Biology subjects

Coyle, R.

Publications and source records attributed to Coyle, R..

5 recordsLinked to original sources

bioRxiv2026

Dual plasmepsin IX and X inhibitors are refractory to development of resistance

Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, but emerging resistance threatens their efficacy. The potential for the development of drug resistance against plasmepsin X (PMX)-selective inhibitors and dual plasmepsin IX/X (PMIX/X) inhibitors was investigated in Plasmodium falciparum. A series of PMX-selective (WM4, WM76, WM92) and PMIX/X dual inhibitors (WM382, WM09, WM42) were characterised for potency against parasite growth and enzyme inhibition. In vitro selection experiments showed that all compounds had a high barrier to resistance, although parasites with reduced sensitivity to PMX-selective inhibitors could still be selected. Resistance mechanisms involved pmx gene amplification and point mutations (D245N, S315P, S359P, I363L) that alter inhibitor binding. Recombinant expression and Michaelis-Menten kinetics demonstrated that these mutations impair drug binding whilst preserving PMX catalytic function. Reverse genetics confirmed that introducing these mutations into the pmx gene resulted in decreased potency of the inhibitors. In this study, resistance to the PMIX/X dual inhibitors evaluated here could not be selected, despite prolonged selection pressure. Antimalarial Resistome Barcoding (AReBar) assays confirmed the absence of pre-existing resistance to either inhibitor class. Critically, PMIX/X dual inhibitors maintained efficacy against parasites with decreased sensitivity to PMX-selective compounds. These findings demonstrate that dual PMIX/X inhibitors present a substantially higher barrier to resistance than PMX-selective inhibitors, informing antimalarial drug development strategies and highlighting dual-target inhibition as a promising approach to mitigate resistance risks.

microbiology↗

bioRxiv2025

The Human Chk1 Inhibitor CHIR-124 Shows Multistage Activity Against Plasmodium falciparum via Dual Inhibition of PfArk1 and Hemozoin Formation

The high burden of malaria and growing resistance to frontline antimalarials demand new drug target combinations with reduced propensities for conferring parasite resistance. An attractive approach for circumventing antimalarial drug resistance is target repurposing in which known drugs that act through protein targets of human origin that are also active against the human malaria parasite Plasmodium falciparum are exploited to identify novel antimalarial drug targets. Here we show that the human checkpoint kinase 1 (Chk1) inhibitor CHIR-124 is active in vitro against both drug-sensitive and drug-resistant asexual blood stage parasites and competitively binds to several Plasmodium kinases. The compound also shows moderate activity against both the liver and gametocyte forms of the parasite. Further target investigation of CHIR-124 via conditional knockdown experiments confirmed that P. falciparum Aurora-related kinase 1 (PfArk1) is implicated in its parasiticidal activity. Notably, CHIR-124 also inhibits {beta}-hematin (synthetic hemozoin) formation and causes a dose-dependent increase in free heme that correlates with inhibition of parasite growth. These findings suggest that polypharmacology is involved in the activity of CHIR-124 against P. falciparum via the dual inhibition of Plasmodium PfArk1 and hemozoin formation, both essential for parasite proliferation. This is further supported by in vitro drug combination experiments, morphological studies and resistance generation attempts. This study validates the feasibility of dual Plasmodium kinase/hemozoin formation inhibitors active against resistant strains with decreased resistance risks in the fight against malaria.

microbiology↗

bioRxiv2025

Isomerized Aβ in the brain can distinguish the status of amyloidosis in the Alzheimer's Disease spectrum

Extracellular amyloid plaques, the pathognomonic hallmark of Alzheimers Disease (AD), are also observed in cognitively unimpaired subjects in the preclinical stages. Progressive accumulation of fibrillar amyloid-{beta} (A{beta}) as plaques and perivascular deposits occur two decades prior to clinical onset, making A{beta} a long-lived peptide. To characterize the amyloid plaques biochemically, both the A{beta}-load as well the post-translational modifications (PTMs) could serve as markers for distinguishing the pre-clinical stage compared to later prodromal and clinical stages of AD. Recently, we described the presence of extensive isomerization of the A{beta} N-terminus in AD post-mortem brains that are significantly increased compared to the age-matched non-AD control brains with A{beta} aggregates in the frontal cortex. In this report, we used targeted mass spectrometry to conduct a quantitative analysis of the most common PTMs associated with A{beta}; pyroglutamation, citrullination, N-terminal truncation (A{beta}4-x), C-terminal truncation (A{beta}42 and A{beta}40), and isomerization of aspartic acid residues (Asp-1 and Asp-7) in postmortem human brain tissue from pathologically negative (no A{beta} plaques) controls, controls with A{beta} plaques, Parkinsons disease (PD) with and without A{beta} accumulation/plaques and symptomatic AD. The AD cases contained statistically significant amounts of Asp-1and Asp-7 isomerized A{beta}1-15 ([~] 90 %) compared to controls (preclinical AD) and PD brains with fibrillar A{beta} aggregates/deposits. We find that ratio of isomerized N-terminus A{beta} (A{beta}1-15) species in the brain detergent soluble pool differentiates older fibrillar A{beta} deposits in symptomatic AD brain compared to A{beta} deposits detected in preclinical AD and PD. Citrullinated A{beta}3pglu-15 was increased only in symptomatic AD, highlighting this A{beta} PTM is a unique feature of parenchymal plaques in advanced AD. Our results have implications for early therapeutic targeting of these modified species as well potential for better biofluid biomarker development for drug efficacy monitoring.

neuroscience↗

bioRxiv2024

A novel chemogenomic screening platform for scalable antimalarial drug target identification

Large-scale chemical-genetic screening, or chemogenomics, can faciliate rapid and scalable drug target identification. To establish a chemogenomic screen for antimalarial drug target identification, we leveraged [~]600 Plasmodium berghei artificial chromosomes (PbACs) encoding potential drug targets to generate a systematic overexpression library. PbACs were engineered with DNA barcodes, enabling their quantification within mixed pools using next generation sequencing (barcode sequencing or BarSeq). Pooled transfection of PbACs into the highly genetically tractable Plasmodium knowlesi demonstrated efficient vector uptake and transcription of encoded P. berghei genes. Parasite pools were exposed to antimalarial candidates, with pilot screens probing for known gene-compound associations identifying their targets with high sensitivity. Screening antimalarial inhibitors with unknown mechanisms of action successfully identified pi4k as the target for one novel compound, which was subsequently validated using in vitro evolution in Plasmodium falciparum parasites. This sensitive and scalable chemogenomics platform therefore represents a valuable early-stage tool for antimalarial target identification.

microbiology↗

bioRxiv2020

Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up. AUTHOR SUMMARYMalaria parasites cause disease after invading human red blood cells, implying that a vaccine that interrupts this process could play a significant role in malaria control. Multiple Plasmodium parasite species can cause malaria in humans, and most malaria outside Africa is caused by Plasmodium vivax. There is currently no effective vaccine against the blood stage of any malaria parasite, and progress in P. vivax vaccine development has been particularly hampered because this parasite species cannot be cultured for prolonged periods of time in the lab. We explored whether a related species, P. knowlesi, which can be propagated in human red blood cells in vitro, can be used to screen for potential P. vivax vaccine targets. We raised antibodies against selected P. vivax proteins and testedtheir ability to recognize and prevent P. knowlesi parasites from invading human red blood cells, thereby identifying multiple novel vaccine candidates.

microbiology↗