Biology subjects

Spry, C.

Publications and source records attributed to Spry, C..

8 recordsLinked to original sources

bioRxiv2026

The heteromeric Plasmodium falciparum pantothenate kinase has only one active site and does not require Pf14-3-3I for activity

Coenzyme A (CoA) is an essential molecule for the intraerythrocytic stage of Plasmodium falciparum. Pantothenate kinase (PanK) catalyses the first step of the CoA biosynthesis pathway and functions as a homodimer in most organisms investigated thus far. P. falciparum possesses a novel heteromeric PanK complex composed of PfPanK1, PfPanK2 and Pf14-3-3I. Using a mutagenesis approach, we generated 10 PfPanK mutants and demonstrate that the PfPanK complex has only one functional active site, with both PfPanK1 and PfPanK2 required for activity by the complex. We also show that PfPanK2 is essential for normal intraerythrocytic parasite proliferation using a conditional knockdown system. 14-3-3 binding motifs generally contain a phosphoserine/threonine residue. Mass spectrometry analyses of phospho-peptide enriched, immunoprecipitated PfPanK samples revealed phosphorylation sites in both PfPanK1 and PfPanK2 that were additional to the previously reported sites. To investigate the role of specific sites in PfPanK1 and PfPanK2 that may be involved in Pf14-3-3I binding, five additional mutants were generated. Mutagenesis of four predicted Pf14-3-3I binding sites in PfPanK1 resulted in a significant reduction in the amount of Pf14-3-3I bound to the PfPanK complex, with S334 being the most likely binding site. Heterologous expression of the PfPanK complex in an insect cell system yielded a small amount of soluble protein that assembled in situ into a functional complex. Combined results from heterologous expression and P. falciparum mutagenesis suggest that Pf14-3-3I may not be essential for PfPanK activity but may be important for stabilising the PfPanK complex.

biochemistry↗

bioRxiv2024

Mutation of the P. falciparum flavokinase confers resistance to roseoflavin and 8-aminoriboflavin

We previously found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilisation. To determine the mechanism of action of roseoflavin in P. falciparum, we generated roseoflavin-resistant parasites by in vitro evolution over 27 weeks. The roseoflavin-resistant parasites were found to be four times more resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Resistant parasites were subjected to whole genome sequencing and a missense mutation (T2015A), leading to an amino acid exchange (L672H), was detected in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin (vitamin B2) into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the observed phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene via a single-crossover recombination. The IC50 values for roseoflavin (RoF) and 8-aminoriboflavin against the RoF-resistant parasites created through in vitro evolution were indistinguishable from the IC50 values for parasites in which the missense mutation was specifically introduced into the native PfFK. To investigate this mutation, we generated two parasite lines episomally-expressing GFP-tagged versions of either the wild type or mutant forms of flavokinase. We found that PfFK-GFP localises to the parasite cytosol and that immunopurified PfFK-GFP was active and phosphorylated riboflavin into flavin mononucleotide. The L672H mutation caused a reduction of the binding affinity, especially for the substrate RoF, which explains the resistance phenotype. The mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonised by increasing the extracellular concentration of riboflavin, consistent with the compound also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite growth by inhibiting FMN production, in addition to the generation of toxic flavin cofactor analogues.

microbiology↗

bioRxiv2022

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites

With the advent of resistance to existing treatments, new drugs are needed to combat apicomplexan parasites such as the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). To identify new inhibitors of the mitochondrial electron transport chain (ETC) in these parasites, we developed a Seahorse XFe96 flux analyzer approach to screen compounds from the Medicines for Malaria Venture Pathogen Box for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC of T. gondii, five of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. We pinpoint the molecular targets of these inhibitors, demonstrating that all target ETC Complex III, with MMV688853 additionally targeting a kinase with a key role in parasite invasion of host cells. Most of the compounds remain effective inhibitors of parasites that are resistant to the clinically used Complex III inhibitor atovaquone. In sum, we have developed a versatile screening approach to identify and characterize new inhibitors of the ETC in apicomplexan parasites.

microbiology↗

bioRxiv2021

A novel heteromeric pantothenate kinase complex in apicomplexan parasites

Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.

biochemistry↗

bioRxiv2020

Exploring heteroaromatic rings as a replacement for the labile amide of antiplasmodial pantothenamides

The Plasmodium parasites that cause malaria are adept at developing resistance to antimalarial drugs, necessitating the search for new antiplasmodials. Although several amide analogs of pantothenate (pantothenamides) show potent antiplasmodial activity, hydrolysis by pantetheinases (or vanins) present in blood rapidly inactivates them. We report herein the facile synthesis and biological activity of a small library of pantothenamide analogs in which the labile amide group is replaced with a variety of heteroaromatic rings. Several of the new analogs display antiplasmodial activity in the nanomolar range against P. falciparum and/or P. knowlesi in the presence of pantetheinase. A previously reported triazole and an isoxazole derivative presented here were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although we show here that the two compounds fail to suppress proliferation of P. berghei in vivo, pharmacokinetic and contact time data presented provide a benchmark for the compound profile required to achieve antiplasmodial activity in mice and should facilitate lead optimization.

pharmacology and toxicology↗

bioRxiv2020

Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 M and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2019

Inhibiting Mycobacterium tuberculosis CoaBC by targeting a new allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent inhibitors of M. tuberculosis CoaB, which we show to bind to a novel cryptic allosteric site within CoaB.

biochemistry↗

bioRxiv2017

Mutations in the pantothenate kinase of the malaria parasite P. falciparum confer resistance or hypersensitivity to diverse pantothenate analogues

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.

pharmacology and toxicology↗