Drug discovery considerations in the development of covalent inhibitors

In recent years, the number of drug candidates with a covalent mechanism of action progressing through clinical trials or being approved by the FDA has increased significantly. And as interest in covalent inhibitors has increased, the technical challenges for characterizing and optimizing these inhibitors have become evident. A number of new tools have been developed to aid this process, but these have not gained wide-spread use. This review will highlight a number of methods and tools useful for prosecuting covalent inhibitor drug discovery programs.     

The Isolation and Characterization of the Acid-Soluble Nucleotides from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The acid-soluble nucleotides were extracted from the tubers of Jerusalem artichoke with percbloric acid, and separated and purified by means of adsorption on and elution from active charcoal, repeated chromatography on columns of Dowex I (Cl^-), followed by paper chromatography. The following nucleotides have been characterized and/or identified: 5′-AMP, 3′-AMP, ADP, ATP, 5′-GMP, 2′-GMP, 3′-GMP, 2′,3′-cyclic GMP, GDP, GTP, 5′-UMP, UDP, UTP, NADP, UDP-glucose, UDP-galactose, UDP-fructose, UDP-N-acetylhexosamine and GDP-mannose.^** Neither cytosine ribonucleotides nor deoxyribonucleotides have been detected. The significance of these observations is discussed.     

Light-induced inhibitors from intact and cultured caps of Zea roots

Growth inhibitors were assayed from extracts of intact (attached) and of excised (cultured) root caps of Zea mays L., cv. Merit, the roots of which show a positive geotropic response only after exposure to light. If caps are intact at the time of illumination, at least two inhibitory substances are produced, an acid inhibitor and a neutral inhibitor, whereas if caps are detached from roots, placed in culture and then illuminated only the neutral inhibitor is formed. Cycloheximide retards inhibitor production in both intact and cultured caps. When [¹⁴C]mevalonic acid is included in the culture medium and the caps are illuminated, 15–25% of the recoverable ¹⁴C cochromatographs with the neutral inhibitor, whereas in caps cultured in the dark, this radiolabelling pattern is not observed. Cyloheximide in the light reduces the incorporation of ¹⁴C into compounds cochromatographing at the R_f of the neutral inhibitor. It is suggested that the neutral inhibitor may be important in the light-induced bending of roots.Abbreviations ABA abscisic acid - CH cycloheximide     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region     

Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2

MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutSα or MutSβ complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

Biochemical Analysis of the Yeast Proteinase Inhibitor (IC) Homolog ICh and Its Comparison with IC

Carboxypeptidase Y (CPY) inhibitor (I^C) and its homologous protein (I^Ch) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I^C and its inhibition mode toward CPY were recently reported, but I^Ch has not been characterized. The molecular mass of I^Ch was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I^Ch is folded as a monomeric β-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I^Ch was exclusively expressed in the early log phase, whereas I^C was expressed in the logarithmic and stationary phase. The intracellular localization of I^Ch was different from that of I^C. These findings provide insights into the physiological functions of I^Ch.     

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.Abbreviation ssRubisco small subunit of ribulose-1,5-bis-phosphate carboxylase     

An Inhibitor of S-Hemolysin,SHI, Produced by Streptomyces sp. Strain No. 6288

Streptomyces sp. strain No. 6288 produces S-Hemolysin, which is a unique phospholipase C with a high substrate specificity for sphingomyelin. Moreover, the strain also produced two kinds of phospholipase inhibitors, designated as SHI and S-PLI, in different phases of cultivation. The purified SHI was shown to be a basic substance containing an amino group and glycoside moiety, and it was a more effective inhibitor of S-Hemolysin and sphingomyelinase from Streptomyces sp. with a higher substrate specificity for sphingomyelin than α-toxin from Clostridium perfringens.     

Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian DNA polymerase inhibitors

We reported previously that sulfo-glycolipids such as sulfoquinovosyl-diacylglycerol (SQDG) and sulfoquinovosyl-monoacylglycerol (SQMG) are potent inhibitors of DNA polymerase alpha and beta and antineoplastic agents. Then, we succeeded in synthesizing SQDG and SQMG chemically, including their stereoisomers, glucopyranosyl-diacylglycerol (GDG) and glucopyranosyl-monoacylglycerol (GMG). In this study, we demonstrated the structure-function relationship of the synthetic sulfo-glycolipids to DNA polymerase alpha and beta and their relationship to the cytotoxic activity. Both SQDG and SQMG inhibited the activity of mammalian DNA polymerase alpha with IC(50) values of 3-5 microM, but GMG only moderately inhibited it. GDG, diacylglycerol (DG), and monoacylglycerol (MG) did not influence any of the DNA polymerase activities. The sulfate moiety in the quinovose was important in inhibiting the enzyme activity. The one-fatty-acid-sulfo-glycolipids, SQMG, GMG, and MG, prevented the growth of NUGC-3 human gastric cancer cells and induced apoptotic cell death, but the two-fatty-acid-sulfo-glycolipids, SQDG, GDG, and DG, did not. SQMG and GMG could halt the cell cycle at the G1 phase, but the cell cycle was not changed by MG. The relationship between the DNA polymerase inhibition and the cell growth effect by these compounds are discussed.     

SERPINE家族在纤维化疾病中作用的研究进展

丝氨酸蛋白酶抑制剂(Serine Protease Inhibitor,Serpin)是一类丝氨酸蛋白酶活性调节剂,在人体中被分为A~I9个亚家族,其中SERPINE(Serpin Peptidase Inhibitor,Clade E)家族参与调节生物体内多个重要的生命过程。本文通过介绍SERPINE家族中两个重要成员SERPINE1与SERPINE2的理化性质、作用机制以及调控因素,阐述SERPINE家族在纤维化相关疾病中的作用及研究进展。     

Phenylephrine,a small molecule,inhibits pectin methylesterases

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1?μM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

不同菌株白僵菌代谢产物对大鼠肝线粒体单胺氧化酶的抑制作用研究

对8株白僵菌[Beauveriasp.]代谢产物的抑制单胺氧化酶的活性进行测定,发现菌株Ba02和Ba05的乙酸乙酯提取物在终浓度为55μg/mL时,对单胺氧化酶A型(MAO＿＿A)有较强的抑制活性,其抑制率分别为95.9%和83.4%;Ba02及Ba03菌株脂溶性成分在终浓度为55μg/mL时对单胺氧化酶B型(MAO＿B)有较强的抑制活性,其抑制率分别为94.8%和84.1%。浓度和抑制活性关系研究表明在一定浓度范围内Ba02菌株脂溶性成分对MAO的抑制活性呈量效关系,经计算得出其对MAO＿A、MAO＿B抑制的IC50分别为18.3μg/mL、28.2μg/mL;抑制特征曲线表明Ba02菌株脂溶性成分对MAO＿A呈竞争型抑制,对MAO＿B为混合型抑制,Km值分别为0.47×10-5mol/L,0.11×10-5mol/L。     

Joint effects of acetochlor and urea on germinating characteristics of crop seeds

Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC5o was from 0.1 μmol/L to 3 μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The result indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.     

Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5–14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 (‘Leu-in’ to ‘Leu-out’) by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.     

蛋白酶体调节颗粒的结构生物学特征及其功能

蛋白酶体调节颗粒(regulatory particle,RP)参与调控许多重要信号通路的蛋白质降解,在维持细胞稳态中发挥重要作用.近年来,真核细胞蛋白酶体在癌症治疗中的作用机制及药物研发已引起了广泛关注,并有3种蛋白酶体抑制剂已用于临床治疗.随着蛋白酶体功能研究的不断深入,以及晶体学和冷冻电镜技术在其结构生物学研究中...     

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus

Virologica Sinica - Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic...