Dataset Information

Phosphonic Acid Analogues of Phenylglycine as Inhibitors of Aminopeptidases: Comparison of Porcine Aminopeptidase N, Bovine Leucine Aminopeptidase, Tomato Acidic Leucine Aminopeptidase and Aminopeptidase from Barley Seeds.

ABSTRACT: The inhibitory activity of 14 racemic phosphonic acid analogs of phenylglycine, substituted in aromatic rings, towards porcine aminopeptidase N (pAPN) and barley seed aminopeptidase was determined experimentally. The obtained patterns of the inhibitory activity against the two enzymes were similar. The obtained data served as a basis for studying the binding modes of these inhibitors by pAPN using molecular modeling. It was found that their aminophosphonate fragments were bound in a highly uniform manner and that the difference in their affinities most likely resulted from the mode of substitution of their phenyl rings. The obtained binding modes towards pAPN were compared, with these predicted for bovine lens leucine aminopeptidase (blLAP) and tomato acidic leucine aminopeptidase (tLAPA). The performed studies indicated that the binding manner of the phenylglycine analogs to biLAP and tLAPA are significantly similar and differ slightly from that predicted for pAPN.

SUBMITTER: Wanat W

PROVIDER: S-EPMC6789573 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Phosphonic Acid Analogues of Phenylglycine as Inhibitors of Aminopeptidases: Comparison of Porcine Aminopeptidase N, Bovine Leucine Aminopeptidase, Tomato Acidic Leucine Aminopeptidase and Aminopeptidase from Barley Seeds.

Wanat Weronika W Talma Michał M Pawełczak Małgorzata M Kafarski Paweł P

Pharmaceuticals (Basel, Switzerland) 20190917 3

The inhibitory activity of 14 racemic phosphonic acid analogs of phenylglycine, substituted in aromatic rings, towards porcine aminopeptidase N (pAPN) and barley seed aminopeptidase was determined experimentally. The obtained patterns of the inhibitory activity against the two enzymes were similar. The obtained data served as a basis for studying the binding modes of these inhibitors by pAPN using molecular modeling. It was found that their aminophosphonate fragments were bound in a highly unifo ...[more]

PMID: 31533309

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Project description:Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

Project description:BACKGROUND: Leucine aminopeptidase (LAP) has been known to be a housekeeping protease, DNA-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. LAP activity was consistently detected in overnight cultures of Burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. The intra- and inter-species nucleotide and deduced amino acid sequence variation of LAP encoding gene (pepA) was determined. A pepA/PCR-RFLP assay was designed to facilitate the identification of major LAP sequence types amongst clinical and environmental isolates of B. pseudomallei. RESULTS: LAP activity was detected in B. pseudomallei culture supernantants by zymographic analysis. Optimum activity was at pH 9 and stable at 50[degree sign]C. Enhanced enzymatic activity was observed in the presence of metallic ions Mg2+, Ca2+, Na+ and K+. LAP activity was inhibited by EDTA, 1,10-phenanthroline, amastatin, Mn2+ and Zn2+. Sequence analysis of the complete nucleotide and deduced amino acid sequences of LAP-encoding (pepA) gene showed close genetic relatedness to B. mallei (similarity 99.7%/99.6%), but not with B. thailandensis (96.4%/96.4%). Eight pepA sequence types were identified by comparison with a 596 bp DNA fragment encompassing central regions of the pepA gene. A pepA/PCR-RFLP was designed to differentiate pepA sequence types. Based on restriction analysis with StuI and HincII enzymes of the amplified pepA gene, clinical and environmental isolates showed different predominant RFLP types. Type I was the most predominant type amongst 71.4% (65/91) of the clinical isolates, while Type II was predominant in 55.6% (5/9) of the environmental isolates. CONCLUSIONS: This study showed that LAP is a secretory product of B. pseudomallei with features similar to LAP of other organisms. Identification of major LAP sequence types of B. pseudomallei was made possible based on RFLP analysis of the pepA gene. The high LAP activity detected in both B. pseudomallei and B. thailandensis, suggests that LAP is probably a housekeeping enzyme rather than a virulence determinant.

Dataset Information

Phosphonic Acid Analogues of Phenylglycine as Inhibitors of Aminopeptidases: Comparison of Porcine Aminopeptidase N, Bovine Leucine Aminopeptidase, Tomato Acidic Leucine Aminopeptidase and Aminopeptidase from Barley Seeds.

Publications

Phosphonic Acid Analogues of Phenylglycine as Inhibitors of Aminopeptidases: Comparison of Porcine Aminopeptidase N, Bovine Leucine Aminopeptidase, Tomato Acidic Leucine Aminopeptidase and Aminopeptidase from Barley Seeds.

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