Flavopereirine-An Alkaloid Derived from Geissospermum vellosii-Presents Leishmanicidal Activity In Vitro.

Chemotherapy is limited in the treatment of leishmaniasis due to the toxic effects of drugs, low efficacy of alternative treatments, and resistance of the parasite. This work assesses the in vitro activity of flavopereirine on promastigote cultures of Leishmania amazonensis. In addition, an in silico evaluation of the physicochemical characteristics of this alkaloid is performed. The extract and fractions were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid identified by NMR. The antileishmanial activity and cytotoxicity were assayed by cell viability test (MTT). The theoretical molecular properties were calculated on the Molinspiration website. The fractionation made it possible to isolate a beta-carboline alkaloid (flavopereirine) in the alkaloid fraction. Moreover, it led to obtaining a fraction with greater antileishmanial activity, since flavopereirine is very active. Regarding the exposure time, a greater inhibitory effect of flavopereirine was observed at 24 h and 72 h (IC50 of 0.23 and 0.15 µg/mL, respectively). The extract, fractions, and flavopereirine presented low toxicity, with high selectivity for the alkaloid. Furthermore, flavopereirine showed no violation of Lipinski's rule of five, showing even better results than the known inhibitor of oligopeptidase B, antipain, with three violations. Flavopereirine also interacted with residue Tyr-499 of oligopeptidase B during the molecular dynamics simulations, giving a few insights of a possible favorable mechanism of interaction and a possible inhibitory pathway. Flavopereirine proved to be a promising molecule for its antileishmanial activity.

Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems - Diffusion through an inhibitor barrier.

A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Isolation and characterization of a novel and potent inhibitor of Arg-gingipain from Streptomyces sp. strain FA-70.

Arg-gingipain (Rgp) is a major cysteine proteinase produced by the oral bacterium Porphyromonas gingivalis, which is a major pathogen of advanced periodontal diseases. This enzyme is important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. The development of Rgp inhibitors thus provides new therapeutic approaches to periodontal diseases. In this study, we first isolated and purified a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. This compound was found to be an antipain analog composed of phenylalanyl-ureido-citrullinyl-valinyl-cycloarginal (C27H43N9O7). The Ki value was calculated to be 4.5x10(-9) M when benzyloxycarbonyl-phenylalanyl-arginine-4-methly-coumaryl-7-amide was used as a substrate. This compound also inhibited cathepsins B, L, and H, though their Ki values were much higher than that of Rgp. FA-70C1 had little or no inhibitory activity on Lys-gingipain, another cysteine proteinase of P. gingivalis. The Rgp-induced degradation of various human proteins was completely blocked by this inhibitor. Disruption of both the bactericidal activity of polymorphonuclear leukocytes and the viability of human fibroblasts and umbilical vein endothelial cells induced by the culture supernatant of P. gingivalis was suppressed by the inhibitor in a dose-dependent manner. The enhancement of vascular permeability induced by in vivo administration of the culture supernatant of P. gingivalis was strongly inhibited by the inhibitor. Furthermore, the growth of P. gingivalis was suppressed by FA-70C1 in a dose-dependent manner. These results strongly suggest that FA-70C1 is a useful tool to prevent the virulence of P. gingivalis.

Matrix metalloprotease 2-mediated activation of Ca(2+)-ATPase by superoxide radical (O2*-) in plasma membrane of bovine pulmonary vascular smooth muscle.

The role of the matrix metalloprotease-2 (MMP-2) in regulating Ca(2+)-ATPase activity in bovine pulmonary artery smooth muscle plasma membranes during treatment with the O2*- generating system, hypoxanthine (HPX) plus xanthine oxidase (XO) has been studied. The smooth muscle membranes possess matrix metalloprotease (MMP) activity in gelatin zymogram, having an apparent molecular mass of 72 kDa; the activity is inhibited by the tissue inhibitor of metalloprotease-2 (TIMP-2). Since both protease and MMP-2 have same molecular mass and are inhibited by TIMP-2, it may, therefore, be suggested that the protease is the MMP-2. Treatment of the smooth muscle membrane suspension with the O2*- generating system stimulates MMP-2 activity, as evidenced by an apparent increase in the intensity of the protease activity. O2*- also enhances [14C]-gelatin degradation and Ca(2+)-ATPase activity. The increase in MMP activity, assessed by [14C]-gelatin degradation and Ca(2+)-ATPase activity are inhibited upon pretreatment with superoxide dismutase (SOD). The O2*- triggered MMP and Ca(2+)-ATPase activities in the membrane are found to be inhibited by TIMP-2. The stimulation of the MMP and Ca(2+)-ATPase activities remain unaffected by the inhibitors of serine, thiol and cysteine groups of proteases such as phenylmethylsulfonylfluoride (PMSF), Bowman Birk inhibitor (BBI), chymostatin, N-ethylmaleimide, leupeptin, antipain and pepstatin. Adding pure bovine MMP-2 to the smooth muscle membrane suspension causes an increase in Ca(2+)-ATPase activity, but the pretreatment with TIMP-2 inhibits the increase in the enzyme activity.

Peptide aldehyde complexes with wheat serine carboxypeptidase II: implications for the catalytic mechanism and substrate specificity.

The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD-WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and -170 degrees. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 A resolution for data collected at room temperature. The -170 degrees C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 A resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S1 through S4 sites, while density is apparent only in S1 and S2 for antipain. In the S1 site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P1 phenylalanyl side-chain of chymostatin. The P1 arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S1 site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approximation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseus proteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness.