Protonectin peptides target lipids, act at the interface and selectively kill metastatic breast cancer cells while preserving morphological integrity.

Despite the need for innovative compounds as antimicrobial and anticancer agents, natural sources of peptides remain underexplored. Protonectin (PTN), a cationic dodecapeptide of pharmacological interest, presents large hydrophobicity that is associated with the tendency to aggregate and supposedly influences bioactivity. A disaggregating role was assigned to PTN' N-terminal fragment (PTN1-6), which enhances the bioactivity of PTN in a 1:1 mixture (PTN/PTN1-6). Spectroscopic techniques and model membranes (phospholipid bilayers and SDS micelles) revealed that environment-dependent aggregation is reduced for PTN/PTN1-6, but cytotoxicity of PTNs on MDA-MB-231 breast cancer showed the same CC50 values around 16 µM and on MCF-10A epithelial breast cells 6 to 5-fold higher values, revealing a selective interaction. Since PTN1-6 lacks activity on breast cells, its presence should differently affect PTN activity, suggesting that aggregation could modulate activity depending on the membrane characteristics. Indeed, increased partitioning and lytic activity of PTN/PTN1-6 were found in model membranes independently of charge density, but affected by the curvature tendency. PTN and PTN/PTN1-6 do not alter morphology and roughness of cancer cells, indicating a superficial interaction with membranes and consistent with results obtained in NMR experiments. Our results indicate that aggregation of PTNs depends on the membrane characteristics and modulates the activity of the peptides.

Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine.

Crotamine is a polypeptide isolated from the venom of a South American rattlesnake. Among the properties and biological activities of crotamine, the most extraordinary is its ability to enter cells with unique selective affinity and cytotoxic activity against actively proliferating cells, such as tumor cells. This peptide is also a cargo carrier, and anticipating commercial application of this native polypeptide as a potential theranostic compound against cancer, we performed here a side-by-side characterization of a chemically synthesized full-length crotamine compared with its native counterpart. The structural, biophysical, and pharmacological properties were evaluated. Comparative NMR studies showed structural conservation of synthetic crotamine. Moreover, similarly to native crotamine, the synthetic polypeptide was also capable of inhibiting tumor growth in vivo, increasing the survival of mice bearing subcutaneous tumor. We also confirmed the ability of synthetic crotamine to transfect and transport DNA into eukaryotic cells, in addition to the importance of proteoglycans on cell surface for its internalization. This work opens new opportunities for future evaluation of chimeric and/or point-mutated analogs of this snake polypeptide, aiming for improving crotamine properties and applications, as well as possibly diminishing its potential toxic effects. KEY MESSAGES: • Synthetic crotamine showed ex vivo and in vivo activities similar to native peptide. • Synthetic crotamine structure conservation was demonstrated by NMR analysis. • Synthetic crotamine is able to transfect and transport DNA into eukaryotic cells. • Synthetic crotamine shows tumor growth inhibition in vivo. • Synthetic crotamine increases survival of mice bearing tumor.

Membrane targeting peptides toward antileishmanial activity: Design, structural determination and mechanism of interaction.

BACKGROUND: Leishmaniasis threatens poor areas population worldwide, requiring new drugs less prone to resistance development. Antimicrobial peptides with antileishmanial activity are considered among fulfilling alternatives, but not much is known about the mode of action of membrane-targeting peptides, considering promastigote and infected macrophage membranes. In a previous work, structural features of very active known peptides were prospected using molecular dynamics simulations. METHODS: Combining sequences of these peptides, analogs were designed. The structure of analog DecP-11 was validated by NMR. In vitro bioassays determined the peptide cytotoxicity toward mammalian cells, IC50 values on promastigotes and amastigotes, and membranolytic activity compared to Decoralin, one of the parent peptides. With biophysical methods, the mechanism of interaction with membrane mimetic systems was investigated. RESULTS: The designed peptide exhibits potent cytolytic and membrane permeabilizing activities, and decreased antileishmanial activity compared to the parent peptide. Interactions with lipid bilayers mimicking those of promastigotes, infected macrophage and mammalian cells showed that these peptides strongly bind to vesicles with intense lytic activity at low concentrations. Additionally, circular dichroism and light scattering experiments showed changes in the secondary structure of peptides and in vesicle size, depending on vesicles compositions. Altogether they suggest that DecP-11 antileishmanial activity is impaired by the aggregation and that aminophospholipids are probably involved. CONCLUSIONS: DecP-11 potent cytolytic and membranolytic activities with lack of selectivity toward promastigote model membranes warrant further structural studies to improve selectivity. GENERAL SIGNIFICANCE: Strong interactions of peptides with aminophospholipids, abundant in parasite membranes, potentially lead to aggregated forms impairing activity.

MD simulations and multivariate studies for modeling the antileishmanial activity of peptides.

Leishmaniasis, a protozoan-caused disease, requires alternative treatments with minimized side-effects and less prone to resistance development. Antimicrobial peptides represent a possible choice to be developed. We report on the prospection of structural parameters of 23 helical antimicrobial and leishmanicidal peptides as a tool for modeling and predicting the activity of new peptides. This investigation is based on molecular dynamic simulations (MD) in mimetic membrane environment, as most of these peptides share the feature of interacting with phospholipid bilayers. To overcome the lack of experimental data on peptides' structures, we started simulations from designed 100% α-helices. This procedure was validated through comparisons with NMR data and the determination of the structure of Decoralin-amide. From physicochemical features and MD results, descriptors were raised and statistically related to the minimum inhibitory concentration against Leishmania by the multivariate data analysis technique. This statistical procedure confirmed five descriptors combined by different loadings in five principal components. The leishmanicidal activity depends on peptides' charge, backbone solvation, volume, and solvent-accessible surface area. The generated model possesses good predictability (q2  = 0.715, r2  = 0.898) and is indicative for the most and the least active peptides. This is a novel theoretical path for structure-activity studies combining computational methods that identify and prioritize the promising peptide candidates.

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis.

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

New catalytic mechanism for human purine nucleoside phosphorylase.

Human purine nucleoside phosphorylase has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Recently, several structures of human PNP have been reported, which allowed redefinition of the active site and understanding of the structural basis for inhibition of PNP by acyclovir and immucillin-H. Based on previously solved human PNP structures, we proposed here a new catalytic mechanism for human PNP, which is supported by crystallographic studies and explains previously determined kinetic data.

Crystal structure of human PNP complexed with hypoxanthine and sulfate ion.

Purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme, which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. Human PNP has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Here we report the crystal structure of human PNP in complex with hypoxanthine, refined to 2.6A resolution. The intermolecular interaction between ligand and PNP is discussed.

Crystal structure of hemoglobin from the maned wolf (Chrysocyon brachyurus) using synchrotron radiation.

Crystal structure of hemoglobin isolated from the Brazilian maned wolf (Chrysocyon brachyurus) was determined using standard molecular replacement technique and refined using maximum-likelihood and simulated annealing protocols to 1.87A resolution. Structural and functional comparisons between hemoglobins from the Chrysocyon brachyurus and Homo sapiens are discussed, in order to provide further insights in the comparative biochemistry of vertebrate hemoglobins.