Structure-Activity Relationship Studies of 9-Alkylamino-1,2,3,4-tetrahydroacridines against Leishmania (Leishmania) infantum Promastigotes.

Leishmaniasis is one of the most neglected diseases in modern times, mainly affecting people from developing countries of the tropics, subtropics and the Mediterranean basin, with approximately 350 million people considered at risk of developing this disease. The incidence of human leishmaniasis has increased over the past decades due to failing prevention and therapeutic measures-there are no vaccines and chemotherapy, which is problematic. Acridine derivatives constitute an interesting group of nitrogen-containing heterocyclic compounds associated with numerous bioactivities, with emphasis to their antileishmanial potential. The present work builds on computational studies focusing on a specific enzyme of the parasite, S-adenosylmethionine decarboxylase (AdoMet DC), with several 1,2,3,4-tetrahydro-acridines emerging as potential inhibitors, evidencing this scaffold as a promising building block for novel antileishmanial pharmaceuticals. Thus, several 1,2,3,4-tetrahydroacridine derivatives have been synthesized, their activity against Leishmania (Leishmania) infantum promastigotes evaluated and a structure-activity relationship (SAR) study was developed based on the results obtained. Even though the majority of the 1,2,3,4-tetrahydroacridines evaluated presented high levels of toxicity, the structural information gathered in this work allowed its application with another scaffold (quinoline), leading to the obtention of N1,N12-bis(7-chloroquinolin-4-yl)dodecane-1,12-diamine (12) as a promising novel antileishmanial agent (IC50 = 0.60 ± 0.11 µM, EC50 = 11.69 ± 3.96 µM and TI = 19.48).

From thiol-subtilisin to omniligase: Design and structure of a broadly applicable peptide ligase.

Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations. Mutation A225N in the S1' pocket and F189W of the S2' pocket increased the synthesis to hydrolysis (S/H) ratio and overall coupling efficiency, whereas the I107V mutation was added to S4 pocket to increase the reaction rate. The final omniligase variants appeared to have a very broad substrate range, coupling more than 250 peptides in a 400-member library of acyl acceptors, as indicated by a high-throughput FRET assay. Crystal structures and computational modelling could rationalize the exceptional properties of omniligase-1 in peptide synthesis.

Efficient Enzymatic Cyclization of Disulfide-Rich Peptides by Using Peptide Ligases.

Disulfide-rich macrocyclic peptides-cyclotides, for example-represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof, as well as of the θ-defensin RTD-1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi-gram scale. The existence of several ligation sites for each macrocycle makes this approach highly flexible and facilitates both the larger-scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.

Evaluation of a flavonoids library for inhibition of pancreatic α-amylase towards a structure-activity relationship.

α-Amylase has been considered an important therapeutic target for the management of type 2 diabetes mellitus (T2DM), decreasing postprandial hyperglycaemia (PPHG). In the present work, a panel of 40 structurally related flavonoids was tested, concerning their ability to inhibit α-amylase activity, using a microanalysis screening system, an inhibitory kinetic analysis and molecular docking calculations. From the obtained results, it was possible to observe that the flavone with a -Cl ion at 3-position of C-ring, an -OH group at 3'- and 4'- positions of B-ring and at 5- and 7- positions of A-ring and the C2 = C3 double bond, was the most active tested flavonoid, through competitive inhibition. In conclusion, some of the tested flavonoids have shown promising inhibition of α-amylase and may be considered as possible alternatives to the modulation of T2DM.

α-Glucosidase inhibition by flavonoids: an in vitro and in silico structure-activity relationship study.

α-Glucosidase inhibitors are described as the most effective in reducing post-prandial hyperglycaemia (PPHG) from all available anti-diabetic drugs used in the management of type 2 diabetes mellitus. As flavonoids are promising modulators of this enzyme's activity, a panel of 44 flavonoids, organised in five groups, was screened for their inhibitory activity of α-glucosidase, based on in vitro structure-activity relationship studies. Inhibitory kinetic analysis and molecular docking calculations were also applied for selected compounds. A flavonoid with two catechol groups in A- and B-rings, together with a 3-OH group at C-ring, was the most active, presenting an IC50 much lower than the one found for the most widely prescribed α-glucosidase inhibitor, acarbose. The present work suggests that several of the studied flavonoids have the potential to be used as alternatives for the regulation of PPHG.

Cholesterol Biosynthesis: A Mechanistic Overview.

Cholesterol is an essential component of cell membranes and the precursor for the synthesis of steroid hormones and bile acids. The synthesis of this molecule occurs partially in a membranous world (especially the last steps), where the enzymes, substrates, and products involved tend to be extremely hydrophobic. The importance of cholesterol has increased in the past half-century because of its association with cardiovascular diseases, which are considered one of the leading causes of death worldwide. In light of the current need for new drugs capable of controlling the levels of cholesterol in the bloodstream, it is important to understand how cholesterol is synthesized in the organism and identify the main enzymes involved in this process. Taking this into account, this review presents a detailed description of several enzymes involved in the biosynthesis of cholesterol. In this regard, the structure and catalytic mechanism of the enzymes involved in cholesterol biosynthesis, from the initial two-carbon acetyl-CoA building block, will be reviewed and their current pharmacological importance discussed. We believe that this review may contribute to a deeper level of understanding of cholesterol metabolism and that it will serve as a useful resource for future studies of the cholesterol biosynthesis pathway.

Receptor-based virtual screening protocol for drug discovery.

Computational aided drug design (CADD) is presently a key component in the process of drug discovery and development as it offers great promise to drastically reduce cost and time requirements. In the pharmaceutical arena, virtual screening is normally regarded as the top CADD tool to screen large libraries of chemical structures and reduce them to a key set of likely drug candidates regarding a specific protein target. This chapter provides a comprehensive overview of the receptor-based virtual screening process and of its importance in the present drug discovery and development paradigm. Following a focused contextualization on the subject, the main stages of a virtual screening campaign, including its strengths and limitations, are the subject of particular attention in this review. In all of these stages special consideration will be given to practical issues that are normally the Achilles heel of the virtual screening process.

Detection of farnesyltransferase interface hot spots through computational alanine scanning mutagenesis.

In this study, we present a detailed characterization of the full α/ß interface in the farnesyltransferase (FTase) enzyme, an important target in drug design efforts. This characterization is presented in terms of hot spots, warm spots, and null spots and is based on the application of an improved variation of the computational alanine scanning mutagenesis methodology, complemented with extensive solvent-accessible surface area and interfacial hydrogen-bonding analysis. A total of 130 interface amino acid residues were considered in this analysis, a number that represents 16.0% of the total of 814 amino acid residues in the full enzyme. Globally, the results provide important clues on the most important structural and energetic determinants for dimer formation, suggesting several key targets at the subunit interface for the development of new molecules that aim to inhibit FTase activity through blocking the formation of the fully active FTase dimer, yielding useful indications for future drug design efforts.

Mechanism of formation of the internal aldimine in pyridoxal 5'-phosphate-dependent enzymes.

In this paper we studied the mechanism of formation of the internal aldimine, a common intermediate to most pyridoxal 5'-phosphate (PLP)-dependent enzymes. A large model based on the crystal structure from the human ornithine decarboxylase (ODC) enzyme was constructed and in total accounts for 504 atoms. The reaction mechanism was investigated using the ONIOM methodology (B3LYP/6-31G(d)//AM1), and the final energies were calculated with the M06/6-311++G(2d,2p)//B3LYP/6-31G(d) level of theory. It was demonstrated that the reaction is accomplished in three sequential steps: (i) the nucleophilic attack of Lysine69 to PLP, (ii) the carbinolamine formation, and (iii) a final dehydration step. For the carbinolamine formation, several mechanistic hypotheses were explored, and the preferred pathway assigns a key role for the conserved active site Cys360. The overall reaction is exergonic in -9.1 kcal/mol, and the rate-limiting step is the dehydration step (E(a) = 13.5 kcal/mol). For the first time, we provide an atomistic portrait of this mechanism in an enzymatic environment. Moreover, we were able to assign a novel role to Cys360 in the ODC reaction mechanism that was never proposed.

Evolução clínica de pacientes com lesões de tronco de coronária esquerda não-protegido submetidos a angioplastia coronária com implante de stents farmacológicos / Clinical follow-up of patients with unprotected left main disease undergoing percutaneous coronary intervention with drug-eluting stents

Introdução: Recentemente a intervenção coronária percutânea (ICP) com stents farmacológicos (SFs) tem se mostrado uma opção viável em pacientes selecionados com lesão de tronco de coronária esquerda não-protegido (TCE-NP). Este estudo teve como objetivo avaliar a efetividade e a segurança da ICP com SFs em lesões de TCE-NP da prática diária, analisando a ocorrência combinada de eventos cardíacos adversos maiores (ECAM) a longo prazo. Métodos: Foram tratados 142 pacientes consecutivos, com média de seguimento clínico de 917 + 743 dias. A decisão de utilizar um ou dois stents e inibidor da glicoproteína IIb/IIIa ficou a critério do operador. Angiografia coronária no seguimento não foi realizada de rotina, mas deixada a critério clínico. Resultados: A média de idade foi de 67,5 + 16 anos, três quartos dos pacientes eram do sexo masculino, 29% eram portadores de diabetes e 39% apresentavam angina instável. Foram utilizados 2,75 + 1,25 stents por paciente. Lesões com comprometimento da bifurcação foram identificadas em 90,1% e as técnicas mais frequentemente utilizadas foram o provisional stent em 36% e o small crush em 29% dos pacientes. Ultrassom intracoronário foi realizado em 92,3% dos pacientes, e reintervenção ocorreu em 21,3% dos stents, por apresentarem aposição incompleta de suas hastes após o implante. ECAM na evolução tardia ocorreram em 15,4%, óbito cardíaco ocorreu em 3,6%, revascularização do vaso-alvo em 11,2% e trombose definitiva / provável do stent em 1,4%. Conclusões: A ICP com SFs em lesões de TCE-NP neste estudo mostrou ser segura e eficazna evolução tardia, com baixas taxas de óbito cardíaco e de trombose do stent.

Background: Recently, percutaneous coronary intervention (PCI) with drug-eluting stents (DES) has proven to be a feasible option in selected patients with unprotected left main (LM) disease. This study was aimed at assessing the efficacy and safety of PCI with DES in LM lesions in the daily practice, analyzing the long-term occurrence of major adverse cardiac events (MACE). Methods: A total of 142 consecutivepatients were treated with a mean follow-up of 917 + 743 days. The decision to use one or two stents or glycoprotein IIb/IIIa inhibitors was left to the operator’s discretion. Coronaryangiography was not performed routinely in the follow-up. Results: Mean age was 67.5 + 16 years, 75% of the patients were male, 29% had diabetes and 39% had unstable angina. A total of 2.75 + 1.25 stents were implanted per patient. Bifurcation lesions were identified in 90.1% and the most frequent techniques were the provisional stent in 36% and small crush in 29% of the patients. Intravascular ultrasound was performed in 92.3% of the patients and reinterventions in 21.3% of the stents due to incomplete appositionof the struts after implantation. In the late follow-up, MACE was observed in 15.4% of the patients, cardiac deathin 3.6%, target-vessel revascularization in 11.2% and definitive/probably stent thrombosis in 1.4%. Conclusions: In thisstudy, PCI with DES proved to be safe and effective in the late follow-up of LM lesions, with low cardiac death and stentthrombosis rates.

Brain nitric oxide production by a proline-rich decapeptide from Bothrops jararaca venom improves baroreflex sensitivity of spontaneously hypertensive rats.

Baroreflex sensitivity is disturbed in many people with cardiovascular diseases such as hypertension. Brain deficiency of nitric oxide (NO), which is synthesized by NO synthase (NOS) in the citrulline-NO cycle (with argininosuccinate synthase (ASS) activity being the rate-limiting step), contributes to impaired baroreflex. We recently showed that a decapeptide isolated from Bothrops jararaca snake venom, denoted Bj-PRO-10c, exerts powerful and sustained antihypertensive activity. Bj-PRO-10c promoted vasodilatation dependent on the positive modulation of ASS activity and NO production in the endothelium, and also acted on the central nervous system, inducing the release of GABA and glutamate, two important neurotransmitters in the regulation of autonomic systems. We evaluated baroreflex function using the regression line obtained by the best-fit points of measured heart rate (HR) and mean arterial pressure (MAP) data from spontaneously hypertensive rats (SHRs) treated with Bj-PRO-10c. We also investigated molecular mechanisms involved in this effect, both in vitro and in vivo. Bj-PRO-10c mediated an increase in baroreflex sensitivity and a decrease in MAP and HR. The effects exerted by the peptide include an increase in the gene expression of endothelial NOS and ASS. Bj-PRO-10c-induced NO production depended on intracellular calcium fluxes and the activation of a G(i/o)-protein-coupled metabotropic receptor. Bj-PRO-10c induced NO production and the gene expression of ASS and endothelial NOS in the brains of SHRs, thereby improving baroreflex sensitivity. Bj-PRO-10c may reveal novel approaches for treating diseases with impaired baroreflex function.

The central nervous system as target for antihypertensive actions of a proline-rich peptide from Bothrops jararaca venom.

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (

The Central Nervous System as Target forAntihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO- 10c (

Argininosuccinate synthetase is a functional target for a snake venom anti-hypertensive peptide: role in arginine and nitric oxide production.

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.