Recent progress in the discovery of inhibitors targeting coronavirus proteases

Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. The fatal outbreak of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) highlights the threat posed by this unique virus subfamily. However, no specific drugs have been approved to treat CoV-associated diseases to date. The CoV proteases, which play pivotal roles in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, are attractive targets for drug design. This review summarizes the recent advances in biological and structural studies, together with the development of inhibitors targeting CoV proteases, particularly main proteases (M^pros), which could help develop effective treatments to prevent CoV infection.

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Herbicidal Activity of Some Pyridazine Derivatives

3-Phenoxypyridazines and related compounds were evaluated their pre-emergence activities. Of these, 3-phenoxy-, 3-(2-methylphenoxy)-, and 3-(2-ethylphenoxy)-pyridazines (III, IV and VII) showed powerful effects on barnyardgrass and spikerush, whereas they gave no injury on rice plants. Furthermore, susceptibility of various kinds of cultivated plants as well as of weeds to III and IV were examined. Growth regulating activity of III was compared with that of maleic hydrazide.     

Comparison of Target Pocket Similarity and Progress into Research on Inhibitors of Picornavirus 3C Proteases

The 3C protease (3C Pro) plays a significant role in the life cycle of picornaviruses from replication to translation, making it an attractive target for structure-based design of drugs against picornaviruses. The structurally related 3C-like protease (3CL Pro) is an important protein involved in the replication of coronaviruses. With the emergence of COVID-19 and consequent intensive research into 3CL Pro, development of 3CL Pro inhibitors has emerged as a popular topic. This article compares the similarities of the target pockets of various 3C and 3CL Pros from numerous pathogenic viruses. This article also reports several types of 3C Pro inhibitors that are currently undergoing extensive studies and introduces various structural modifications of 3C Pro inhibitors to provide a reference for the development of new and more effective inhibitors of 3C Pro and 3CL Pro.     

A copy of exon 3-intron 3 from the barley aleurain gene is present on chromosome 2

A genomic clone fromHordeum vulgare L. cv. Himalaya contains 700 bp of DNA that is homologous with a high degree of nucleotide sequence similarity to exon 3-intron 3 from the gene for the thiol protease, aleurain. Genomic Southern blot mapping data indicate that this clone in phage had not undergone rearrangement, and no other sequences homologous to aleurain are present on it. Although exon 3 in aleurain encodes the polypeptide region cleaved during proteolytic processing of the proenzyme to its mature form, we do not know if the copy is expressed in some other protein. We have mapped the aleurain gene to chromosome 1, and this copy of exon 3-intron 3 to chromosome 2.     

杠柳杀虫组分F3-28对东方粘虫和小地老虎幼虫中肠消化酶活性的影响

F3-28是从杀虫植物杠柳Periploca sepium Bunge根皮中分离的一个具有杀虫活性的馏分,其主要成分为杠柳苷A、杠柳苷E和杠柳苷X。为了探索F3-28的初始作用部位, 为深入研究其作用机理奠定基础, 本研究采用经典的昆虫消化酶活性测定方法, 比较了F3-28对小地老虎Agrotis ypsilon和东方粘虫Mythimna separata 5龄幼虫消化酶系活性的影响。结果表明：对F3-28不敏感的小地老虎幼虫摄食F3-28后,其中肠蛋白酶、淀粉酶、脂肪酶的活性无显著变化。对F3-28敏感的东方粘虫幼虫摄食F3-28后2,4,8,10,12,24,48 h, 其中肠总蛋白酶活性分别为对照组的0.76,2.53,1.45,1.88,1.54,1.46,1.70倍,且和药物浓度呈依赖关系; 类胰蛋白酶的活性分别为对照组的1.60, 1.75,1.60,1.12,1.39,1.16,1.15倍（以BAPNA为底物）或1.68,1.95,1.53,1.26,1.15,1.13,1.14倍（以TAME为底物）,且和药物浓度呈依赖关系; 类胰凝乳蛋白酶活性分别为对照组的0.50,1.66,1.44,1.18,1.54,1.08和1.03 倍,但和药物浓度无依赖关系; 淀粉酶的活性分别为对照组的1.60,1.35,1.27,1.31,1.23和1.20 倍,但和药物浓度无依赖关系;对脂肪酶活性无明显影响。这些结果说明,杠柳杀虫活性组分F3-28的作用机理可能涉及对试虫中肠蛋白酶的激活,特别是对类胰蛋白酶的激活。     

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.     

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC₅₀ of 200 nM with a therapeutic index of >1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. C_max and absolute bioavailability of 34S were higher and half-life and time above EC₉₅ were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.     

Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in "rigid-body" proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1A). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, "rigid-body" proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.     

Gene structure and expression of the gene from Beauveria bassiana encoding bassiasin I, an insect cuticle-degrading serine protease

Genomic and cDNA encoding Beauveria bassiana bassiasin I, a potential cuticle-degrading serine protease, were isolated and analysed. Bassiasin I gene is comprised of 1137 bp (379 aa) and 3 introns which are 69, 62 and 68 bp long. The comparison of a deduced amino acid sequence with Metarhizium anisopliae Pr1, B. bassiana Pr1, and proteinase K showed high homology. When the cDNA including the intact signal peptide was expressed in E. coli, a clear proteolytic-degraded zone on LB-skimmed milk plates was observed.     

Examination of the genus Bordetella for specific human IgA protease activity

Abstract Three laboratory strains and 3 clinical isolates of Bordetella pertussis were found to have no IgA protease activity when incubated with radio-labelled IgA. In addition, no IgA protease activity was detected in the Second British Reference Preparation for Pertussis Vaccine, an acellular vaccine produced in Japan, or one strain each of Bordetella parapertussis and Bordetella bronchiseptica .     

Development of potent inhibitors of the coxsackievirus 3C protease

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.     

HIV Gag CAP2NC噬菌体蛋白酶切割模型的构建

HIV蛋白酶(protease,PR)耐药突变的大量出现严重地影响了AIDS的治疗.应用突变PR对展示HIVPR靶序列随机文库的噬菌体进行切割筛选,可获得突变PR的敏感噬菌体,该噬菌体可用于针对HIVPR耐药突变株的蛋白酶抑制剂(protease inhibitor,PI)新药筛选.为了探索这一可能性,将包含HIVPR靶位点P2/NC序列的Gag蛋白CAP2NC片段展示于噬菌体表面,并在该片段的N端连接一可与人免疫球蛋白分子特异结合的固相化标签序列LD3,将该噬菌体固定于人免疫球蛋白包被的酶标板上,用HIVSF2PR进行切割,用抗M13噬菌体酶标抗体ELISA法检测未被切割的剩余噬菌体以反映切割效果.结果表明,所构建的噬菌体能被HIVPR有效切割,最大切割效应可达80%以上,其ELISA检测值明显下降,并且该切割效应与HIVPR呈明显的量效关系,能被PI类药物Indinavir(IDV)特异抑制.首次成功构建了展示HIV Gag CAP2NC片段的噬菌体蛋白酶切割模型,不仅可为研究HIVPR的耐药性变异及其靶序列的适应性变异提供一新的研究平台,同时也为构建一种全新的PI类药物,尤其是针对耐药的PI类药物大规模体外噬菌体筛选模型打下基础.     

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography

Recombinant wild-type protease of human immunodeficiency virus, type [(HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, and they were purified as the wild-type protease. A third mutant protease (Arg-87 → Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.     

Purification and Properties of Trypsin Inhibitor from Hakuhenzu Bean (Dolichos lablab)

A highly purified trypsin inhibitor was obtained from the oriental plant Hakuhenzu bean (Dolichos lablab) by column chromatography on DEAE-Sephadex and gel-filtration on Sephadex G–75. The purified Hakuhenzu bean trypsin inhibitor (HTI) was obtained as a chemically homogeneous protein, and was stable to heat and to enzymes such as pepsin. It shows no obvious maximum at 280 nm in the ultraviolet absorption spectrum, and it contains more than 20% carbohydrate as galactose and 10% hexosamine as glucosamine. The molecular weight of this inhibitor was determined to be approximately 9,500 by gel-filtration. The protein contained 59 residures of amino acids; Lys₃, His₄, Arg₁, Asp₈, Thr₃, Ser₉, Glu₆, Pro₅, Gly₁, Ala₃, l/2Cys₁₀, Val₁, Ile₁, Leu₂, Tyr₁, Phe₁, from which a molecular weight of 6,400 is obtained. No methionine and tryptophan were found in the amino acid composition of the inhibitor. This inhibitor showed inhibitory activity against α-chymotrypsin in addition to trypsin.     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

The Conformational Analysis of Serine Protease Inhibitors and Its Applications for Drug Design

Abstract

Introduction

In our laboratory we have focused on several enzymes, among them β-glucuronidase, which have been shown to be released in abnormal quantities during chronic inflammatory diseases such as Rheumatoid Arthritis.^1,2 This enzyme, which is involved in the catalysis of β-glucuronides, has been characterized in mammalian tissues.³ In previous studies^4,5 we have demonstrated that it was inhibitied by a number of synthetic antiinflammatory gold (I) complexes, such as gold (I) thiomalate (Myochrisin) and gold (I) thiosulfate (Solganol). although the mechanism of inhibition has not been verified we have suggested from these and previous studies⁶ that a thiol group on the enzyme may be the primary site of binding to the gold complex.     

Functional interaction among catalytic residues in subtilisin BPN'

Variants of the serine protease, subtilisin BPN', in which the catalytic triad residues (Ser-221, His-64, and Asp-32) are replaced singly or in combination by alanine retain activities with the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPF-pna) that are at least 10(3) to 10(4) above the non-enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564-568, 1988]. A possible source of the residual activity was the hydrogen bond with the N delta 2 of Asn-155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild-type enzyme. Replacing Asn-155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF-pna by 150-fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5-fold greater than for the S221A enzyme. Thus, the catalytic role of Asn-155 is dependent upon the presence of Ser-221. The residual activity of the N155G:S221A enzyme (approximately 10(4)-fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. The mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The extracellular proteases of Myxococcus xanthus

Abstract A zymogram technique was used to resolve the proteases from the culture supernatants of three strains derived from Myxococcus xanthus FB. Of the 8 bands obtained, 3 were possibly proteolytic artefacts, and another may be derived from membrane vesicles or fragments. 3 of the bands were tentatively identified as serine proteases by affinity labelling. A non-motile, non-fruiting strain, M. xanthus DZ1, differed from 2 wild-type strains, NCIB9412 and DK101, in the relative intensity of certain bands, and all 3 strains differed qualitatively from M. xanthus XK, which is not FB-derived.