Development of Inhibitors of SAICAR Synthetase (PurC) from <i>Mycobacterium abscessus</i> Using a Fragment-Based Approach

Charoensutthivarakul, Sitthivut; Thomas, S.E.; Curran, A.H.; Brown, Karen; Belardinelli, Juán Manuel; Whitehouse, Andrew; Acebrón-García-de-Eulate, Marta; Sangan, Jaspar; Govindan, Subramanian; Jackson, Mary; Mendes, V.; Floto, R. Andres; Blundell, Tom L.; Coyne, Anthony G.; Abell, Chris

doi:10.1021/acsinfecdis.1c00432

Cited by 11 publications

(10 citation statements)

References 53 publications

(111 reference statements)

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“…Substrate/product-based inhibitors of PAICS homologues have been synthesized in the past as tools in enzymatic analysis 10 and recent efforts have been made to develop inhibitors of prokaryotic PAICS homologues to combat antibiotic resistance. 11 …”

Section: Introductionmentioning

confidence: 99%

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations

et al. 2022

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Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N -succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO 2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation–condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C–C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation–condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.

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Section: Introductionmentioning

confidence: 99%

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations

et al. 2022

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“…Structures of M. tuberculosis PurN, PurH, and PurF, which are involved in de novo purine synthesis, revealed structural distinctions from the equivalent human enzymes, suggesting that specific inhibitors could be designed against these proteins [57-59]. Mycobacterium abscessus PurC is also structurally distinct from the human ortholog, and PurC inhibitors that prevent M. tuberculosis growth in vitro were recently described [60]. An indazole sulfonamide compound that inhibits GuaB2 was cidal against replicating M. tuberculosis in vitro but lacked activity in infected mice due to either low drug concentration in lung lesions, slower growth of M. tuberculosis in chronically infected mice, or high levels of guanine in host tissues that antagonize the drug [61].…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium tuberculosisrequires conditionally essential metabolic pathways for infection

Block,

Wiegert,

Namugenyi

et al. 2023

Preprint

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New drugs are needed to shorten and simplify treatment of tuberculosis caused by Mycobacterium tuberculosis. Metabolic pathways that M. tuberculosis requires for growth or survival during infection represent potential targets for anti-tubercular drug development. Genes and metabolic pathways essential for M. tuberculosis growth in standard laboratory culture conditions have been defined by genome-wide genetic screens. However, whether M. tuberculosis requires these essential genes during infection has not been comprehensively explored because mutant strains cannot be generated using standard methods. Here we show that M. tuberculosis requires functional phenylalanine (Phe) and de novo purine and thiamine biosynthetic pathways for mammalian infection. We used a defined collection of M. tuberculosis transposon (Tn) mutants in essential genes, which we generated using a custom nutrient-rich medium, and transposon sequencing (Tn-seq) to identify multiple central metabolic pathways required for fitness in a mouse infection model. We confirmed by individual retesting and complementation that mutations in pheA (Phe biosynthesis) or purF (purine and thiamine biosynthesis) cause death of M. tuberculosis in the absence of nutrient supplementation in vitro and strong attenuation in infected mice. Our findings show that Tn-seq with defined Tn mutant pools can be used to identify M. tuberculosis genes required during mouse lung infection. Our results also demonstrate that M. tuberculosis requires Phe and purine/thiamine biosynthesis for survival in the host, implicating these metabolic pathways as prime targets for the development of new antibiotics to combat tuberculosis.

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“…From a structural perspective, both the FtsZ nucleotide and the allosteric binding sites show annex cavities available for fragment binding ( Figure 1 and Figure 6 ). This suggests the possibility of applying HT X-ray crystallographic screening of fragment libraries and further structure-guided fragment-based approaches [ 82 ] to develop new FtsZ inhibitors. Several NMR methods may be also applied to search for FtsZ-binding fragments, including ligand-based fluorine NMR screening [ 83 ].…”

Section: Outlook For Other Methods Vs and Machine Learning Approaches...mentioning

confidence: 99%

The Search for Antibacterial Inhibitors Targeting Cell Division Protein FtsZ at Its Nucleotide and Allosteric Binding Sites

et al. 2022

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The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed.

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Development of Inhibitors of SAICAR Synthetase (PurC) from Mycobacterium abscessus Using a Fragment-Based Approach

Cited by 11 publications

References 53 publications

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations

Mycobacterium tuberculosisrequires conditionally essential metabolic pathways for infection

The Search for Antibacterial Inhibitors Targeting Cell Division Protein FtsZ at Its Nucleotide and Allosteric Binding Sites

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