10 research outputs found
Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth
A class of hydrazones are active against non-replicating Mycobacterium tuberculosis.
There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations of Mycobacterium tuberculosis. We previously identified a series of hydrazones active against M. tuberculosis. We selected five representative compounds for further analysis. All compounds were active against non-replicating M. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of < 4 and demonstrated an inoculum-dependent effect against aerobically replicating bacteria. Bacterial kill kinetics demonstrated a faster rate of kill against non-replicating bacilli generated by nutrient starvation. Compounds had limited activity against other bacterial species. In conclusion, we have demonstrated that hydrazones have some attractive properties in terms of their anti-tubercular activity
Mechanism and Catalytic Strategy of the Prokaryotic Specific GTP Cyclohydrolase IB
GTP cyclohydrolase I catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine modified tRNA nucleosides in bacteria and archaea. The type IB GTP cyclohydrolase (GCYH-IB) is a prokaryotic-specific enzyme found in a number of pathogens. GCYH-IB is structurally distinct from the canonical type IA GTP cyclohydrolase involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB, and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxo-GTP, and one with a TRIS molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP reveals an inverted mode of binding of the inhibitor ribosyl moiety and, together with site-directed mutagenesis data, shows that the two enzymes utilize different strategies for catalysis. Notably, the inhibitor interacts with a conserved active site Cys149, and this residue is S-nitrosylated in the structures. This is the first structural characterization of a biologically S-nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S-nitrosylation maintains enzyme activity, suggesting a potential role of the S-nitrosothiol in catalysis
Functional Modular Dissection of DEBS1-TE Changes Triketide Lactone Ratios and Provides Insight into Acyl Group Loading, Hydrolysis, and ACP Transfer
The DEBS1-TE fusion protein is comprised of the loading
module,
the first two extension modules, and the terminal TE domain of the <i>Saccharopolyspora erythraea</i> 6-deoxyerythronolide B synthase.
DEBS1-TE produces triketide lactones that differ on the basis of the
starter unit selected by the loading module. Typical fermentations
with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate
to isobutyrate-derived triketide lactones. Functional dissection of
the loading module from the remainder of DEBS1-TE results in 50% lower
titers of triketide lactone and a dramatic shift in the production
to a 1:4 ratio of propionate to isobutyrate-derived products. A series
of radiolabeling studies of the loading module has shown that transfer
from the AT to the ACP occurs much faster for propionate than for
isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate
such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP
is the kinetic product, while isobutyryl-ACP is the thermodynamic
product. A slowed transfer from the loading domain ACP to first-extension
module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored
equilibrium to be realized and likely accounts for the observed shift
in triketide lactone products