A new class of peptides from wasp venom: a pathway to antiepileptic/neuroprotective drugs.

The ability of venom-derived peptides to disrupt physiological processes in mammals provides an exciting source for pharmacological development. Our research group has identified a new class of neuroactive peptides from the venom of a Brazilian social wasp, Polybia occidentalis, with the potential pharmacological profile to treat epilepsies. The study was divided into five phases: Phase 1 concerned the extraction, isolation and purification of Occidentalin-1202(n) from the crude venom, followed by the synthesis of an identical analogue peptide, named Occidentalin-1202(s). In Phase 2, we described the effects of both peptides in two acute models of epilepsy-kainic acid and pentylenetetrazole-induced model of seizures-and measured estimated ED50 and therapeutic index values, electroencephalographic studies and C-fos evaluation. Phase 3 was a compilation of advanced tests performed with Occidentalin-1202(s) only, reporting histopathological features and its performance in the pilocarpine-induced status epilepticus. After the determination of the antiepileptic activity of Occidentalin-1202(s), Phase 4 consisted of evaluating its potential adverse effects, after chronic administration, on motor coordination (Rotarod) and cognitive impairment (Morris water maze) tests. Finally, in Phase 5, we proposed a mechanism of action using computational models with kainate receptors. The new peptide was able to cross the blood-brain barrier and showed potent antiseizure effects in acute (kainic acid and pentylenetetrazole) and chronic (temporal lobe epilepsy model induced by pilocarpine) models. Motor and cognitive behaviour were not adversely affected, and a potential neuroprotective effect was observed. Occidentalin-1202 can be a potent blocker of the kainate receptor, as assessed by computational analysis, preventing glutamate and kainic acid from binding to the receptor's active site. Occidentalin-1202 is a peptide with promising applicability to treat epilepsy and can be considered an interesting drug model for the development of new medicines.

Work ability psychological variables in workers of the pharmaceutical industry.

BACKGROUND AND AIM: Internal Marketing is also positively related to job satisfaction. However, there is a gap in the literature on the influence of Internal Marketing (IM) on Work Ability (WA) in the workers of the Pharmaceutical Industry. The aim of our study is to assess this interaction, demonstrating evidence that the Internal Marketing's five dimensions affect, positively or negatively the WA in workers of the Pharmaceutical Industry. METHODS: We collected data (N = 150)  from workers of Pharmaceutical Industry mean age of 37.86 years (SD=10.788), through internal advertisements at the university posted on information dissemination panels, e-mail databases and through the social LinkedIn network. The Statistical Package R-Studio program was used for statistical analysis. RESULTS: We verified that the group with Poor WA had a lower average value of Perceived IM than the group with Good WA. CONCLUSIONS: This study seems to indicate that promoting IM protocols helps increase WA among pharmaceutical industry workers.

Structural insight into the catalytic mechanism and inhibitor binding of aminopeptidase A.

Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases BP in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2' subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2' subsite of human APA established various types of interactions in major part with the P2' residue but also with the P1' residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

NI956/QGC006, a Potent Orally Active, Brain-Penetrating Aminopeptidase A Inhibitor for Treating Hypertension.

Brain renin-angiotensin system hyperactivity has been implicated in the development and maintenance of hypertension. We have shown that aminopeptidase A is involved in the formation of brain angiotensin III, which exerts tonic stimulatory control over blood pressure in hypertensive deoxycorticosterone acetate-salt rats and spontaneously hypertensive rats. We have also shown that injection of the specific and selective aminopeptidase A inhibitor, (3S)-3-amino-4-sulfanyl-butane-1-sulfonic acid (EC33), by central route or its prodrug, RB150/firibastat, by oral route inhibited brain aminopeptidase A activity and blocked the formation of brain angiotensin III, normalizing blood pressure in hypertensive rats. These findings identified brain aminopeptidase A as a potential new therapeutic target for hypertension. We report here the development of a new aminopeptidase A inhibitor prodrug, NI956/QGC006, obtained by the disulfide bridge-mediated dimerization of NI929. NI929 is 10× more efficient than EC33 at inhibiting recombinant mouse aminopeptidase A activity in vitro. After oral administration at a dose of 4 mg/kg in conscious deoxycorticosterone acetate-salt rats, NI956/QGC006 normalized brain aminopeptidase A activity and induced a marked decrease in blood pressure of -44±13 mm Hg 4 hours after treatment ( P<0.001), sustained over 10 hours (-21±12 mm Hg; P<0.05). Moreover, NI956/QGC006 decreased plasma arginine-vasopressin levels, and increased diuresis and natriuresis, that may participate to the blood pressure decrease. Finally, NI956/QGC006 did not affect plasma sodium and potassium concentrations. This study shows that NI956/QGC006 is a best-in-class central-acting aminopeptidase A inhibitor prodrug. Our results support the development of hypertension treatments targeting brain aminopeptidase A.

Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues.

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-ß-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis.

Mycobacterium tuberculosis Prolyl Oligopeptidase Induces In vitro Secretion of Proinflammatory Cytokines by Peritoneal Macrophages.

Tuberculosis (TB) is a disease that leads to death over 1 million people per year worldwide and the biological mediators of this pathology are poorly established, preventing the implementation of effective therapies to improve outcomes in TB. Host-bacterium interaction is a key step to TB establishment and the proteases produced by these microorganisms seem to facilitate bacteria invasion, migration and host immune response evasion. We presented, for the first time, the identification, biochemical characterization, molecular dynamics (MDs) and immunomodulatory properties of a prolyl oligopeptidase (POP) from Mycobacterium tuberculosis (POPMt). POP is a serine protease that hydrolyzes substrates with high specificity for proline residues and has already been characterized as virulence factor in infectious diseases. POPMt reveals catalytic activity upon N-Suc-Gly-Pro-Leu-Gly-Pro-AMC, a recognized POP substrate, with optimal activity at pH 7.5 and 37°C. The enzyme presents KM and Kcat/KM values of 108 µM and 21.838 mM-1 s-1, respectively. MDs showed that POPMt structure is similar to that of others POPs, which consists of a cylindrical architecture divided into an α/ß hydrolase catalytic domain and a ß-propeller domain. Finally, POPMt was capable of triggering in vitro secretion of proinflammatory cytokines by peritoneal macrophages, an event dependent on POPMt intact structure. Our data suggests that POPMt may contribute to an inflammatory response during M. tuberculosis infection.

Identification of novel Trypanosoma cruzi prolyl oligopeptidase inhibitors by structure-based virtual screening.

We have previously demonstrated that the secreted prolyl oligopeptidase of Trypanosoma cruzi (POPTc80) is involved in the infection process by facilitating parasite migration through the extracellular matrix. We have built a 3D structural model where POPTc80 is formed by a catalytic α/ß-hydrolase domain and a ß-propeller domain, and in which the substrate docks at the inter-domain interface, suggesting a "jaw opening" gating access mechanism. This preliminary model was refined by molecular dynamics simulations and next used for a virtual screening campaign, whose predictions were tested by standard binding assays. This strategy was successful as all 13 tested molecules suggested from the in silico calculations were found out to be active POPTc80 inhibitors in the micromolar range (lowest K i at 667 nM). This work paves the way for future development of innovative drugs against Chagas disease.

New binding site conformations of the dengue virus NS3 protease accessed by molecular dynamics simulation.

Dengue fever is caused by four distinct serotypes of the dengue virus (DENV1-4), and is estimated to affect over 500 million people every year. Presently, there are no vaccines or antiviral treatments for this disease. Among the possible targets to fight dengue fever is the viral NS3 protease (NS3PRO), which is in part responsible for viral processing and replication. It is now widely recognized that virtual screening campaigns should consider the flexibility of target protein by using multiple active conformational states. The flexibility of the DENV NS3PRO could explain the relatively low success of previous virtual screening studies. In this first work, we explore the DENV NS3PRO conformational states obtained from molecular dynamics (MD) simulations to take into account protease flexibility during the virtual screening/docking process. To do so, we built a full NS3PRO model by multiple template homology modeling. The model comprised the NS2B cofactor (essential to the NS3PRO activation), a glycine flexible link and the proteolytic domain. MD simulations had the purpose to sample, as closely as possible, the ligand binding site conformational landscape prior to inhibitor binding. The obtained conformational MD sample was clustered into four families that, together with principal component analysis of the trajectory, demonstrated protein flexibility. These results allowed the description of multiple binding modes for the Bz-Nle-Lys-Arg-Arg-H inhibitor, as verified by binding plots and pair interaction analysis. This study allowed us to tackle protein flexibility in our virtual screening campaign against the dengue virus NS3 protease.

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases.

BACKGROUND: Pathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease. RESULTS: The enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH. CONCLUSIONS: LAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.