Crystal structure of dihydroneopterin aldolase from Mycobacterium tuberculosis associated with 8-mercaptoguanine, and development of novel S8-functionalized analogues as inhibitors: Synthesis, enzyme inhibition, in vitro toxicity and antitubercular activity

Alexia de Matos Czeczot; Mauro Neves Muniz; Marcia Alberton Perelló; Éverton Edésio Dinis Silva; Luís Fernando Saraiva Macedo Timmers; Andresa Berger; Laura Calle Gonzalez; Guilherme Arraché Gonçalves; Sidnei Moura; Pablo Machado; Cristiano Valim Bizarro; Luiz Augusto Basso

doi:10.1080/14756366.2024.2388207

Crystal structure of dihydroneopterin aldolase from Mycobacterium tuberculosis associated with 8-mercaptoguanine, and development of novel S8-functionalized analogues as inhibitors: Synthesis, enzyme inhibition, in vitro toxicity and antitubercular activity

J Enzyme Inhib Med Chem. 2024 Dec;39(1):2388207. doi: 10.1080/14756366.2024.2388207. Epub 2024 Aug 14.

Authors

Alexia de Matos Czeczot^{1

2}, Mauro Neves Muniz¹, Marcia Alberton Perelló¹, Éverton Edésio Dinis Silva¹, Luís Fernando Saraiva Macedo Timmers³, Andresa Berger^{1

4}, Laura Calle Gonzalez^{1

4}, Guilherme Arraché Gonçalves^{1

2}, Sidnei Moura⁵, Pablo Machado^{1

2

4}, Cristiano Valim Bizarro^{1

4}, Luiz Augusto Basso^{1

2

4}

Affiliations

¹ Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil.
² Programa de Pós-Graduação em Medicina e Ciências da Saúde, Escola de Medicina, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil.
³ Programa de Pós-Graduação em Biotecnologia, Universidade do Vale do Taquari, Lajeado, Brazil.
⁴ Programa de Pós-Graduação em Biologia Celular e Molecular, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil.
⁵ Laboratório de Biotecnologia de Produtos Naturais e Sintéticos, Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, Rio Grande do Sul, Brazil.

Abstract

The crystallographic structure of the FolB enzyme from Mycobacterium tuberculosis (MtFolB), complexed with its inhibitor 8-mercaptoguanine (8-MG), was elucidated at a resolution of 1.95 Å. A novel series of S8-functionalized 8-MG derivatives were synthesised and evaluated as in vitro inhibitors of dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of MtFolB. These compounds exhibited IC₅₀ values in the submicromolar range. Evaluation of the activity for five compounds indicated their inhibition mode and inhibition constants. Molecular docking analyses were performed to determine the enzyme-inhibitor intermolecular interactions and ligand conformations upon complex formation. The inhibitory activities of all compounds against the M. tuberculosis H37Rv strain were evaluated. Compound 3e exhibited a minimum inhibitory concentration in the micromolar range. Finally, Compound 3e showed no apparent toxicity in both HepG2 and Vero cells. The findings presented herein will advance the quest for novel, specific inhibitors targeting MtFolB, an attractive molecular target for TB drug development.

Keywords: 8-mercaptoguanine; Dihydroneopterin aldolase inhibitors; Mycobacterium tuberculosis; SAR.

MeSH terms

Aldehyde-Lyases* / antagonists & inhibitors
Aldehyde-Lyases* / chemistry
Aldehyde-Lyases* / metabolism
Animals
Antitubercular Agents* / chemical synthesis
Antitubercular Agents* / chemistry
Antitubercular Agents* / pharmacology
Chlorocebus aethiops
Crystallography, X-Ray
Dose-Response Relationship, Drug*
Enzyme Inhibitors* / chemical synthesis
Enzyme Inhibitors* / chemistry
Enzyme Inhibitors* / pharmacology
Guanine / analogs & derivatives
Guanine / chemical synthesis
Guanine / chemistry
Guanine / pharmacology
Hep G2 Cells
Humans
Microbial Sensitivity Tests*
Models, Molecular
Molecular Docking Simulation
Molecular Structure
Mycobacterium tuberculosis* / drug effects
Mycobacterium tuberculosis* / enzymology
Structure-Activity Relationship
Vero Cells

Substances

Antitubercular Agents
Enzyme Inhibitors
dihydroneopterin aldolase
Aldehyde-Lyases
Guanine

Grants and funding

This work was supported by Banco Nacional de Desenvolvimento Econômico e Social (BNDES) [grant number 14.2.0914.1] and the National Institute of Science and Technology on Tuberculosis (CNPq-FAPERGS-CAPES) [grant numbers: 421703–2017-2 and 17–1265-8]. C. V. Bizarro, L. A. Basso, and P. Machado are Research Career Awardees of the National Research Council of Brazil (CNPq). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior, Brasil (CAPES), Finance Code 001.