Structure-based drug design for dihydrofolate reductase
Date of Completion
January 2008
Keywords
Biology, Molecular
Degree
Ph.D.
Abstract
Fungal diseases such as Candida infections and bacterial infections of methicillin-resistant S. aureus (MRSA) have emerged as important public health problems. There is an urgent need for new drugs that target these pathogens due to the growing resistance to the current antibiotics. Dihydrofolate reductase (DHFR) is an enzyme that plays an important role in the folate biosynthesis pathway and is crucial for cell viability, which makes it a potential target for antifungal and antibacterial drugs. Since DHFR is essential to all cells, inhibitors targeting pathogenic organisms must be selective. The objective of this thesis is to find potent and selective inhibitors against pathogen DHFRs through high-resolution structural characterization. ^ In this thesis, we describe a full cycle of structure-based drug design resulting in new lead compounds effective against DHFR from Candida glabrata. Initial assays of a series of DHFR inhibitors, which are characterized by a propargyl-based linker between the 2, 4-diaminopyrimidine and substituted aryl ring, identified several compounds with moderate potency and antifungal activity. The high resolution structure of C. glabrata DHFR complexed with one of these initial leads, UCP 120B, was determined by molecular replacement. The structure revealed an additional hydrophobic pocket that could be exploited for increased potency and selectivity for the pathogenic enzyme. Based on the first known C. glabrata DHFR structure, we designed the second generation compounds which have a newly introduced biphenyl group. Several biphenyl compounds displayed subnanomolar potency, thousand-fold levels of selectivity and superior antifungal activity at levels observed with clinically employed antifungal therapeutics. This work demonstrated the hypothesis that DHFR could be effectively targeted in a strategy to inhibit the growth of C. glabrata. ^ Subsequently, the structures of C. glabrata DHFR bound to four of the second generation compounds were determined. New information of inhibitor binding was obtained, which is helpful for the further optimization of lead compounds. The propargyl-linked compounds were also screened as inhibitors against C. albicans DHFR both in enzyme and antifungal assays. A number of compounds are found having IC50 values lower than 50 nM and moderate selectivity. ^ To explore the potential of DHFR as a drug target against S. aureus and MRSA, a group of propargyl-linked inhibitors were investigated in enzyme and antibacterial assays. Several compounds have been recognized as potent inhibitors with modest antibacterial activity. Ternary structures of S. aureus DHFR and trimethoprim-resistant S. aureus DHFR complexed with the cofactor NADPH and three potent inhibitors were determined. The structures revealed that the propargyl-linked compounds bind in the active sites of S. aureus and MRSA DHFRs flexibly with various binding conformations. ^
Recommended Citation
Liu, Jieying, "Structure-based drug design for dihydrofolate reductase" (2008). Doctoral Dissertations. AAI3334963.
https://digitalcommons.lib.uconn.edu/dissertations/AAI3334963