Maday Alonso del Rivero

Crystal Structure of Novel Metallocarboxypeptidase Inhibitor from Marine Mollusk Nerita versicolor in Complex with Human Carboxypeptidase A4

Background: Only a few proteinaceous inhibitors of metallocarboxypeptidases have been characterized structurally in depth. Results: The structure of human carboxypeptidase A4 in complex with a Nerita versicolor inhibitor (NvCI) was derived at 1.7 Å. Conclusion: NvCI displays a different protein fold that inhibits carboxypeptidases in a substrate-like manner. Significance: We deciphered the structural determinants for picomolar inhibition constants for A-type carboxypeptidases, the most potent by now. NvCI is a novel exogenous proteinaceous inhibitor of metallocarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 Å resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.

Intensity fading MALDI-TOF mass spectrometry and functional proteomics assignments to identify protease inhibitors in marine invertebrates

Proteases and their inhibitors have become molecules of increasing fundamental and applicative value. Here we report an integrated strategy to identify and analyze such inhibitors from Caribbean marine invertebrates extracts by a fast and sensitive functional proteomics-like approach. The strategy works in three steps: i) multiplexed enzymatic inhibition kinetic assays, ii) Intensity Fading MALDI-TOF MS to establish a link between inhibitory molecules and the related MALDI signal(s) detected in the extract(s), and iii) ISD-CID-T3 MS fragmentation on the parent MALDI signals selected in the previous step, enabling the partial or total top-down sequencing of the molecules. The present study has allowed validation of the whole approach, identification of a substantial number of novel protein protease inhibitors, as well as full or partial sequencing of reference molecular species and of many unknown ones, respectively. Such inhibitors correspond to six protease subfamilies (metallocarboxypeptidases-A and -B, pepsin, papain, trypsin and subtilisin), are small (1-10KDa) disulfide-rich proteins, and have been found at diverse frequencies among the invertebrates (13 to 41%). The overall procedure could be tailored to other enzyme-inhibitor and protein interacting systems, analyzing samples at medium-throughput level and leading to the functional and structural characterization of proteinaceous ligands from complex biological extracts. SIGNIFICANCE Invertebrate animals, and marine ones among, display a remarkable diversity of species and contained biomolecules. Many of their proteins-peptides have high biological, biotechnological and biomedical potential interest but, because of the lack of sequenced genomes behind, their structural and functional characterization constitutes a great challenge. Here, looking at the small, disulfide-rich, proteinaceous inhibitors of proteases found in them, it is shown that such problem can be significatively facilitated by integrative multiplexed enzymatic assays, affinity-based Intensity-Fading (IF-) MALDI-TOF mass spectrometry (MS), and on-line MS fragmentation, in a fast and easy approach.

KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC50=18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.

203 The crystal structure of a tri domain BPTI Kunitz inhibitor in complex with human carboxypeptidase A4 revealed a novel and non canonical inhibition mechanism.

References Grossman, L. & Yeung, A. T. (1990). The UvrABC endonuclease system of Escherichia coli—A view from Baltimore. Mutation Research/DNA Repair, 236, 213–221. Munikumar, M., Priyadarshini, I.V., Pradhan, D., Swargam, S., & Umamaheswari, A. (2013). 177 T-cell vaccine design for Streptococcus pneumoniae: An in silico approach. Journal of Biomolecular Structure and Dynamics, 31, 114–115. Priyadarshini, I. V., Pradhan, D., Munikumar, M., Swargam, S., Umamaheswari, A., & Rajasekhar, D. (2014). Genomebased approaches to develop epitope-driven subunit vaccines against pathogens of infective endocarditis. Journal of Bimolecular Structure and Dynamics. doi:10.1080/ 07391102.2013.79587