Jarbas M. Resende

Study of angiotensin-(1-7) vasoactive peptide and its β-cyclodextrin inclusion complexes : Complete sequence-specific NMR assignments and structural studies

We report the complete sequence-specific hydrogen NMR assignments of vasoactive peptide angiotensin-(1-7) (Ang-(1-7)). Assignments of the majority of the resonances were accomplished by COSY, TOCSY, and ROESY peak coordinates at 400MHz and 600MHz. Long-side-chain amino acid spin system identification was facilitated by long-range coherence transfer experiments (TOCSY). Problems with overlapped resonance signals were solved by analysis of heteronuclear 2D experiments (HSQC and HMBC). Nuclear Overhauser effects (NOE) results were used to probe peptide conformation. We show that the inclusion of the angiotensin-(1-7) tyrosine residue is favored in inclusion complexes with beta-cyclodextrin. QM/MM simulations at the DFTB/UFF level confirm the experimental NMR findings and provide detailed structural information on these compounds in aqueous solution.

The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments.

Solid-state NMR spectroscopy is a powerful technique for the investigation of membrane-associated peptides and proteins as well as their interactions with lipids, and a variety of conceptually different approaches have been developed for their study. The technique is unique in allowing for the high-resolution investigation of liquid disordered lipid bilayers representing well the characteristics of natural membranes. Whereas magic angle solid-state NMR spectroscopy follows approaches that are related to those developed for solution NMR spectroscopy the use of static uniaxially oriented samples results in angular constraints which also provide information for the detailed analysis of polypeptide structures. This review introduces this latter concept theoretically and provides a number of examples. Furthermore, ongoing developments combining solid-state NMR spectroscopy with information from solution NMR spectroscopy and molecular modelling as well as exploratory studies using dynamic nuclear polarization solid-state NMR will be presented.

Epitope mapping and protective immunity elicited by adenovirus expressing the Leishmania amastigote specific A2 antigen: Correlation with IFN-γ and cytolytic activity by CD8+ T cells

A2 was identified as an amastigote virulence factor of Leishmania (Leishmania) donovani and as a candidate antigen for vaccine development against visceral leishmaniasis. Here, predicted hydrophilic, class I and II MHC-binding synthetic peptides were used to define epitopes recognized by A2-specific antibodies, CD8+ T and CD4+ T cells, respectively. Immunization of BALB/c mice with adenovirus expressing A2 (AdA2) resulted in low antibody response, contrasting with high levels of IFN-gamma producing CD4+ T and CD8+ T cells specific for A2. Further, A2-specific CD8+ T cells from immunized mice were capable of lysing sensitized target cells in vivo. Finally, we demonstrated an association of A2-specific T cell responses and reduced parasitism in both liver and spleen from mice immunized with AdA2 and challenged with L. (L.) chagasi.

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy

The heterodimeric antimicrobial peptide distinctin is composed of 2 linear peptide chains of 22- and 25-aa residues that are connected by a single intermolecular S-S bond. This heterodimer has been considered to be a unique example of a previously unrecorded class of bioactive peptides. Here the 2 distinctin chains were prepared by chemical peptide synthesis in quantitative amounts and labeled with 15N, as well as 15N and 2H, at selected residues, respectively, and the heterodimer was formed by oxidation. CD spectroscopy indicates a high content of helical secondary structures when associated with POPC/POPG 3:1 vesicles or in membrane-mimetic environments. The propensity for helix formation follows the order heterodimer >chain 2 >chain 1, suggesting that peptide-peptide and peptide-lipid interactions both help in stabilizing this secondary structure. In a subsequent step the peptides were reconstituted into oriented phospholipid bilayers and investigated by 2H and proton-decoupled 15N solid-state NMR spectroscopy. Whereas chain 2 stably inserts into the membrane at orientations close to perfectly parallel to the membrane surface in the presence or absence of chain 1, the latter adopts a more tilted alignment, which further increases in the heterodimer. The data suggest that membrane interactions result in considerable conformational rearrangements of the heterodimer. Therefore, chain 2 stably anchors the heterodimer in the membrane, whereas chain 1 interacts more loosely with the bilayer. These structural observations are consistent with the antimicrobial activities when the individual chains are compared to the dimer.

Membrane structure and interactions of human catestatin by multidimensional solution and solid-state NMR spectroscopy

Catestatin is a natural peptide of higher organisms including humans, with a wide variety of biological functions involved in catecholamine inhibition, cardiovascular regulation, control of blood pressure, inflammation, and innate immunity. It is derived from the natural processing of chromogranin A, induced in the skin after injury, and produced by chromaffin cells and neutrophils. With neutrophils, the peptide enters the cell by crossing the plasma membrane where it interacts with internal targets to induce calcium influx. Therefore, we investigated the membrane interactions and structure of several catestatin‐derived peptides. Whereas fluorescence dye release experiments are indicative of membrane permeabilization, multidimensional solution NMR and circular dichroism spectroscopies show that catestatin adopts α‐helical conformations between Ser‐6 and Tyr‐12 in the presence of dodecylphosphocholine micelles. Furthermore, proton‐decoupled 15N solid‐state NMR spectroscopy of sequences labeled with 15N and reconstituted into oriented lipid bilayers indicates that this domain is aligned in a strongly tilted to inplanar alignment. Proton‐decoupled 31P NMR spectra of the same samples are indicative of conformational and/or orientational heterogeneity at the level of the lipid bilayer head groups due to the presence of catestatin. The sequence and 3‐dimensional structure of catestatin exhibit homologies with penetratin, which is suggestive that they both enter the cells by related mechanisms to target internal structures.—Sugawara, M., Resende, J. M., Moraes, C. M., Marquette, A., Chich, J.‐F., Metz‐Boutigue, M.‐H., Bechinger, B. Membrane structure and interactions of human catestatin by multidimensional solution and solid‐state NMR spectroscopy. FASEB J. 24, 1737–1146 (2010). www.fasebj.org

Synthesis of a New Peptide–Coumarin Conjugate:A Potential Agent against Cryptococcosis

Antimicrobial peptides (AMPs) are currently being investigated as potential sources of novel therapeutics against an increasing number of microorganisms resistant to conventional antibiotics. The conjugation of an AMP to other bioactive compounds is an interesting approach for the development of new derivatives with increased antimicrobial efficiency and broader spectra of action. In this work, the synthesis of a new peptide-coumarin conjugate via copper(I)-catalyzed azide-alkyne cycloaddition is described. The conjugate was assayed for in vitro cytotoxicity and displayed antifungal activity against Cryptococcus gattii and Cryptococcus neoformans. Additionally, the conjugate exhibited increased antifungal efficacy when compared with the individual peptide, coumarin, or triazole moieties. Treatment of C. gattii with the peptide-coumarin conjugate enhanced the production of reactive oxygen species, suggesting that the oxidative burst plays an important role in the mechanism of action of the conjugate.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

On the search for potential antimycobacterial drugs: synthesis of naphthoquinoidal, phenazinic and 1,2,3-triazolic compounds and evaluation against mycobacterium tuberculosis

Fifteen naphthoquinones, sixteen phenazines and fifteen aryl triazoles were synthesized and evaluated against Mycobacterium tuberculosis. Twenty five substances are reported here for the first time and, among all of the compounds evaluated, six presented MIC (minimal inhibitory concentration) values < 6.25 µg mL-1. These substances are promising antimycobacterial prototypes.

Chemical interactions study of antiretroviral drugs efavirenz and lamivudine concerning the development of stable fixed-dose combination formulations for AIDS treatment

Lamivudine and efavirenz are among the most worldwide used drugs for acquired immune deficiency syndrome (AIDS) treatment. Solid state nuclear magnetic resonance (ssNMR), Fourier-transformed infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermo-optical analysis (TOA) were used to study possible interactions between these drugs, aiming the development of a fixed-dose drug combination. DSC and TOA have evidenced significant shifts on the melting points of both drugs in the mixture, which may be due to interaction between them. Although DSC and TOA results indicated incompatibility between the drugs, FTIR spectra were mostly unmodified due to overlapping peaks. The ssNMR analyses showed significant changes in chemical shifts values of the mixture when compared with spectra of pure drugs, especially in the signals relating to the deficient electron carbon atoms of both drugs. These results confirm the interactions suggested by DSC and TOA, which is probably due to acid-base interactions between electronegative and deficient electron atoms of both lamivudine and efavirenz.

Structure and membrane interactions of the homodimeric antibiotic peptide homotarsinin

Antimicrobial peptides (AMPs) from amphibian skin are valuable template structures to find new treatments against bacterial infections. This work describes for the first time the structure and membrane interactions of a homodimeric AMP. Homotarsinin, which was found in Phyllomedusa tarsius anurans, consists of two identical cystine-linked polypeptide chains each of 24 amino acid residues. The high-resolution structures of the monomeric and dimeric peptides were determined in aqueous buffers. The dimer exhibits a tightly packed coiled coil three-dimensional structure, keeping the hydrophobic residues screened from the aqueous environment. An overall cationic surface of the dimer assures enhanced interactions with negatively charged membranes. An extensive set of biophysical data allowed us to establish structure-function correlations with antimicrobial assays against Gram-positive and Gram-negative bacteria. Although both peptides present considerable antimicrobial activity, the dimer is significantly more effective in both antibacterial and membrane biophysical assays.