Ghizal Siddiqui

Spore-forming bacteria produce two distinct cell types: vegetative cells and resilient spores. While antibiotic resistance is typically associated with vegetative cells, spores play a critical role in disseminating resistance genes due to their durability and transmissibility. We previously demonstrated that cephamycin antibiotics target the conserved spore-specific protein SpoVD, significantly reducing spore formation in pathogens including Clostridioides difficile . Here, we show that when C. difficile acquires CdmecA , a homologue of Staphylococcus aureus mecA , one of the most globally burdensome resistance genes, the anti-sporulation effect of cephamycins is bypassed. Cd MecA functionally replaces Cd SpoVD, restoring sporulation and producing phenotypically distinct spores. We further show that mecA is prevalent across C. difficile strains and other pathogenic, gut, and environmental spore-formers. Since SpoVD is conserved, MecA may broadly co-opt sporulation; we confirm this in Clostridium perfringens . This work reveals an unusual resistance mechanism with unexpected physiological consequences, reshaping our understanding of antibiotic resistance within the context of sporulation and microbial adaptation.

Plasmepsin V (PMV), an essential aspartyl protease, plays a critical role during the asexual blood stage of infection of Plasmodium by enabling the export of parasite proteins into the host red blood cell. This export is vital for parasite survival and pathogenesis, making PMV an attractive target for antimalarial drug development. Peptidomimetic inhibitors designed to mimic the natural substrate of PMV have demonstrated potent parasite-killing activity by blocking protein export. While these compounds have been instrumental in validating PMV as a bona fide antimalarial target, inconsistencies between their biochemical potency and cellular activity have raised questions regarding their precise mechanism of action. In this study, we employed chemoproteomic approaches, including solvent-induced protein precipitation and intact-cell thermal profiling, to demonstrate PMV target engagement by the peptidomimetics. To further support these findings, we generated parasite lines exhibiting reduced sensitivity to peptidomimetics. Through whole-genome sequencing of these parasite lines, a single nucleotide variant within the pmv gene was revealed. This mutation was later validated using reverse genetics, confirming its role in mediating resistance. Together, these data provide strong evidence that the peptidomimetics exert their antimalarial activity by directly targeting PMV. These findings further support the potential of PMV as a validated and promising target for future antimalarial drug development.

Background/Objectives: Monoclonal antibody (mAb) stability is critical not only during manufacturing but also at the point of clinical administration. For therapies like tislelizumab (Tevimbra), a programmed death-1 (PD-1) targeting IgG mAb, delays in dosing often result in prepared infusions being discarded, contributing to substantial drug waste despite being engineered for improved stability. Methods: To evaluate the physicochemical in-use stability of tislelizumab in a ready-to-administer format, we mapped degradation pathways, including post-translational modifications (PTMs); peptide alterations; pH and solution characteristics—under 12-month storage (ultra-long), under 1-month storage (0, 7, 14, 21, 28 and 31 days), and under exposure-related forced degradation conditions including room temperature, elevated temperature, pH (acidic/basic), oxidation and UV exposure. Structural analysis was contextualised to the known PD-1 binding site, making stability assessment relevant to tislelizumab’s mechanism-of-action in blocking PD-1. To assess solution stability, a validated size-exclusion chromatography (SEC) assay was applied to all conditions. Results: Aggregation was identified as the primary degradation pathway during ultra-long-term storage. SEC and chemical assessment revealed no measurable changes in protein quantity, aggregation, peptide integrity, or PTM profile over 31 days at 2–8 °C in polyolefin intravenous bags (1.6 mg/mL). Conclusions: These results support the structural and physicochemical stability of tislelizumab under refrigerated conditions.

Thienotriazolodiazepines, including (+)-JQ1 (4), are well-known inhibitors of the bromodomain (BD) and extra-terminal domain (BET) family of proteins. Despite the suboptimal physicochemical properties as a drug candidate, such as poor solubility and half-life, (+)-JQ1 (4) has proven as an effective chemical probe with high target potency and selectivity. (+)-JQ1 (4) and (+)-JQ1-derived chemical probes have played a vital role in chemical biology and drug discovery over the past decade, which is demonstrated by the high number of impactful research studies published since the disclosure of (+)-JQ1 (4) in 2010. In this review, we discuss the development of (+)-JQ1-derivatized chemical probes over the past decade and their significant contribution to scientific research. Specifically, we will summarize the development of innovative label-free and labeled (+)-JQ1-derivatized chemical probes, such as bivalent, covalent, and photoaffinity probes as well as protein degraders, with a focus on the design of these chemical probes.

Over the last two decades, global malaria cases caused by Plasmodium falciparum have declined due to the implementation of effective treatments and the use of insecticides. However, the COVID-19 pandemic caused major disruption in the timely delivery of medical goods and diverted public health resources, impairing malaria control. The emergence of resistance to all existing frontline antimalarials underpins an urgent need for new antimalarials with novel mechanisms of action. Furthermore, the need to reduce malaria transmission and/or prevent malaria infection has shifted the focus of antimalarial research towards the discovery of compounds that act beyond the symptomatic blood stage and also impact other parasite life cycle stages. Phenotypic screening has been responsible for the majority of new antimalarial lead compounds discovered over the past 10 years. This review describes recently reported novel antimalarial hits that target multiple parasite stages and were discovered by phenotypic screening during the COVID-19 pandemic. Their modes of action and targets in blood stage parasites are also discussed.

Artemisinin (ART) is a quick-killing and effective antimalarial activated by the haem derived from haemoglobin digestion. Mutations in the parasite’s Kelch 13 (K13) protein compromise the efficacy of this drug. Recent studies indicate an undefined role for K13 in haemoglobin uptake. Here, we show that K13 is associated with the collar that constricts cytostomal invaginations required for the parasite to ingest host cytosol. Induced mislocalisation of K13 led to the formation of atypical invaginations lacking the cytostomal ring and constricted neck normally associated with cytostomes. Moreover, the levels of haemoglobin degradation products, haem and haemozoin, are decreased when K13 is inactivated. Our findings demonstrate that K13 is required for normal formation and/or stabilisation of the cytostome, and thereby the parasite’s uptake of haemoglobin. This is consistent with perturbation of K13 function leading to decreased activation of ART and consequently, reduced killing.

Artemisinin (ART) is a quick-killing and effective antimalarial activated by the haem derived from haemoglobin digestion. Mutations in the parasite’s Kelch 13 (K13) protein compromise the efficacy of this drug. Recent studies indicate an undefined role for K13 in haemoglobin uptake. Here, we show that K13 is associated with the collar that constricts cytostomal invaginations required for the parasite to ingest host cytosol. Induced mislocalisation of K13 led to the formation of atypical invaginations lacking the cytostomal ring and constricted neck normally associated with cytostomes. Moreover, the levels of haemoglobin degradation products, haem and haemozoin, are decreased when K13 is inactivated. Our findings demonstrate that K13 is required for normal formation and/or stabilisation of the cytostome, and thereby the parasite’s uptake of haemoglobin. This is consistent with perturbation of K13 function leading to decreased activation of ART and consequently, reduced killing. Significance Statement: Artemisinin-resistant parasites contain mutations in the gene encoding the Kelch 13 protein (K13). How K13 mutations result in artemisinin resistance is unclear. Here, we present evidence that normal K13 is required for the formation of the cytostome, a specialised parasite feeding apparatus used to endocytose host cell haemoglobin. Our results suggest that artemisinin resistance is due to a decrease in artemisinin activation brought about by a decrease in efficiency of haemoglobin uptake and consequently reduced production of haem.

Plasmodium falciparum, the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the host’s main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here, we use both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that loss of PfA-M17 results in parasites exhibiting multiple digestive vacuoles at the trophozoite stage. In contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.