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1.
Biochemistry ; 61(19): 2088-2105, 2022 10 04.
Article in English | MEDLINE | ID: mdl-36193631

ABSTRACT

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.


Subject(s)
Chagas Disease , Trypanosoma cruzi , Antiparasitic Agents , Guanine/metabolism , Guanosine Monophosphate , Humans , Hypoxanthine Phosphoribosyltransferase/chemistry , Hypoxanthine Phosphoribosyltransferase/genetics , Hypoxanthine Phosphoribosyltransferase/metabolism , Inosine Monophosphate , Isoenzymes , Purines/metabolism , Purines/pharmacology
2.
Bioorg Med Chem ; 74: 117038, 2022 11 15.
Article in English | MEDLINE | ID: mdl-36209571

ABSTRACT

Phosphate groups play essential roles in biological processes, including retention inside biological membranes. Phosphodiesters link nucleic acids, and the reversible transfer of phosphate groups is essential in energy metabolism and cell-signalling processes. Phosphorylated metabolic intermediates are known targets for metabolic and disease-related disorders, and the enzymes involved in these pathways recognize phosphate groups in their catalytic sites. Therapeutics that target these enzymes can require charged (ionic) entities to capture the binding energy of ionic substrates. Such compounds are not cell-permeable and require pro-drug strategies for efficacy as therapeutics. Protozoan parasites such as Plasmodium and Trypanosoma spp. are unable to synthesise purines de novo and rely on the salvage of purines from the host cell to synthesise free purine bases. Purine phosphoribosyltransfereases (PPRTases) play a crucial role for purine salvage and are potential target for drug development. Here we present attempts to design inhibitors of PPRTases that are non-ionic and show affinity for the nucleotide 5'-phosphate binding site. Inhibitor design was based on known potent ionic inhibitors, reported phosphate mimics and computational modelling studies.


Subject(s)
Parasites , Plasmodium , Animals , Phosphates , Purines/pharmacology , Purines/metabolism , Hypoxanthine Phosphoribosyltransferase
3.
Int J Mol Sci ; 22(4)2021 Feb 23.
Article in English | MEDLINE | ID: mdl-33672312

ABSTRACT

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.


Subject(s)
Bacterial Proteins/metabolism , Hydroxymethyl and Formyl Transferases/metabolism , Mixed Function Oxygenases/metabolism , Siderophores/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Catalytic Domain , Hydroxymethyl and Formyl Transferases/genetics , Hydroxymethyl and Formyl Transferases/isolation & purification , Mixed Function Oxygenases/genetics , Mutagenesis, Site-Directed , Oligopeptides/biosynthesis , Protein Interaction Maps , Protein Stability , Pseudomonas aeruginosa/metabolism
4.
J Org Chem ; 82(10): 5317-5327, 2017 05 19.
Article in English | MEDLINE | ID: mdl-28440639

ABSTRACT

A tandem deprotection-cyclization reaction of 1,1-diacylcyclopropanes is described which allows rapid access to structurally diverse 2,3-disubstituted chromones in good yields, and with straightforward purification. The utility of this reaction is showcased by the construction of the potent antibacterial marine natural product bromophycoic acid E scaffold.

5.
J Org Chem ; 79(5): 2111-4, 2014 Mar 07.
Article in English | MEDLINE | ID: mdl-24517486

ABSTRACT

The first total synthesis of asterredione was efficiently accomplished over five linear steps and in 21.5% overall yield. As the crucial step, the 2-quaternary 1,3-cyclopentenedione skeleton of asterredione was readily achieved using the Darzens/ring-expansion strategy developed in our laboratory. The structure of synthesized asterredione was fully confirmed by X-ray crystallography.


Subject(s)
Cyclopentanes/chemistry , Indoles/chemical synthesis , Crystallography, X-Ray , Cyclopentanes/chemical synthesis , Indoles/chemistry , Molecular Structure , Stereoisomerism
6.
Org Lett ; 21(8): 2872-2875, 2019 04 19.
Article in English | MEDLINE | ID: mdl-30924665

ABSTRACT

A rapid and facile synthesis of benzannulated 6,5-spiroketals from vinyl 1,1-diacylcyclopropanes is reported. The method utilizes mild reaction conditions with good to excellent yields and high diastereoselectivity. This methodology was then used to construct the core of berkelic acid.

7.
Chem Asian J ; 14(8): 1102-1105, 2019 Apr 15.
Article in English | MEDLINE | ID: mdl-30552751

ABSTRACT

The selective mono-allylation of 1,3-diketone containing compounds is described. The reaction proceeds under mild reaction conditions and in moderate to high yield (66-99 %). Using this procedure to access the key mono-allylated intermediate, the hitherto difficult to access 3-allyl chromones were synthesized in excellent yield (87-98 %). Finally, the utility of this newly developed procedure was showcased through the rapid synthesis of the scaffold of the xyloketal family of natural products.

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