Classification of Congeneric and QSAR of Homologous Antileukemic S–Alkylcysteine Ketones

Based on a set of six vector properties, the partial correlation diagram is calculated for a set of 28 S-alkylcysteine diazomethyl- and chloromethyl-ketone derivatives. Those with the greatest antileukemic activity in the same class correspond to high partial correlations. A periodic classification is performed based on information entropy. The first four characteristics denote the group, and the last two indicate the period. Compounds in the same period and, especially, group present similar properties. The most active substances are situated at the bottom right. Nine classes are distinguished. The principal component analysis of the homologous compounds shows five subclasses included in the periodic classification. Linear fits of both antileukemic activities and stability are good. They are in agreement with the principal component analysis. The variables that appear in the models are those that show positive loading in the principal component analysis. The most important properties to explain the antileukemic activities (50% inhibitory concentration Molt-3 T-lineage acute lymphoblastic leukemia minus the logarithm of 50% inhibitory concentration Nalm-6 B-lineage acute lymphoblastic leukemia and stability k) are ACD logD, surface tension and number of violations of Lipinski’s rule of five. After leave-m-out cross-validation, the most predictive model for cysteine diazomethyl- and chloromethyl-ketone derivatives is provided.

Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ∼1–2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIaHis). Crystal structures were determined of MBP-βFXIIaHisin complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of βFXIIaHisin isolation. The βFXIIaHisstructure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2–P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHisstructure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.

The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Digestive proteolytic activity in Apodiphus amygdali Germar (Hemiptera: Pentatomidae): effect of endogenous inhibitors

The digestive proteolytic profile of <em>Apodiphus amygdali</em> was determined by using several substrates and specific inhibitors. Analysis of optimal pH and temperature showed the highest enzymatic activity at the pH range of 6-7 and temperature of 40°C when azocasein was used as a substrate. By using a negative control, the presence of several specific proteases were determined including tryspin-like, chymotrypsinlike, elastase, cathepsin B, cathepsin L, amino- and carboxypeptidases in the midgut content of <em>A. amygdali</em>, with the highest and the lowest activities of cathepsin L and carboxypeptidase, respectively. pH dependency of specific proteases revealed optimal pHs of 9, 8 and 9 for trypsin-, chymotrypsin-like, 6 for cathepsins and 5-6 for carboxy- and aminopeptidases, respectively. Specific inhibitors, including phenylmethylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, Ntosyl- L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinylleucylamido-(4-guanidino)-butane, phenanthroline and ethylendiamidetetraacetic acid, significantly decreased proteolytic activity, indicating the presence of different proteases in the midgut of <em>A. amygdali</em>. Extracted inhibitors from the midgut demonstrated significant inhibition of specific proteolytic activities of <em>A. amygdali</em> except for cathepsin B and aminopeptidase. The results indicated that determination of digestive proteolytic activity could be helpful to clarify digestion process in insects. Moreover, understanding the nature of digestive proteases might be used to develop several inhibitors for providing resistant crop varieties against pests.