and biological effects of progestins are frequently investigated in various in vitro mammalian cell line and tissue models. However, whether progestogens are differentially metabolised in different cell types in vivo or in vitro is unknown. For nine mammalian cell lines commonly used to investigate progestogen mechanisms of action, we developed and validated an ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) protocol for simultaneously quantifying the metabolism of the above-mentioned steroids. We show for the first time that, while 50-100% of P4 was metabolised within 24 h in all cell lines, the metabolism of the progestins is progestin- and cell line-specific. We also show that MPA and NET are significantly metabolised in human cervical tissue, but to a lesser extent than P4. Taken together, our findings suggest that differential progestogen metabolism may play a role in cell-specific therapeutic and side-effects. Relative affinities for binding to steroid receptors as well as potencies, efficacies and biocharacters for transcriptional activity of progestins, relative to P4, are most frequently determined using some of the cell lines investigated. Our results, however, suggest that differential metabolism of progestins and P4 may confound these results. In particular, metabolism may under-estimate the receptor-mediated intrinsic in vitro binding and dose-response values and predicted endogenous physiological effects of P4.

Chemical Proteomics and Super-resolution Imaging Reveal That Chloroquine Interacts with Plasmodium falciparum Multidrug Resistance-Associated Protein and Lipids.

It is well established that chloroquine, a quinoline antimalarial, inhibits hemozoin formation in the malaria parasite. Counterintuitively, this archetypal antimalarial is also used in the treatment of diseases in which hemozoin biocrystallization does not play a role. Hence, we decided to investigate whether chloroquine possesses binding targets other than Fe(III) protoporphyrin IX in blood stage Plasmodium falciparum parasites and whether these are related to sites of accumulation within the parasite other than the digestive vacuole. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled fluorescent derivative of chloroquine, especially sensitive to regions outside the digestive vacuole and retaining the antiplasmodial pharmacophore, was synthesized to investigate subcellular localization in the parasite. Super-resolution microscopy revealed association with membranes including the parasite plasma membrane, the endoplasmic reticulum, and possibly also the mitochondrion. A drug-labeled affinity matrix was then prepared to capture protein binding targets of chloroquine. SDS-PAGE revealed a single prominent band between 200 and 250 kDa from the membrane-associated fraction. Subsequent proteomic analysis revealed that this band corresponded to P. falciparum multidrug resistance-associated protein (PfMRP1). Intrigued by this finding, we demonstrated pull-down of PfMRP1 by matrices labeled with Cinchona alkaloids quinine and quinidine. While PfMRP1 has been implicated in resistance to quinolines and other antimalarials, this is the first time that these drugs have been found to bind directly to this protein. Based on previous reports, PfMRP1, the only prominent protein found to bind to quinolines in this work, is likely to modulate the activity of these antimalarials in P. falciparum rather than act as a drug target.

Medroxyprogesterone acetate, unlike norethisterone, increases HIV-1 replication in human peripheral blood mononuclear cells and an indicator cell line, via mechanisms involving the glucocorticoid receptor, increased CD4/CD8 ratios and CCR5 levels.

High usage of progestin-only injectable contraceptives, which include the intramuscular injectables depo-medroxyprogesterone acetate (DMPA-IM, Depo-Provera) and norethisterone (NET) enanthate (NET-EN or Nur-Isterate), correlates worldwide with areas of high HIV-1 prevalence. Epidemiological data show a significant association between usage of DMPA-IM and increased HIV-1 acquisition but no such association from limited data for NET-EN. Whether MPA and NET have similar effects on HIV-1 acquisition and pathogenesis, and the relationship between these effects and the dose of MPA, are critical issues for women's health and access to suitable and safe contraceptives. We show for the first time that MPA, unlike NET, significantly increases HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and a cervical cell line model. The results provide novel evidence for a biological mechanism whereby MPA, acting via the glucocorticoid receptor (GR), increases HIV-1 replication by at least in part increasing expression of the CCR5 HIV-1 coreceptor on target T-lymphocytes. MPA, unlike NET, also increases activation of T-cells and increases the CD4/CD8 ratio, suggesting that multiple mechanisms are involved in the MPA response. Our data offer strong support for different biological mechanisms for MPA versus NET, due to their differential GR activity. The dose-dependence of the MPA response suggests that significant effects are observed within the range of peak serum levels of progestins in DMPA-IM but not NET-EN users. Dose-response results further suggest that effects of contraceptives containing MPA on HIV-1 acquisition and disease progression may be critically dependent on dose, time after injection and intrinsic factors that affect serum concentrations in women.