Structural, functional characterization, and fragment screening for dihydrofolate reductase from invasive pathogenic fungi

• Grant number: 24/22643-5
• Support Opportunities: Regular Research Grants
• Start date: June 01, 2025
• End date: May 31, 2028
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Marcio Vinicius Bertacine Dias
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• City of the host institution: São Paulo
• Associated researchers: Kelly Ishida ; Rafaela Salgado Ferreira ; Roberto Parise Filho
• Associated scholarship(s): 25/24805-5 - Application of a fragment-based drug discovery approach to the dihydrofolate reductase from Candida auris, BP.IC; 25/15258-0 - Development and maintenance of a compound library for the development of drugs based on fragments., BP.TT; 25/13004-1 - Production, crystallization, and crystallographic structure determination of dihydrofolate reductase from Cryptococcus neoformans, BP.IC

Abstract

Infectious diseases are one of the major problems of the current public and worldwide health. Although much has been released about infectious caused by bacteria, those infectious diseases caused by invasive fungi are also alarming and lead to the death of a high number of patients, particularly those from hospital environments or those suffering from immunodeficiency. Furthermore, there are only a few treatment alternatives for invasive fungi infections, which are often associated with strong side effects, and there is an increased number of reports about the emergence of resistant strains to used antifungals. Thus, it is urgent to study molecular targets and also discover new antifungals for the control of these diseases, particularly those caused by Aspergillus fumigatus, Paracoccidioides brasiliensis, Cryptococcus neoformans, and Candida auris. In this project, the main objective is to study the enzyme dihydrofolate reductase (DHFR) from these four pathogenic fungi. This enzyme, which is part of tetrahydrofolate (THF) biosynthesis, is essential for these microorganisms since this coenzyme is crucial for the biosynthesis of DNA and several amino acids. Thus, we aim to determine the three-dimensional structures and obtain structural and functional data about the interaction of these DHFRs with antifolates. Additionally, we will apply a fragment-based drug discovery approach to identify molecules that could have novel chemical scaffolds that interact with these enzymes and could lead to the development of entirely new inhibitors for these targets. To achieve our objectives, we will perform the heterologous expression of the interest enzymes in the bacterial system and purify the enzymes using affinity and molecular exclusion chromatography. The different enzymes will be characterized using biophysical and biochemical techniques, including protein crystallography, thermal shift assay, isothermal titration calorimetry (ITC), and activity assay based on NADPH consumption. For the fragment screening, we will use the thermal shift assay against a library of about 600 molecules in our laboratory. The orthogonal validation of the fragment binding will be performed using nuclear magnetic resonance (NMR), ITC, and NADHP consumption. The binding mode of those molecules validated to Interact with the targets will be obtained by protein crystallography. Based on the fragment obtained, strategies to obtain compounds with higher affinity will be applied, including SAR by catalog, methods of artificial intelligence, and organic chemistry. The best obtained compounds will be tested against the different pathogenic fungi targets of this project to determine the effect of in vivo growth and the determination of the minimal inhibitory concentration. At the end of the project, we expect to obtain crucial information about the antifolate's binding mode and affinity parameters against pathogenic fungi and identify molecules that could be the starting point for developing new antifungals. (AU)