Isatin compounds as noncovalent SARS coronavirus 3C-like protease inhibitors

A series of isatin derivatives were synthesized and tested against SARS CoV 3C-like protease. Substitutions at the N-1 and C-5 positions were examined to elucidate the differences in substrate binding sites of the rhinovirus 3C protease and SARS CoV 3C-like protease. Compound 5f shows significant inhibition with an IC(50) of 0.37 microM. Further study showed that, unlike the irreversible covalent binding of isatin derivatives to human rhinovirus 3C protease, the compounds tested in this study are all noncovalent reversible inhibitors.     

Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity

The human noradrenaline transporter (NET) and 5-hydroxytryptamine (5-HT) transporter (SERT) are inhibited by antidepressants and psychoactive drugs such as cocaine. Both substrates and inhibitors bind in the transmembrane core of the protein, but molecular divergence at the binding site is not sufficient to account for the NET-selective and SERT-selective inhibition of the antidepressants, desipramine and citalopram, respectively. We considered that the poorly conserved third extracellular loop may contribute to these differences. We substituted single amino acid residues of the third extracellular loop in NET for equivalents from SERT, transiently transfected COS-7 cells with WT NET, 13 mutant NETs and WT SERT, and measured [³H]noradrenaline uptake, [³H]nisoxetine binding and [³H]5-HT uptake. Mutants F299W, Y300Q, R301K and K303L, at the C-terminal end of EL3, all showed significantly decreased [³H]nisoxetine binding, indicative of a reduced cell surface expression. Most mutants differed little, if at all, from WT NET regarding [³H]noradrenaline uptake; however, the I297P mutant showed no significant uptake activity despite intact cell surface expression, and the A293F mutant showed a significantly slower transporter turnover than WT NET in addition to [³H]5-HT uptake that was significantly greater than that of WT NET. The A293F mutation also decreased desipramine potency and increased the inhibition of [³H]noradrenaline uptake by citalopram compared to WT NET. These results suggest that the third extracellular loop allosterically regulates the ability of the transmembrane domains to transport substrates and bind inhibitors and thus contributes to the selectivity of substrates and antidepressants for NET and SERT.     

Crystal structures of phosphodiesterases and implications on substrate specificity and inhibitor selectivity

Crystal structures of seven phosphodiesterase families (PDE1-5, 7, 9) show a conserved core catalytic domain that contains about 300 amino acids and fourteen alpha-helices. The catalytic domains of the PDE families 1-4, 7, and 9 have a uniform conformation. However, the H-loop at the active site of PDE5 shows four different conformations upon binding of inhibitors, probably implying a special mechanism for recognition of substrates and inhibitors by PDE5. The active site of all PDE families contains two divalent metal ions: zinc and probably magnesium. The PDE4-AMP and PDE5-GMP structures reveal the conserved interactions of the phosphate groups of the products AMP and GMP, and thus suggest a universal mechanism of nucleophilic attack for all PDE families. The substrate specificity has not been well understood. This review will comment on the early proposal, "glutamine switch", on basis of the recent biochemical and structural information. The PDE-inhibitor structures have identified a common subpocket for non-selective binding of all inhibitors and potential elements for recognition of family-selective inhibitors. The kinetic analysis on the mutations of PDE7 to PDE4 suggests that the multiple elements must work together to define inhibitor selectivity.     

Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor

A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.     

A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate

The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex; it is a reversible competitive inhibitor with a K(i) comparable to the K(m) of the parent substrate. Low concentrations (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.     

Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

AIM: To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CL(pro)) and its four site-directed mutants. METHODS: Based on the fluorescence resonance energy transfer (FRET) principle using 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CL(pro) proteolytic activity. RESULTS: The kinetic parameters of the fluorogenic substrate have been determined as Km=404 micromol.L(-1), kcat=1.08 min(-1), and kcat/Km=2.7 mmol(-1).L.min(-1). SARS-CoV 3CL(pro) showed substantial pH and temperature-triggered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CL(pro) revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity. CONCLUSION: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CL(pro). This FRET-based assay might supply an ideal approach for the exploration SARS-CoV 3CL(pro) putative inhibitors.     

Cysteine proteinase inhibitors as therapy for parasitic diseases: advances in inhibitor design

Clan CA (papain-like) cysteine proteinases of protozoan parasites are validated targets for the rational design of new anti-parasitic chemotherapies. Peptidyl and peptidomimetic proteinase inhibitors of differing chemistries limit parasite survival in vitro and in vivo. Also, the development of activity-based affinity labels has enabled the identification and characterization of potential cysteine proteinase targets in situ. This article reviews the biology and physicochemistry of parasite proteinases and the ongoing design of peptidyl and non-peptidyl inhibitors to generate anti-parasitic compounds of greater efficacy with decreased toxicity to the host.     

Structure-based design, synthesis, and evaluation of peptide-mimetic SARS 3CL protease inhibitors

The design and evaluation of low molecular weight peptide-based severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL) protease inhibitors are described. A substrate-based peptide aldehyde was selected as a starting compound, and optimum side-chain structures were determined, based on a comparison of inhibitory activities with Michael type inhibitors. For the efficient screening of peptide aldehydes containing a specific C-terminal residue, a new approach employing thioacetal to aldehyde conversion mediated by N-bromosuccinimide was devised. Structural optimization was carried out based on X-ray crystallographic analyses of the R188I SARS 3CL protease in a complex with each inhibitor to provide a tetrapeptide aldehyde with an IC(50) value of 98 nM. The resulting compound carried no substrate sequence, except for a P(3) site directed toward the outside of the protease. X-ray crystallography provided insights into the protein-ligand interactions.     

Chemically modified tetracycline-3 (CMT-3): A novel inhibitor of the serine proteinase, elastase

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α₁-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases.     

The role of structure in kinase-targeted inhibitor design

Protein kinases are attractive targets for rational drug design against a wide range of diseases. From detailed knowledge of the structure-function relationships underlying protein kinase activity and regulation, a number of methods for achieving kinase inhibition have been suggested and explored using structure-aided drug discovery. Attaining selective protein kinase inhibition in a cellular context, and converting the large number of known potent kinase inhibitors into effective drugs, are outstanding problems in this area and, from a structural perspective, the challenges presented by modulating pharmacokinetics and minimizing the incidence of resistant mutations in the target are of particular interest.     

Characteristics of aztreonam as a substrate, inhibitor and inducer for beta-lactamases

Aztreonam was investigated as to its characteristics as a substrate, inhibitor and inducer for the well-defined beta-lactamases of Gram-negative bacteria, and its antibacterial efficacy as to bacterial cells producing eight types of beta-lactamases was also evaluated. Aztreonam was hydrolyzed at measurable rates by class A beta-lactamases, a TEM-2 type penicillinase and the Proteus vulgaris cephalosporinase with a broad substrate range. However, the affinity of aztreonam for the class A enzymes was low, this property being well reflected by its high antibacterial activity toward producers of class A beta-lactamases. Aztreonam was extremely stable as to the typical class C cephalosporinase of Citrobacter freundii, and acted as a competitive and progressive inhibitor for the beta-lactamase. While the MICs of aztreonam in the cases of the constitutive producers of class C beta-lactamases were evidently affected by enzyme production. An experiment involving aztreonam as a inhibitor in combination with a hydrolyzable beta-lactam gave ambiguous results, however, a strong synergistic effect was found in combination with mecillinam. Using Pseudomonas aeruginosa, aztreonam was confirmed to be a poor inducer of beta-lactamases.     

病毒3CL蛋白酶三维结构模型及其抑制剂的虚拟筛选(英文)

目的:构建SARS病毒类3C(3CL)蛋白酶的三维结构模型,根据这一模型设计3CL蛋白酶的抑制剂。方法:用生物信息学方法从GenBank和PDB库中搜寻出具有晶体结构并与SARS病毒3CL蛋白酶有较高同源型的蛋白质,以此为模板,用同源蛋白模建方法构建SARS病毒3CL蛋白酶的三维结构模型;针对模建的三维结构模型,进行高通量虚拟筛选,从现有小分子数据库中获得具有抑制SARS病毒3CL蛋白酶活性的化合物。结果:同源性分析表明SARS病毒3CL蛋白酶与遗传性肠胃炎主蛋白酶(TGEV M~(pro)),有较高的同源性,组成底物结合口袋残基的同源性更高。因此,可以根据TGEV M~(pro)的晶体结构为模板模建SARS病毒3CL蛋白酶的三维结构。 三维结构模型表明,ARS病毒3CL蛋白酶的结构与TGEV M~(pro)的结构非常相象,两个蛋白酶活性口袋的结构和形状儿乎一样。虚拟筛选测试研究表明,MRRD数据库中的73个蛋白酶抑制剂能与两个蛋白同时作用。结论:无论是SARS病毒3CL蛋白酶还是TGEV M~(pro)的晶体结构均可以作为设计抗SARS药物的结构模型。从现有的蛋白酶抑制剂中筛选抗SARS药物可能是一条好的途径。     

Structural determinants of PERK inhibitor potency and selectivity

The unfolded protein response (UPR) is a coordinated program that promotes cell survival under conditions of endoplasmic reticulum stress and is required in tumor progression as well. To date, no specific small molecule inhibitor targeting this pathway has been identified. Pancreatic endoplasmic reticulum kinase (PERK), one of the UPR transducers, is an eIF2α kinase. Compromising PERK function inhibits tumor growth in mice, suggesting that PERK may be a cancer drug target, but identifying a specific inhibitor of any kinase is challenging. The goal of this study was to identify some pair-wise receptor-ligand atomic contacts that confer selective PERK inhibition. Compounds selectively inhibiting PERK-mediated phosphorylation in vitro were identified using an initial virtual library screen, followed by structure-activity hypothesis testing. The most potent PERK selective inhibitors utilize three specific kinase active site contacts that, when absent from chemically similar compounds, abrogates the inhibition: (i) a strong van der Waals contact with PERK residue Met7, (ii) interactions with the N-terminal portion of the activation loop, and (iii) groups providing electrostatic complementarity to Asp144. Interestingly, the activation loop contact is required for PERK selectivity to emerge. Understanding these structure-activity relationships may accelerate rational PERK inhibitor design.     

Multiple pharmacophores for the investigation of human UDP-glucuronosyltransferase isoform substrate selectivity

The UDP-glucuronosyltransferase (UGT) enzyme 'superfamily' contributes to the metabolism of a myriad of drugs, nondrug xenobiotic agents, and endogenous compounds. Although the individual UGT isoforms exhibit distinct but overlapping substrate selectivities, structural features of substrates that confer selectivity remain largely unknown. Using methods developed for pharmacophore fingerprinting combined with optimization and pattern recognition techniques, subsets of pharmacophores associated with the substrates and nonsubstrates of 12 human UGT isoforms were selected to generate predictive models of substrate selectivity and to elucidate the chemical and structural features associated with substrates and nonsubstrates. For all 12 UGT isoforms, the pharmacophore model generated showed predictive ability, as determined by a test set comprising 30% of the available data for each isoform. Models for UGT1A6, -1A7, -1A9, and -2B4 displayed the best predictive ability (>75% of test set predicted correctly) and were further analyzed to interpret the pharmacophores selected as important. The individual pharmacophores differed among isoforms but generally represented relatively simple structural and chemical features. For example, an aromatic ring attached to the nucleophilic group was found to increase the likelihood of glucuronidation by UGT1A6, UGT1A7 and UGT1A9. A large hydrophobic region close to the nucleophile and a hydrogen bond acceptor 10 A from the nucleophile were found to be common to most UGT2B4 substrates. The pharmacophores further suggest that the environment immediately adjacent to the nucleophilic site of conjugation is an important determinant of metabolism by a particular UGT.     

Structural bases for substrate and inhibitor recognition by matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which are involved in the proteolytic processing of several components of the extracellular matrix. As a consequence, MMPs are implicated in several physiological and pathological processes, like skeletal growth and remodelling, wound healing, cancer, arthritis, and multiple sclerosis, raising a very widespread interest toward this class of enzymes as potential therapeutic targets. Here, structure-function relationships are discussed to highlight the role of different MMP domains on substrate/inhibitor recognition and processing and to attempt the formulation of advanced guidelines, based on natural substrates, for the design of inhibitors more efficient in vivo.     

Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5

To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL(int) estimates using CYP3A5 compared with CYP3A4 including midazolam (CL(int) = 3.4 versus 3.3 microl min(-1) pmol(-1)). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL(int) = 10.3 versus 2.4 microl min(-1) pmol(-1)), and ritonavir (CL(int) = 0.76 versus 0.47 microl min(-1) pmol(-1)). The majority of compounds studied showed a greater inhibitory potential (IC(50)) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.     

Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5

To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL_int estimates using CYP3A5 compared with CYP3A4 including midazolam (CL_int?=?3.4 versus 3.3?µl?min^–1?pmol^–1). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL_int?=?10.3 versus 2.4?µl?min^–1?pmol^–1), and ritonavir (CL_int?=?0.76 versus 0.47?µl?min^–1?pmol^–1). The majority of compounds studied showed a greater inhibitory potential (IC₅₀) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.     

3C样蛋白酶抑制剂博福特韦的研发实践和策略

博福特韦是一种靶向新冠病毒3C样蛋白酶的小分子抑制剂,具有注射和雾化吸入两种给药形式,可单药给药,不需要联用肝药酶抑制剂。目前数据显示,博福特韦具有广谱抗冠状病毒活性、高耐药屏障、呼吸道和肺部中的药物浓度比血药浓度更高,在动物模型中可显著抑制肺部和脑部病毒复制等特点。简介了博福特韦的特点及其临床开发经验,旨在为抗COVID-19药物的开发提供思路与参考。     

Differential substrate selectivity of murine hepatic cytosolic and microsomal epoxide hydrolases

The initial rates of hydration of sixteen epoxides in the presence of cytosolic and microsomal fractions of mouse liver were determined. 1,2-Disubstituted trans-epoxides were found to be excellent, selective substrates for the cytosolic epoxide hydrolase, while 1,2-cis-epoxides were poorly hydrated when one or more substituents was a phenyl moiety. Epoxides of cyclic systems including benzo[a]pyrene 4,5-oxide, and two cyclodiene analogs were hydrated almost exclusively by the microsomal epoxide hydrolase while monosubstituted epoxides were hydrated by both systems. Some epoxides which were mediocre substrates proved to be reasonable inhibitors of the cytosolic epoxide hydrolase, indicating that the structural requirements for substrate binding and turnover are different. Some reagents known to interact with sulfhydryl groups, including styrene oxide, proved to be good inhibitors. This work facilitates the design of radiochemical and spectrophotometric assays for both major forms of epoxide hydrolase as well as prediction of potential intrinsic substrates. Also such data may be meaningful in assessing the risk involved in human exposure to epoxidized xenobiotics.