Structure-activity relationship studies on 1-(5-carboxyindol-1-yl)-propan-2-one inhibitors of human cytosolic phospholipase A2α: variation of the activated ketone moiety

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be potent inhibitors of human cytosolic phospholipase A₂α (cPLA₂α). In course of structure-activity relationship studies, we investigated the effect of the substitution of the electrophilic ketone group in the middle part of the molecule by other polar residues, such as hydroxyimino, azido, acyloxy, acylamino, urea and carbamate, on enzyme inhibition. With an IC₅₀ of 1.7 μM against cPLA₂α from human platelets, the 4-fluorophenylcarbamate derivative 23f was the most active of the compounds tested.     

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.     

A polyisoprenylated ketone from Calophyllum enervosum

A polyisoprenylated ketone named enervosanone has been isolated from the stem bark of Calophyllum enervosum together with three known compounds, cambogin, osajaxanthone and epicatechin. Their structures were determined by spectroscopic analysis. The antimicrobial evaluations of the isolated compounds were also reported.     

A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.     

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.     

Mcl-1 fragment induces apoptosis through direct interaction with Bax

Mcl-1 full-length (Mcl-1^1-350), a tightly regulated protein, plays an important role in protecting cells against apoptosis. Cleavage of Mcl-1 at Asp127 by caspase (Mcl-1^C1) contributes to the regulation of Mcl-1 expression, but its pro-apoptotic function remains controversial. Here, we reported that Mcl-1^128-350 expression induced caspase-dependent apoptosis. We demonstrated that Mcl-1^128-350 but not Mcl-1^1-350 interacts with Bax. This interaction required an intact BH3 Mcl-1^128-350 domain and leads to Bax activation and translocation to mitochondria. The silencing of Bax, but not of Bak, prevented Mcl-1^128-350 induced apoptosis. In conclusion, Mcl-1^128-350 exerts a pro-apoptotic function governed by its capacity to interact with Bax.

Structured summary

MINT-7306752: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0915) with BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7306728: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0914) with BAX (uniprotkb:Q07812) and BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7307171: F1 ATPase (uniprotkb:Q5TC12), Mcl-1 (uniprotkb:Q07820) and BAX (uniprotkb:Q07812) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

Asymmetric reduction of alpha, beta-unsaturated ketone to (R) allylic alcohol by Candida chilensis

A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.     

Plasmodium falciparum: biochemical characterization of the cysteine protease falcipain-2'

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential antimalarial drug targets. The falcipain-2' gene was identified recently and is nearly identical in sequence to falcipain-2. The product of this gene has not been studied previously. The mature protease domain of falcipain-2' was expressed in Escherichia coli, purified, and refolded to active enzyme. Functional analysis revealed similar biochemical properties to those of falcipain-2, including pH optima (pH 5.5-7.0), reducing requirements, and substrate preference. Studies with cysteine protease inhibitors showed similar inhibition of falcipain-2 and falcipain-2', although specificities were not identical. Considering activity against the presumed biological substrate, both enzymes readily hydrolyzed hemoglobin. Our results confirm that falcipain-2' is an active hemoglobinase and suggest that falcipain-2 and falcipain-2' play similar roles in erythrocytic parasites but that, for promising cysteine protease inhibitors, it will be important to confirm activity against this additional target.     

Candida tenuis Xylose Reductase Catalyzed Reduction of Aryl Ketones for Enantioselective Synthesis of Active Oxetine Derivatives

Candida tenuis xylose reductase shows high catalytic efficiencies in carbonyl reduction of acetophenone and 1‐phenyl‐1‐propanone derivatives. The quite low substrate solubility in aqueous buffer systems is circumvented by addition of methanol or by two‐phase solvent systems. In the latter, methanol improves the substrate phase transfer as solvent mediator and leads to reasonable space/time yields. Resulting enantiomerically pure chiral alcohols are key intermediates for synthesis of active pharmaceutical ingredients. (R)‐Atomoxetine is exemplarily synthesized in four steps, and the further use for generation of other oxetine derivatives and a polo‐like kinase 1 inhibitor are discussed. Chirality 00:000–000, 2012. © 2012 Wiley Periodicals, Inc.