Structure-based design, synthesis, and study of potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, beta-ketoacyl-ACP synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Small molecules that inhibit FabH enzymatic activity have the potential to be candidates within a novel class of selective, nontoxic, broad-spectrum antibacterials. Using crystallographic structural information on these highly conserved active sites and structure based drug design principles, a benzoylaminobenzoic acid series of compounds was developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity against Gram-positive and selected Gram-negative organisms.     

猪肾中纯化的非组蛋白是furin样酶的自杀性底物抑制剂(英文)

目的:从猪肾脏中寻找并纯化furin样酶的天然抑制剂。方法:通过酵母分泌表达系统获得furin样酶的重组Kexin。从猪肾丙酮粉中抽提并纯化抑制剂组分。抑制剂活力在荧光分光光度计上,用荧光底物Boc-Arg-Val-Arg-Arg-MCA测定。结果:纯化到的抑制剂组分是一等电点超过9.5的碱性蛋白。测定了其N末端22个残基的序列。该序列与非组蛋白HMG-17高度同源,后者含有4个易被furin样酶裂解的双碱性氨基酸位点。因此,该非组蛋白可与荧光底物强烈竞争。若将酶与非组蛋白长时间温育,其抑制剂活力将最终丧失。结论:猪肾中纯化的非组蛋白是furin样酶的自杀性底物抑制剂。     

Bacterial beta-ketoacyl-acyl carrier protein synthase III (FabH): an attractive target for the design of new broad-spectrum antimicrobial agents

The emergence of drug resistant strains of important human pathogens has made urgent the necessity of finding new targets and novel antimicrobial agents. One of the most promising targets is FabH. In this review we summarize the progress made in the design of FabH inhibitors and the role played by the 3D-structure of the enzyme in the drug design process.     

Structure-based drug design and the discovery of aliskiren (Tekturna): perseverance and creativity to overcome a R&D pipeline challenge

At Ciba-Geigy (now Novartis), the clinical development of the CGP38560 renin inhibitor was halted due to insufficient pharmacokinetics. This indicated that the peptidomimetic approach to the development of antihypertensive agents was improper. Real non-peptide drug candidates were then expected to provide the necessary framework for obtaining the desired properties. For this purpose a homology model of the enzyme was used to characterize the binding mode of CGP38560 in complex with the renin model and served as a basis for the four chemistry laboratories that were assigned to this project. The renin team worked in a full structure-based perspective with this model, and four chemically-unrelated non-peptide series were discovered acting as renin inhibitors at the 1-3 nanomolar level. One of these leads was selected for further development and led to Aliskiren, which has been just approved by the FDA. Here is presented the successful structure-based strategy that enabled the discovery of several non-peptide inhibitors and the recent launch of a new drug that will be commercialized in the United States under the name Tekturna (for the treatment of high blood pressure as monotherapy or in combination with other high blood pressure medications).     

Potent, Plasmodium-selective farnesyltransferase inhibitors that arrest the growth of malaria parasites: structure-activity relationships of ethylenediamine-analogue scaffolds and homology model validation

New chemotherapeutics are urgently needed to combat malaria. We previously reported on a novel series of antimalarial, ethylenediamine-based inhibitors of protein farnesyltransferase (PFT). In the current study, we designed and synthesized a series of second generation inhibitors, wherein the core ethylenediamine scaffold was varied in order to examine both the homology model of Plasmodium falciparum PFT (PfPFT) and our predicted inhibitor binding mode. We identified several PfPFT inhibitors (PfPFTIs) that are selective for PfPFT versus the mammalian isoform of the enzyme (up to 136-fold selectivity), that inhibit the malarial enzyme with IC50 values down to 1 nM, and that block the growth of P. falciparum in infected whole cells (erythrocytes) with ED50 values down to 55 nM. The structure-activity data for these second generation, ethylenediamine-inspired PFT inhibitors were rationalized by consideration of the X-ray crystal structure of mammalian PFT and the homology model of the malarial enzyme.     

Design,synthesis, and biological evaluation of chrysin derivatives as potential FabH inhibitors

New series of chrysin derivatives ( 4a – 4t ) were designed and synthesized by introducing different substituted piperazines at C‐7 position. Their inhibitory effects on FabH were evaluated using two Gram‐negative bacterial strains, Escherichia coli and Pseudomonas aeruginosa, and two Gram‐positive bacterial strains, Bacillus subtilis and Staphylococcus aureus. To our delight, most of these compounds exhibited a dramatic increase in inhibitory potency, compared with the control positive drugs. Among them, compound 4s exhibited the most potent inhibitory activity with IC₅₀ values of 5.78 ± 0.24 μm inhibiting E. coli FabH and potent antibacterial activity against S. aureus and E. coli with MIC of 1.25 ± 0.01, 1.15 ± 0.12 μg/mL, respectively, comparing to the control positive drugs penicillin G (7.56 ± 0.30 μm ). Docking simulation was performed to position compound 4s into the FabH active site, and the result showed that compound 4s could bind well with the FabH as potent FabH inhibitor.     

Discovery of a potent and selective protein kinase CK2 inhibitor by high-throughput docking

To assess the potential of protein kinase CK2 as a target for developing new antitumor agents, we have undertaken a search for inhibitors of this enzyme. As part of this effort, we report here the discovery of the potent and selective CK2 inhibitor (5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid. We identified this inhibitor of a novel structural type by high-throughput docking of our corporate compound collection in the ATP binding site of a homology model of human CK2, using an appropriate protocol. The synthesis of the inhibitor as well as that of related analogues whose CK2 inhibitory activities give support to the binding mode proposed by the docking program is described. The results obtained suggest that virtual screening of a 3D database by molecular docking is a useful approach for lead finding provided that adapted postdocking filtering and reranking procedures are applied to the primary hit list.     

Homology modeling of human Fyn kinase structure: discovery of rosmarinic acid as a new Fyn kinase inhibitor and in silico study of its possible binding modes

Tyrosine phosphorylation represents a unique signaling process that controls metabolic pathways, cell activation, growth and differentiation, membrane transport, apoptosis, neural, and other functions. We present here the three-dimensional structure of Fyn tyrosine kinase, a Src-family enzyme involved in T-cell receptor signal transduction. The structure of Fyn was modeled for homology using the Sybyl-Composer suite of programs for modeling. Procheck and Prosa II programs showed the high quality of the obtained three-dimensional model. Rosmarinic acid, a secondary metabolite of herbal plants, was discovered as a new Fyn kinase inhibitor using immunochemical and in silico methods. Two possible binding modes of rosmarinic acid were evaluated here, i.e., near to or in the ATP-binding site of kinase domain of Fyn. Enzyme kinetic experiments revealed that Fyn is inhibited by a linear-mixed noncompetitive mechanism of inhibition by rosmarinic acid. This indicates that rosmarinic acid binds to the second "non-ATP" binding site of the Fyn tyrosine kinase.     

The major cysteine proteinase of Trypanosoma cruzi: a valid target for chemotherapy of Chagas disease

Trypanosoma cruzi, the causative agent of the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain (also known as cruzain, or GP57/51). The enzyme is a member of the papain C1 family of CPs, with a specificity intermediate between those of cathepsin L and cathepsin B. The enzyme, which is expressed at different levels by different parasite stages, is encoded by a high number of genes (up to 130 in the Tul2 strain), which code for a pre-pro-enzyme. Mature cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of Type I CPs of Trypanosomatids, and absent in all other C1 family CPs described so far. Irreversible inhibitors of cruzipain (peptidyl diazomethylketones, peptidyl fluoromethylketones, peptidyl vinyl sulphones) are able to block the differentiation steps in the parasite's life cycle, and effectively kill the organism. Recently, a vinyl sulphone derivative (N-piperazine-Phe-hPhe-vinyl sulphone phenyl) which is an efficient inhibitor of cruzipain and kills T. cruzi by inducing an accumulation of unprocessed cruzipain in the Golgi cisternae, interfering with the secretory pathway, has been tested in vivo in a mice model (J.H. McKerrow et al.). The curative effects observed, as well as the good bioavailability of the inhibitor and its apparent lack of undesirable side effects, make it a promising lead compound for the development of new drugs for the chemotherapy of Chagas disease.     

Inhibition of BACE, a promising approach to Alzheimer's disease therapy

The first proteolytic step in the processing of amyloid precursor protein (APP) to amyloid-beta (Abeta) in the brain is performed by beta-site APP cleaving enzyme (BACE1). This enzyme is a membrane bound aspartic protease with high homology of the catalytic domain to renin and pepsin and of yet unknown physiologic function. It is a primary drug discovery target for Alzheimer s disease therapy. The first potent inhibitors are based on the sequence of APP around the beta-secretase cleavage site EVNL/DAEF, with the scissile Leu-Asp amide bond being replaced by a hydroxyethylene transition state analogue isostere. In addition, lipophilic sidechains have been incorporated and a crystal structure of such an octapeptidic inhibitor bound in the active site is already available. Recent progress in the field of BACE inhibition is reviewed.     

In silico structural prediction of human steroid 5α-reductase type II

Steroid 5α-reductase type II is a membrane-associated enzyme in an oxidoreductase family. This enzyme, which is found in male sexual organs, plays the important biological actions toward steroid metabolism. Overexpression of 5α-reductase type II has affected the balance between testosterone and dihydrotestosterone, which implicates the androgenic disorders, including prostate cancer, hirsutism, and androgenic alopecia. Lack of single-crystal X-ray structures of 5α-reductase has hindered mechanistic understanding and delayed the discovery and development of an effective inhibitor. Herein, we illustrated a comparative structure of 5α-reductase type II that derived from the homology modeling, employing a membrane-bound protein, isoprenylcysteine carboxyl methyltransferase as a homologous template. A catalytic pocket and the transmembrane site were identified. The resulting in silico structure was validated via Ramachandran plot and confirmed by docking studies of 30 known 5α-reductase type I and type II inhibitors, including finasteride and dutasteride. The comparative docking study of the derived in silico 5α-reductase type II and 5β-reductase, a reported surrogate enzyme, was conducted. Our homology model approximately fitted to the membrane-associated character and showed the reasonable docking results, which depicted the well-defined comparative three-dimensional structure applicable for steroid reductase drug design.     

Expression and purification of lipoprotein-associated phospholipase A(2), a key enzyme involved in atherosclerosis

AIM: To express and purify lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), and to establish a screening model for Lp-PL A(2) inhibitors using the expressed Lp-PLA(2). METHODS: We cloned the full-length cDNA of Lp-PLA(2) from differentiated THP-1 cells, and subcloned the cDNA into the baculovirus transfer vector pFastBac1. In addition, we introduced an N-terminal Kozak sequence for high-level translation initiation and a C-terminal sequence of 6 histidine residues for purification. The fusion enzyme was expressed in Sf9 insect cells and purified by Ni(2+) affinity chromatography. Recombinant Lp-PLA(2) activity was measured using [(3)H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA(2) pretreated with the known Lp-PLA(2) inhibitor SB435495. RESULTS: We successfully cloned the full-length Lp-PLA(2) gene from differentiated THP-1 cells. The fusion enzyme was expressed in Sf9 insect cells at a high level and purified efficiently through a 2-step procedure. The recombinant Lp-PLA(2) activity was measured using [(3)H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA(2) inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA(2) with an IC(50) of 57+/-1 micromol/L. CONCLUSION: We expressed and purified Lp-PLA(2) at a high level in insect cell-baculovirus expression system. The yield ratio was much greater than that obtained from human plasma and we established a screening model for Lp-PL A(2) inhibitors using the expressed Lp-PLA(2).     

Advances in the research of β-ketoacyl-ACP synthase III (FabH) inhibitors

Fatty acid biosynthesis is essential for bacterial survival. In recent years, components of this biosynthetic pathway have aroused wide concern. β-ketoacyl-acyl carrier protein synthase III (FabH) is a particularly attractive target which catalyzes the initial step of fatty acid biosynthesis. In this review, fatty acid biosynthesis, recent advances in the research of FabH as well as related inhibitors are reviewed. Finally, we also discuss the prospect and developmental trend of FabH inhibitors as anti-bacterial agents.     

Free energy calculations on snake venom metalloproteinase BaP1

BaP1 is a snake venom metalloproteinase from the venom of Bothrops asper, showing high structural homology with the catalytic domain of human adamalysins and matrix metalloproteinases. It induces the release of cytokines, like interleukin-1 and tumor necrosis factor alpha. Recently, the high-resolution crystal structure of BaP1 with a bound inhibitor became available, representing an interesting model concerning inhibitor design for medicinally important metalloproteinases such as tumor necrosis factor alpha-converting enzyme and MMP13. We here use computational modeling to gain a better understanding about the binding properties of various ligands to BaP1, with a focus on computing ligand binding free energies. The obtained results should be of general significance for future research on medicinally important metalloproteinases. We have investigated the binding of the original inhibitor in detail and calculated its binding strength using MMP/GBSA free energy calculations. Additionally, the binding strengths of alternative ligands have been computed, and two of them are predicted and experimentally verified to strongly inhibit the enzyme. A suggestion for chemical modifications of BaP1 inhibitors could be made to guide future synthesis efforts. Furthermore, a contribution to the proteolytic reaction mechanism of metzincins is given. The pK value of the catalytically active glutamic acid residue 143 has been found to be significantly raised when compared with a free glutamate side chain. Calculations on other matrix metalloproteinases confirmed that this is not confined to BaP1, but seems to be a common feature of metzincins.     

Novel FabH inhibitors: a patent and article literature review (2000 – 2012)

Introduction: The traditional antimicrobial chemotherapy drugs play their effects mostly via bacterial interference with in vivo amino acids, nucleotides, amino sugars and other small molecule synthesis, or interfering the biochemical processes of these small molecules to synthesize nucleic acids, peptidoglycan and other biological macromolecules. In recent years, enzymes with single function in bacterial fatty acid synthetase system have become the genome-driven novel antibacterial drug targets. Among inhibitors of these targets, FabH inhibitors are distinguished, for their target is different from that of existing antibiotics. Therefore, discovery of FabH inhibitors might be a potential orientation to overcome bacterial resistance.

Areas covered: This review summarized new patents and articles published on FabH inhibitors from 2000 to 2012.

Expert opinion: The review gives a brief understanding about the background and development in the area of FabH inhibitors that aims to solve the bacterial resistance problem. This review puts emphasis on some typical small molecules, which participate in the process of FabH inhibition. Overall, the research scopes of antibacterial agents are getting broad. Fatty acid synthase (FAS) pathway has been proved to be a promising target for the therapy. However, claim of novel antibacterial agents with more active and higher specificity is still continued.     

Phosphodiesterase 1:a unique therapeutic target with multiple potential indications

Cyclic-nucleotide phosphodiesterase 1(PDE1) is a unique enzyme family hydrolyzing both cyclic guanosine monophosphate(cGMP) and cyclic adenosine monophosphate(cAMP) intra-cellular signaling molecules. A unique aspect of this enzyme family is its activation by calcium-calmodulin upon excitation of excitatory cells such as neurons and cardiomyocytes. In chronic degenerative diseases such as Parkinson disease,Alzheimer disease and heart failure,over-stimulation and chronic excessive levels of intra-cellular calcium leads to cell death. Targeting the PDE1 enzyme family with enzyme inhibitors is a novel approach to develop therapeutic agents for degenerative disorders. ITI-214 is a potent,and selective PDE1 inhibitor that has been tested in four human clinical trials. It is safe and well tolerated even at high dose levels that lead to high plasma and cerebral spinal fluid levels. In animal models,ITI-214 has cognitive enhancing properties as demonstrated in the rat novel object recognition model. Using a unilateral 6-hydroxy-dopamine lesion mouse model,the cylinder test readout of front paw use indicated that ITI-214 displays L-DOPA sparing effects ITI-214 reverses catalepsy induced by the potent dopamine D2 receptor antagonist haloperidol,indicating potential applications in Parkinson disease and as an adjunctive treatment in schizophrenia. Aspects of the Intra-Cellular Therapies PDE1 inhibitor program wil be outlined and the potential application to multiple therapeutic areas wil be discussed.     

Characteristics of monkey tryptase purified from cheek pouch vascular tissues

Tryptase purified from rat and dog tissues has been reported, although the characteristics of these enzymes are different from human tryptase. For pathophysiological studies of human tryptase, studies on species that have a similar tryptase to humans is needed. In this study, we purified monkey tryptase from cheek pouch vascular tissues using heparin affinity and gel filtration columns. The monkey tryptase, which had a molecular weight of 130 kDa by gel filtration, consisted of a tetramer of 33 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal sequence showed high homology with tryptases from other species. The optimum pH and temperature were 7.5-9.0 and 25-40 degrees C, respectively. The enzyme was labile in high-KCl buffer, and the optimum KCl concentration was 0.1 M. The enzyme activity was completely inhibited by diisopropyl phosphorofluoridate and leupeptin but not by soybean trypsin inhibitor and alpha-antitrypsin. The enzyme hydrolyzed vasoactive intestinal peptide but did not affect angiotensin I, somatostatin and bradykinin. In the present study, we first isolated monkey tryptase from cheek pouch vascular tissues and showed that the characteristics of monkey tryptase are very similar to those of human tryptase.     

Expression of acetoacetyl-CoA synthetase,a novel cytosolic ketone body-utilizing enzyme,in human brain

Acetoacetyl-CoA synthetase (AACS, acetoacetate-CoA ligase, EC 6.2.1.16) is a ketone body-utilizing enzyme, the physiological role of which remains unclear yet in mammals, particularly has never been studied in human. In order to investigate the tissue distribution of AACS in human, cDNA encoding AACS was isolated from HepG2 cells. Amino acid sequence of human AACS deduced from the open reading frame showed high homology (89.3%) with that of rat AACS and much less homology (43.7%) with that of bacterial AACS. The expression level of the AACS mRNA was high in kidney, heart and brain, but low in liver, and the expression profile of AACS in the human brain was quite similar to that of 3-hydroxy-3-methylglutaryl-CoA reductase.     

Modeling the Met Form of Human Tyrosinase: A Refined and Hydrated Pocket for Antagonist Design

Tyrosinases are type 3 copper proteins involved in melanin biosynthesis, responsible for skin and hair color in mammals. To steer tyrosinase inhibitor discovery for therapeutic and cosmetic purposes, structural information about human tyrosinase is necessary. As this protein has never been crystallized so far, we derived a robust homology model built using structural information from Streptomyces castaneoglobisporus and Ipomea batata catecholoxidase enzymes. The active site containing two copper atoms in co‐ordination with six histidine residues was refined through an optimization protocol based on molecular mechanics parameters for copper co‐ordination and charges calculated by quantum mechanics methods. Five structural water molecules and a hydroxyl ion were found to be essential for optimization. The superimposition of the human homology model on crystallographic structures of tyrosinases from other species revealed similar overall backbone topologies, active site conformations, and conserved water molecules. Phenylthiourea (PTU), the tyrosinase inhibitor of reference, was then docked into the solvated human active pocket. A binding mode consistent with crystallographic information was obtained. Taken together, these findings demonstrated that the human tyrosinase model, deposited in the Protein Model Database, is a reliable structure for future rational inhibitor design projects.