Synthetic conversion of ACAT inhibitor to acetylcholinesterase inhibitor

Natural product acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor pyripyropene A was synthetically converted to acetylcholinesterase (AChE) inhibitor via heterolitic cleavage of the 2-pyrone ring, followed by gamma-acylation/cyclization with several aroyl chlorides. The 4-pyridyl analogue selectively showed AChE inhibitory activity (IC50 7.9 microM) and no ACAT inhibitory activity IC50 = >1000 microM.     

安定受体抑制肽

安定受体抑制肽,又名酰基辅酶A结合蛋白,是一个含86个氨基酸,分子量为10kD的多功能肽,广泛分布于酵母、植物和动物中。在高等动物中可作为前体蛋白加工成各种生理所需片段。主要的生理功能有诱发焦虑和兴奋的产生,促进类固醇的合成和胆囊收缩素的释放,抑制胰岛素分泌,减少水分和食物的摄取,调节细胞的增殖和分化,是中、长链脂肪酸酰基辅酶A的载体,能调节植物生长并促进植物种子油脂的形成。广泛的生理功能显示出它在生物界存在的重要性。     

A metalloproteinase inhibitor as an inhibitor of neovascularization.

Metalloproteinases and their endogenous inhibitors are key components of an enzyme system which is important in a number of fundamental biochemical and cellular processes. Our recent work has focused on the role of a particular metalloproteinase, collagenase, and the role of an endogenous inhibitor of this enzyme in the control of neovascularization. The proteolytic degradation of extracellular matrix components by capillary endothelial cells (EC) has been shown to be one of the key prerequisites of the angiogenic process. As part of a study of the effect(s) of the inhibition of collagenase on neovascularization, we have recently reported the purification, characterization and partial NH2-terminal sequence of a cartilage-derived inhibitor (CDI) of angiogenesis in vivo and in vitro. Evidence is presented which suggests that one means of controlling deregulated vascular growth characteristic of a number of "angiogenic diseases" may be at the level of the control of metalloproteinase activity.     

Small-molecule inhibitor starting points learned from protein-protein interaction inhibitor structure

MOTIVATION: Protein-protein interactions (PPIs) are a promising, but challenging target for pharmaceutical intervention. One approach for addressing these difficult targets is the rational design of small-molecule inhibitors that mimic the chemical and physical properties of small clusters of key residues at the protein-protein interface. The identification of appropriate clusters of interface residues provides starting points for inhibitor design and supports an overall assessment of the susceptibility of PPIs to small-molecule inhibition. RESULTS: We extract Small-Molecule Inhibitor Starting Points (SMISPs) from protein-ligand and protein-protein complexes in the Protein Data Bank (PDB). These SMISPs are used to train two distinct classifiers, a support vector machine and an easy to interpret exhaustive rule classifier. Both classifiers achieve better than 70% leave-one-complex-out cross-validation accuracy and correctly predict SMISPs of known PPI inhibitors not in the training set. A PDB-wide analysis suggests that nearly half of all PPIs may be susceptible to small-molecule inhibition.     

Hepatic proliferation inhibitor

Summary Although a long held tenet of biology has been that endogenous inhibitors can modulate cell proliferation, little progress was made in purifying any such inhibitor. This was largely due to the rarity of non-malignant cell cultures in which regulation of cell division was still operative, and to problems in separating cytotoxic and cytostatic effects in the complex biological extracts which were being studied. During the last decade, hepatic proliferation inhibitors of varying degrees of purity have been isolated using regenerating rat liver or hepatoma cell cultures as test systems. In these early studies, a number of inhibitors with differing molecular weights, physicochemical properties and biological responses were purified from liver cytosol and/or serum. Some of them could inhibit DNA synthesis or mitosis and thus were considered to be G1 or G2 inhibitors. However, experiments which could give precise answers about mechanisms of action could not be done until an inhibitor purified to homogeneity was available.Using well-characterized rat liver diploid epithelial cell cultures, which maintain a number of liver properties and which do not possess any transformation markers or malignant properties, we recently purified an hepatic proliferation inhibitor to a homogenous protein. It has a molecular weight of 26 000 daltons and an isoelectric point of 4.65. It specifically inhibits cell division and DNA synthesis in a number of non-malignant rat liver epithelial cell types, and has no effect on transformed liver cells, or hepatoma cells, in culture. Its effect is not mediated through destruction or sequestration of essential nutrients or calcium ions. Nor have preliminary experiments shown the hepatic proliferation inhibitor to interfere with the binding of epidermal growth factor to its receptors. The majority of the cells treated with the inhibitor are blocked in the G1 phase. Further experiments to study its mechanism of action and the inter-relationship, if any, between the cell cycle block induced by serum or nutrient deprivation, and the inhibitor-induced cycle block are in progress.     

Two-component covalent inhibitor

Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.     

Pectin methylesterase inhibitor

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

The amino-acid sequence of the trypsin-released inhibitor from sheep inter-alpha-trypsin inhibitor

The amino-acid sequence of the inhibitory part of the sheep serum inter-alpha-trypsin inhibitor (ITI) was determined. The inhibitor is composed of two covalently linked Kunitz-type domains. The reactive site of the C-terminal antitryptic domain contains arginine in position 71 (P1) and glycine in position 73 (P'2), whereas ITI derived inhibitors hitherto investigated contain phenylalanine in these positions. The reactive site of the N-terminal elastase inhibiting domain contains leucine in position 15 (P1) and methionine in position 17 (P'2), as in ITI-derived inhibitors of pig and horse.     

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.     

Resveratrol as a kcat type inhibitor for tyrosinase: potentiated melanogenesis inhibitor

Resveratrol exhibited the inhibitory activity against mushroom tyrosinase (EC1.14.18.1) through a k(cat) inhibition. Resveratrol itself did not inhibit tyrosinase but rather was oxidized by tyrosinase. In the enzymatic assays, resveratrol did not inhibit the diphenolase activity of tyrosinase when l-3,4-dihydroxyphenylalanin (L-DOPA) was used as a substrate; however, L-tyrosine oxidation by tyrosinase was suppressed in presence of 100 μM resveratrol. Oxidation of resveratrol and inhibition of L-tyrosine oxidation suggested the inhibitory effects of metabolites of resveratrol on tyrosinase. After the 30 min of preincubation of tyrosinase and resveratrol, both monophenolase and diphenolase activities of tyrosinase were significantly suppressed. This preincubational effect was reduced with the addition of L-cysteine, which indicated k(cat) inhibition or suicide inhibition of resveratrol. Furthermore, investigation was extended to the cellular experiments by using B16-F10 murine melanoma cells. Cellular melanin production was significantly suppressed by resveratrol without any cytotoxicity up to 200 μM. trans-Pinosylvin, cis-pinosylvin, dihydropinosylvin were also tested for a comparison. These results suggest that possible usage of resveratrol as a tyrosinase inhibitor and a melanogenesis inhibitor.     

Characterization of inhibitor binding sites of mitochondrial complex I using fluorescent inhibitor

Recent progress in complex I research suggests that a wide variety of complex I inhibitors share a common large binding domain with partially overlapping sites. To verify this concept, we carried out real-time displacement tests of a fluorescent ligand with various competitors using a novel quinazoline-type inhibitor (aminoquinazoline, AQ). In the presence of an excess amount of the competitors, the binding of AQ to the enzyme was completely suppressed, being in line with the concept mentioned above. However, AQ bound to the enzyme was not displaced by subsequent addition of an increasing amount of competitors in the concentration range expected from the relative magnitude of the K(d) values of AQ and competitors, rather, much higher concentrations of the competitors were needed to displace bound AQ. These results cannot be explained merely by the premise of a common or partially overlapping binding site(s) between AQ and competitors. On the other hand, double-inhibitor titration of steady state complex I activity suggested that additivity of inhibition is not necessarily observed for all pairs of complex I inhibitors. Our results are discussed in light of the cooperativity of the inhibitor binding sites.     

From cosubstrate similarity to inhibitor diversity--inhibitors of ADP-ribosyltransferases from kinase inhibitor screening

ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.     

Ribonuclease-RNAase inhibitor complex from rat testis. Purification of the RNAase inhibitor

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.     

Primary structure of a proteinase inhibitor released from goat serum inter-alpha-trypsin inhibitor

An acid-resistant trypsin inhibitor was released from goat serum inter-alpha-trypsin inhibitor and isolated by affinity chromatography. The primary structure of the inhibitor was established and the inhibitory properties were estimated. The inhibitor designed gIK-14 was characterized as a serine proteinase inhibitor from the family of the double-headed Kunitz-type inhibitors as suggested.