Methionine
aminopeptidases (MetAPs) are essential enzymes that
make them good drug targets in cancer and microbial infections. MetAPs
remove the initiator methionine from newly synthesized peptides in
every living cell. MetAPs are broadly divided into type I and type
II classes. Both prokaryotes and eukaryotes contain type I MetAPs,
while eukaryotes have additional type II MetAP enzyme. Although several
inhibitors have been reported against type I enzymes, subclass specificity
is scarce. Here, using the fine differences in the entrance of the
active sites of MetAPs from Mycobacterium tuberculosis, Enterococcus faecalis, and human,
three hotspots have been identified and pyridinylpyrimidine-based
molecules were selected from a commercial source to target these hotspots.
In the biochemical evaluation, many of the 38 compounds displayed
differential behavior against these three enzymes. Crystal structures
of four selected inhibitors in complex with human MetAP1b and molecular
modeling studies provided the basis for the binding specificity.
Methionine aminopeptidase (MetAP) represents a unique class of proteases that is responsible for removing the N-terminal initiator methionine from newly synthesized proteins. The lone MetAP gene in prokaryotes is essential for the survival of the microorganism suggesting that it could be used as a drug target. Here, we describe the crystal structure of the Enterococcus faecalis MetAP in the apo-form, biochemical characterization, metal affinity and small molecule library screening. Enzyme inhibition and modeling studies of the best inhibitor, 2,2 0 -bipyridine, were performed. Employing the molecular modeling tools, 2,2 0 -bipyridine derivatives were generated that could specifically inhibit class specific MetAPs.
Escherichia coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic domain (Domain II) limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket, while making critical contact with the amino-terminus of the substrate. In addition, the carboxylate of E121 forms a salt bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu-Asn pair in tricon interacting factor F3 (F3) and Gln-Asn pair in human leukotriene A 4 hydrolase (LTA 4 H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7°C, while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3, and LTA 4 H homologs adopted a divergent evolution that includes E121-K319 or its analogous pairs, and these cannot be interchanged.
Flipping out! The X‐ray crystal structure of L‐α,γ‐diaminobutyric acid (DABA, shown in magenta) in complex with an M1 family aminopeptidase reveals that the γ‐amino group displaces the α‐amino group from its original position (as in the complex with lysine, shown in cyan), which in turn results in a peptide flip at Met 260.
Both glycolate oxidase (GO) and lactate
dehydrogenase A (LDHA)
influence the endogenous synthesis of oxalate and are clinically validated
targets for treatment of primary hyperoxaluria (PH). We investigated
whether dual inhibition of GO and LDHA may provide advantage over
single agents in treating PH. Utilizing a structure-based drug design
(SBDD) approach, we developed a series of novel, potent, dual GO/LDHA
inhibitors. X-ray crystal structures of compound 15 bound
to individual GO and LDHA proteins validated our SBDD strategy. Dual
inhibitor 7 demonstrated an IC50 of 88 nM
for oxalate reduction in an Agxt-knockdown mouse
hepatocyte assay. Limited by poor liver exposure, this series of dual
inhibitors failed to demonstrate significant PD modulation in an in vivo mouse model. This work highlights the challenges
in optimizing in vivo liver exposures for diacid
containing compounds and limited benefit seen with dual GO/LDHA inhibitors
over single agents alone in an in vitro setting.
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