Thrombin inhibitors with novel P1 binding pocket functionality: free energy of binding analysis

The high incidence of thrombembolic diseases justifies the development of new antithrombotics. The search for a direct inhibitor has resulted in the synthesis of a considerable number of low molecular weight molecules that inhibit human α-thrombin potently. However, efforts to develop an orally active drug remain in progress as the most active inhibitors with a highly basic P1 moiety exhibit an unsatisfactory bioavailability profile. In our previous work we solved several X-ray structures of human α-thrombin in complexes with (1) novel bicyclic arginine mimetics attached to the glycylproline amide and pyridinone acetamide scaffold and (2) inhibitors with a novel aza scaffold and with charged or neutral P1 moieties. In the present contribution, we correlate the structures of the complex between these inhibitors and the protein with the calculated free energy of binding. The energy of solvation was calculated using the Poisson–Boltzmann approach. In particular, the requirements for successful recognition of an inhibitor at the protein’s active site pocket S1 are discussed. Figure We report here on free energy of binding analysis of thrombin inhibitors with novel aza scaffold and novel bicyclic arginine mimetics in S1 pocket of thrombin     

3-Mercapto-5H-1,2,4-Triazino[5,6-b]Indole-5-Acetic Acid (Cemtirestat) Alleviates Symptoms of Peripheral Diabetic Neuropathy in Zucker Diabetic Fatty (ZDF) Rats: A Role of Aldose Reductase

Peripheral neuropathy is the most prevalent chronic complication of diabetes mellitus. Good glycemic control can delay the appearance of neuropathic symptoms in diabetic patients but it is not sufficient to prevent or cure the disease. Therefore therapeutic approaches should focus on attenuation of pathogenetic mechanisms responsible for the nerve injury. Considering the role of polyol pathway in the etiology of diabetic neuropathy, we evaluated the effect of a novel efficient and selective aldose reductase inhibitor, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (cemtirestat), on symptoms of diabetic peripheral neuropathy in Zucker Diabetic Fatty (ZDF) rats. Since the age of 5 months, male ZDF rats were orally administered cemtirestat, 2.5 and 7.5 mg/kg/day, for two following months. Thermal hypoalgesia was evaluated by tail flick and hot plate tests. Tactile allodynia was determined by a von Frey flexible filament test. Two-month treatment of ZDF rats with cemtirestat (i) did not affect physical and glycemic status of the animals; (ii) partially inhibited sorbitol accumulation in red blood cells and the sciatic nerve; (iii) markedly decreased plasma levels of thiobarbituric acid reactive substances; (iv) normalized symptoms of peripheral neuropathy with high significance. The presented findings indicate that inhibition of aldose reductase by cemtirestat is not solely responsible for the recorded improvement of the behavioral responses. In future studies, potential effects of cemtirestat on consequences of diabetes that are not exclusively dependent on glucose metabolism via polyol pathway should be taken into consideration.

     

Plant latex thrombin-like cysteine proteases alleviates bleeding by bypassing factor VIII in murine model

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.     

Dosimetric accuracy of three dose calculation algorithms for radiation therapy of in situ non-small cell lung carcinoma

BackgroundStudy determines differences in calculated dose distributions for non-small cell lung carcinoma (NSC LC) patients. NSC LC cases were investigated, being the most common lung cancer treated by radiotherapy in our clinical practice.Materials and methodsA retrospective study of 15 NSCLC patient dose distributions originally calculated using standard superposition (SS) and recalculated using collapsed cone (CC ) and Monte Carlo (MC) based algorithm expressed as dose to medium in medium (MCD_m) and dose to water in medium (MCD_w,) was performed so that prescribed dose covers at least 99% of the gross target volume (GTV). Statistical analysis was performed for differences of conformity index (CI), heterogeneity index (HI), gradient index (GI), dose delivered to 2% of the volume (D_2%), mean dose (D_mean) and percentage of volumes covered by prescribed dose (V_70Gy). For organs at risk (OARs), D_mean and percentage of volume receiving 20 Gy and 5Gy (V_20Gy, V_5Gy) were analysed.ResultsStatistically significant difference for GTVs was observed between MCD_w and SS algorithm in mean dose only. For planning target volumes (PTVs), statistically significant differences were observed in prescribed dose coverage for CC, MCD_m and MCD_w. The differences in mean CI value for the CC algorithm and mean HI value for MCD_m and MCD_w were statistically significant. There is a statistically significant difference in the number of MUs for MCD_m and MCD_w compared to SS.ConclusionAll investigated algorithms succeed in managing the restrictive conditions of the clinical goals. This study shows the drawbacks of the CC algorithm compared to other algorithms used.     

Solid-phase peptide synthesis by fragment condensation: Coupling in swelling volume

The condensation of short peptides to resin-bound fragments was examined with respect to high coupling yields with only a small molar excess of a peptide in the reaction solution. The best results were achieved by the addition of reactants (C-unprotected peptide, DIC, and HOBt) dissolved in a so-called swelling volume of an appropriate solvent to a dry resin with an attached N-deprotected peptide chain. Each coupling step was followed by the end-capping of unreacted resin-bound peptide with 2,4-dinitrofluorobenzene. The substituted dinitroaniline chromophore formed in this reaction made the detection and separation of deletion peptides easy. Both conventional and swelling volume methods were compared on parallel syntheses of the HIV-1 protease C-terminal 78–99 fragment. The yields of the isolated heneicosapeptide were 21 and 81% in favor of the swelling volume procedure.