Inhibition of pathogenic bacterial carbonic anhydrases by monothiocarbamates.

Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195-0.959 µM and of 0.149-1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.

Structural Characterization of Thiadiazolesulfonamide Inhibitors Bound to Neisseria gonorrhoeae α-Carbonic Anhydrase.

Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.

Structure-Activity Relationship Studies of Acetazolamide-Based Carbonic Anhydrase Inhibitors with Activity against Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an urgent threat to public health in the United States and around the world. Many of the current classes of antibiotics to treat N. gonorrhoeae infection are quickly becoming obsolete due to increased rates of resistance. Thus, there is a critical need for alternative antimicrobial targets and new chemical entities. Our team has repurposed the FDA-approved carbonic anhydrase inhibitor scaffold of acetazolamide to target N. gonorrhoeae and the bacteria's essential carbonic anhydrase, NgCA. This study established both structure-activity and structure-property relationships that contribute to both antimicrobial activity and NgCA activity. This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 µg/mL equating to an 8- to 16-fold improvement in antigonococcal activity compared to acetazolamide. These analogues were determined to be bacteriostatic against the pathogen and likely on-target against NgCA. Additionally, they did not exhibit any detrimental effects in cellular toxicity assays against both a human endocervical (End1/E6E7) cell line or colorectal adenocarcinoma cell line (Caco-2) at concentrations up to 128 µg/mL. Taken together, this study presents a class of antigonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models.

Puromycin, a selective inhibitor of PSA acts as a substrate for other M1 family aminopeptidases: Biochemical and structural basis.

Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes including human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hydrolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puromycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the entrance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis.

Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Discovery of a new class of type 1 methionine aminopeptidases that have relaxed substrate specificity.

Methionine aminopeptidases (MetAPs) are a class of enzymes evolved to cleave initiator methionine in 60-70% of the total cellular proteins in all living cells. Based on their sequence differences, they are classified into Type 1 and Type 2. Type 1 is further divided into Type 1a, 1a', 1b, 1c and 1d. Irrespective of various classifications, all MetAPs reported till date displayed hydrolytic activity against peptides that contain only methionine on the N-terminus. A cysteine at the top of the active site in all the Type 1 structures is reported to be critical for the specificity. Mutation of this cysteine to serine or asparagine leads to loss of specificity. In the present study, we have identified a class of MetAPs in some of the proteobacteria that have an asparagine at this site. Most of the proteobacteria that contain MetAP1n are pathogenic in nature. Biochemical and structural studies on two proteins, one from each of V. coralliilyticus and K. pneumoniae confirm that these enzymes cleave leucine in addition to methionine. Crystallographic and homology modeling studies suggest that relaxed substrate specificity of this new class of enzymes could be due to the increased flexibility in the active site. Since this new class has an asparagine at the critical position that probably contributes for the relaxed substrate specificity and also differentiates them from other Type 1 MetAPs, we classified them as Type 1n.

Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

Discovery, Structural and Biochemical Studies of a rare Glu/Asp Specific M1 Class Aminopeptidase from Legionella pneumophila.

Aminopeptidases catalyze the hydrolysis of amino acids from the N-terminus of protein or peptide substrates. M1 family aminopeptidases are important for the pathogenicity of bacteria and play critical role in many physiological processes such as protein maturation, regulation of peptide hormone levels in humans. Most of the M1 family aminopeptidases reported till date display broad substrates specificity, mostly specific to basic and hydrophobic residues. In the current study we report the discovery of a novel M1 class aminopeptidase from Legionella pneumophila (LePepA), which cleaves only acidic residues. Biochemical and structural studies reveal that the S1 pocket is polar and positively charged. Bioinformatic analysis suggests that such active site is unique to only Legionella species and probably evolved for special needs of the microbe. Given its specific activity, LePepA could be useful in specific biotechnological applications.

Highly functionalized tetrahydropyridines are cytotoxic and selective inhibitors of human puromycin sensitive aminopeptidase.

Efficient one-pot five-component synthetic protocols for highly functionalized tetrahydropyridines (THPs) and their biological evaluation have been illustrated. Synthesis of novel functionalized tetrahydropyridines containing differential substitutions at 2,6-positions has been achieved via a modified MCR. Cytotoxic studies of 23 synthesized compounds have been carried out against three different cell lines, namely A-549, HeLa and HepG2, wherein some compounds have displayed appreciable cytotoxicity. Further, investigation of enzyme inhibition by the synthesized THPs has been carried out against four members of M1 family aminopeptidases. Several compounds have selectively inhibited only one member of this enzyme family i.e., human puromycin sensitive aminopeptidase (hPSA). Among the compounds; 4b, 9b, 9e and 10a demonstrated best inhibition against hPSA.

Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N.

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

Identification, biochemical and structural evaluation of species-specific inhibitors against type I methionine aminopeptidases.

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.