Abstract
Inosine 5’-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP, which is the rate-limiting step of de novo guanine nucleotide biosynthesis. The differences between mammalian IMPDHs and bacterial IMPDHs make this enzyme a promising antibiotic drug target. Selective inhibitors for bacterial IMPDHs have been developed that bind to the NAD+ site. Some inhibitors display varied potency even though the NAD+ site is highly conserved in IMPDHs from some important pathogens. Here, we hypothesized that protein dynamics, in particular the conformational movements of the active site flap, play an important role in inhibitor binding. In Chapter 2, we used IMPDH from Bacillus anthracis as a model system and introduced various point mutations in the active site flap. The most significant inhibitor-specific potency changes were observed when mutating Leu413 to Ala. Detailed kinetics studies on the wild-type enzyme and the L413A mutant involved both pre-steady-state kinetics and steady-state initial rate experiments. The results show that all inhibitors bind at least as strongly to the covalent intermediate E-XMP* as they do to the non-covalent complex E•IMP. Interactions with Leu413 determine the affinities to both complexes, for some, but not all, inhibitors. I conclude that the conformational dynamics of the flap needs to be considered in the rational design of IMPDH inhibitors.
Tuberculosis (TB) is a worldwide disease caused by bacteria called Mycobacterium tuberculosis (Mtb). The emergence of multidrug- and extensively-drug resistant TB calls for new drugs for TB treatment. While previously developed selective inhibitors for bacterial IMPDHs with different scaffolds were potent to MtbIMPDH, most of them display poor anti-tubercular activity. MtbIMPDH inhibitors with new scaffolds need to be developed. In Chapter 3, we performed screening against MtbIMPDH on compounds selected by an initial virtual HTS and identified three hits with novel scaffold. Further optimization is needed.
IMPDH controls the guanine nucleotide pool in cell. The inhibition of IMPDH causes the inhibition of cell growth. A hca pathway converting coumarate to protocatechuic acid (PCA) originates from Acinetobacter baylyi ADP1. When this pathway was transferred to E. coli, the resulting strain E. coli/hca cannot grow in the presence of coumarate. In Chapter 4, we performed in vitro studies on EcIMPDH and AbIMPDH. The results suggest that 4-Hydroxybenzaldehyde, the intermediateof hca pathway, inhibits EcIMPDH but not AbIMPDH. These results further emphasize the importance of IMPDH in cell growth.