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Discovery of Potent Anilide Inhibitors against the Severe Acute Respiratory Syndrome 3CL Protease
Abstract
A diversified library of peptide anilides was prepared, and their inhibition activities against the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The most potent inhibitor is an anilide derived from 2-chloro-4-nitroaniline, l-phenylalanine and 4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL protease with K(i) = 0.03 muM. The molecular docking experiment indicates that the P1 residue of this anilide inhibitor is distant from the nucleophilic SH of Cys145 in the active site.
... Метаболічний синдром та астма є основними проблемами охорони здоров'я, які швидко зросли за попередні десятиліття [8]. Астма характеризується оборотною епізодичною обструкцією дихальних шляхів з гіперреактивністю, тоді як метаболічний синдром характеризується ожирінням, резистентністю до інсуліну, дисліпідемією, гіпертонією та непереносимістю глюкози [17]. Дослідження вказують на те, що метаболічний синдром пов'язаний з порушенням функції легень може бути чинником формування астми [15]. ...
... Хоча, за даними окремих авторів [5], деякі пацієнти з ожирінням, що страждають алергічною астмою, мають більш важкий перебіг запалення у бронхіальному дереві, ніж особи з нормальною масою тіла, також виявляється значущий фенотип "ожиріння-астма", коли ступінь тяжкості не залежить від клітинного запалення [15,17]. ...
... Тому дослідження регуляторних особливостей, в тому числі кардіореспіраторної системи, при бронхіальній астмі з ожирінням та без нього є достатньо важливим з позицій визначення більш тонких механізмів регуляторних порушень [1,4,5,6,11,13,16,17]. Достатньо важливим з цих позицій є висновок про те, що зниження ВСР у пацієнтів з персистуючим перебігом БА пов'язане як з контрольованістю патологічного процесу, так і з масою тіла хворих. ...
The collection presents reports submitted to the scientific-practical conference "Modern problems of pharmacology, cosmetology and aromamology", dedicated to the 175th anniversary of the birth of the prominent immunologist and microbiologist, Nobel Laureate Ilya Ilyich Mechnikov, and the Pharmacist's Day, which took place at the Odessa Medical Institute of the International Humanitarian University on September 18, 2020.
... The generated pharmacophore model was validated by retrospective screening of a test set database composed of 166 molecules. The test set included 12 SARS-CoV M pro inhibitors collected from literature (Barretto et al., 2005;Jacobs et al., 2013;Jain et al., 2004;Shie et al., 2005;Shimamoto et al., 2015;Thanigaimalai, Konno, Yamamoto, Koiwai, Taguchi, Takayama, Yakushiji, Akaji, Kiso, et al., 2013;Turlington et al., 2013;Xue et al., 2008;H. Z. Zhang et al., 2006;Zhu et al., 2011) which were labelled as actives (Table S1 in Supporting Information). ...
... Z. Zhang et al., 2006;Zhu et al., 2011) which were labelled as actives (Table S1 in Supporting Information). The remaining 154 molecules were labelled as inactives, comprising 10 weak inhibitors of SARS-CoV M pro (B egu e & Bonnet-Delpon, 1991;Gelb et al., 1985;Jain et al., 2004;Shao et al., 2008;Shie et al., 2005) and 144 DUD-E (Mysinger et al., 2012) generated decoys. All molecules were prepared as described in the ligand preparation Section 2.1. ...
... To this end, a small-molecule test set was generated for model validation. Thus, 12 peptide-like M pro inhibitors of diverse structures, that showed high affinity to the protease enzyme and shared a similar binding mode were selected and labeled as actives Jain et al., 2004;Shie et al., 2005;Shimamoto et Table S1 in Supporting Information). The multi-conformation test set compounds were mapped to the pharmacophore model. ...
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an ongoing global health emergency. Repurposing of approved pharmaceutical drugs for COVID-19 treatment represents an attractive approach to quickly identify promising drug candidates. SARS-CoV-2 main protease (Mpro) is responsible for the maturation of viral functional proteins making it a key antiviral target. Based on the recently revealed crystal structures of SARS-CoV-2 Mpro, we herein describe a multi-stage virtual screening protocol including pharmacophore screening, molecular docking and protein-ligand interaction fingerprints (PLIF) post-docking filtration for efficient enrichment of potent SARS-CoV-2 Mpro inhibitors. Potential hits, along with a cocrystallized control were further studied via molecular dynamics. A 150-ns production trajectory was followed by RMSD, free energy calculation, and H-bond analysis for each compound. The applied virtual screening protocol led to identification of five FDA-approved drugs with promising binding modes to key subsites of the substrate-binding pocket of SARS-CoV-2 Mpro. The identified compounds belong to different pharmaceutical classes, including several protease inhibitors, antineoplastic agents and a natural flavonoid. The drug candidates discovered in this study present a potential extension of the recently reported SARS-CoV-2 Mpro inhibitors that have been identified using other virtual screening protocols and may be repurposed for COVID-19 treatment.
... The antiviral effect of niclosamide appears due to the inhibition of E3 ligase S-phase kinase-associated protein 2 (SKP2), which regulates the polyubiquitination of Benclin 1 (BECN1), thereby causing the proteasomal degradation. The inhibition of SKP2 raises BENC1 levels leading to an augmentation of Fig. (3), against SARS-CoV 3CL protease, which serves as a critical enzyme for proteolytic processing and virus propagation [159]. Reportedly, the anilides inhibited SARS-CoV 3CL protease via competitive inhibition via highly stabilized fitting into the active site pocket of the target enzyme. ...
... Reportedly, the anilides inhibited SARS-CoV 3CL protease via competitive inhibition via highly stabilized fitting into the active site pocket of the target enzyme. The docking investigations confirmed that the L-phenylalanine P1 site of the anilides accommodates at 4.95 Å from the -SH group of Cys145 residues, hence preventing the nucleophilic proteolysis [159]. The antiviral profile of anilides (1, 2 and 3; Fig. 3) suggested the therapeutic efficacy of anilides, and amide functionality for the rational development of drugs targeting the coronavirus class, further providing the essential perspectives for using niclosamide and related anilides as potential drug class against the novel SARS-CoV-2. ...
... ). Chemical structures of undervaluation therapeutics for the treatment of SARS-CoV-annihilation of MERS-CoV replication. Similarly, the replication of MERS-CoV witnessed a decline in the presence of niclosamide, mainly due to the modulation in autophagic efflux in infected cells[142].Shie et al., in 2005, investigated the activity of anilides 2 and 3, ...
Background: The newly emerged coronavirus SARS-CoV-2 first reported in December 2019 has infected upwards of five and a half million people globally and resulted in nearly 9063264 deaths until the 24th of June 2020. Nevertheless, the highly contagious virus has instigated an unimaginably rapid response from scientific and medical communities.
Objectives: Pioneering research on molecular mechanisms underlying the viral transmission, molecular pathogenicity, and potential treatments will be highlighted in this review. The development of antiviral drugs specific to SARS-CoV-2 is a complicated and tedious process.To acceleratethe scientfic advancement, researchers are consolidating available data from associated coronaviruses into a single pipeline, which can be readily made available to vaccine developers.
Methods: In order to find studies evaluating the COVID-19 virus epidemiology, repurposed drugs and potential vaccines, web searches and bibliographical bases have been used with keywords matches the content of this review.
Results: The published results of SARS-CoV-2 structures and interactomics have been used to identify potential therapeutic candidates. We illustrate recent publications on SARS-CoV-2, concerning its molecular, epidemiological, and clinical characteristics, and focus oninnovative diagnostics technologies in the production pipeline. This objective of this review is to enhance the comprehension of the unique characteristics of SARS-CoV-2and strengthen future control measures.
Keywords: Epidemiology; SARS-CoV-2; COVID-19; Therapeutics; Pandemics; Therapeutics repurposing; Viruses.
... The antiviral effect of niclosamide appears due to the inhibition of E3 ligase S-phase kinase-associated protein 2 (SKP2), which regulates the polyubiquitination of Benclin 1 (BECN1), thereby causing the proteasomal degradation. The inhibition of SKP2 raises BENC1 levels leading to an augmentation of Fig. (3), against SARS-CoV 3CL protease, which serves as a critical enzyme for proteolytic processing and virus propagation [159]. Reportedly, the anilides inhibited SARS-CoV 3CL protease via competitive inhibition via highly stabilized fitting into the active site pocket of the target enzyme. ...
... Reportedly, the anilides inhibited SARS-CoV 3CL protease via competitive inhibition via highly stabilized fitting into the active site pocket of the target enzyme. The docking investigations confirmed that the L-phenylalanine P1 site of the anilides accommodates at 4.95 Å from the -SH group of Cys145 residues, hence preventing the nucleophilic proteolysis [159]. The antiviral profile of anilides (1, 2 and 3; Fig. 3) suggested the therapeutic efficacy of anilides, and amide functionality for the rational development of drugs targeting the coronavirus class, further providing the essential perspectives for using niclosamide and related anilides as potential drug class against the novel SARS-CoV-2. ...
... ). Chemical structures of undervaluation therapeutics for the treatment of SARS-CoV-annihilation of MERS-CoV replication. Similarly, the replication of MERS-CoV witnessed a decline in the presence of niclosamide, mainly due to the modulation in autophagic efflux in infected cells[142].Shie et al., in 2005, investigated the activity of anilides 2 and 3, ...
Background
The newly emerged coronavirus SARS-CoV-2 first reported in December 2019 has infected upwards of five and a half million people globally and resulted in nearly 9063264 deaths until the 24th of June 2020. Nevertheless, the highly contagious virus has instigated an unimaginably rapid response from scientific and medical communities.
Objectives
Pioneering research on molecular mechanisms underlying the viral transmission, molecular pathogenicity, and potential treatments will be highlighted in this review. The development of antiviral drugs specific to SARS-CoV-2 is a complicated and tedious process.To acceleratethe scientfic advancement, researchers are consolidating available data from associated coronaviruses into a single pipeline, which can be readily made available to vaccine developers.
Methods
In order to find studies evaluating the COVID-19 virus epidemiology, repurposed drugs and potential vaccines, web searches and bibliographical bases have been used with keywords matches the content of this review.
Results
The published results of SARS-CoV-2 structures and interactomics have been used to identify potential therapeutic candidates. We illustrate recent publications on SARS-CoV-2, concerning its molecular, epidemiological, and clinical characteristics, and focus oninnovative diagnostics technologies in the production pipeline. This objective of this review is to enhance the comprehension of the unique characteristics of SARS-CoV-2and strengthen future control measures.
... A diverse series of peptide anilides (29)(30)(31)(32)(33)(34)(35) were reported based on niclosamide ( Figure 11). 58 Unlike typical nitroanilide-based peptides, which are readily hydrolyzed by serine and cysteine protease, 59 these peptides were not efficiently cleaved by SARS-CoV 3CL pro . Niclosamide showed no inhibitory activity at a concentration of 50 µM. ...
... This anilide is a competitive inhibitor of the SARS-CoV 3CL pro with a K i value of 0.03 µM 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Modification of compound 29 in a series of analogues resulted in reduced potency (30)(31)(32)(33)(34)(35). 58 A docking study ( Figure 12, PDB ID: 1UK4) showed that the 2-chloro-4-nitroanilide unit of compound 29 occupies the second preferred pocket. Thus, the nitro group was predicted to be hydrogen bonded with Ala46 and His41, providing a possible key interaction with the catalytic dyad. ...
... The (dimethylamino)phenyl group fit into the cleft formed by Gln189-Gln192 and Met165-Pro68. 58 A docking study also suggested that anilide 29 has the lowest binding energy (-9.1 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 22 kcal/mol) compared to the other derivatives. This experiment supports the observations of the enzymatic assay, which revealed the important roles of 2-chloro-4-nitroaniline and 4-(dimethylamino)benzoic acid residues in effective inhibition. ...
Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 (10%) fatalities in 2003. The causative agent of SARS has been identified as a novel human coronavirus (SARS-CoV), and its viral protease, SARS-CoV 3CLpro, has been shown to be essential for replication and has hence been recognized as a potent drug target for SARS infection. Currently, there is no effective treatment for this epidemic, despite the intensive research that has been undertaken since 2003 (over 3500 publications). This review focuses on the status of various efficacious anti-SARS-CoV 3CLpro chemotherapies discovered during the last 12 years (2003-2015) from all sources, including laboratory synthetic methods, natural products and virtual screening. We describe here mainly peptidomimetic and small molecule inhibitors of SARS-CoV 3CLpro. Attempts have been made to provide an exhaustive compilation of the structural features and binding modes of these inhibitors under many circumstances. Key words: Severe acute respiratory syndrome, coronavirus, 3CL protease, inhibitors, infectious diseases and chemotherapy.
... [8][9][10][11] A fine-tuning of the steric properties of BOX ligands showed that L9 was optimal. Control experiments indicated that essentially no detectable product formed when reactions were conducted without nickel catalyst or chiral ligand (entries [13][14]. The reaction was not very sensitive to oxygen because only a slight loss of yield was observed when the reaction was conducted under air in a capped vial, although the deliberate addition of water (1.0 equiv) significantly reduced the reaction efficiency (entries 15-16). ...
... Similarly, structures 63 and 69 ( Figure 2C), intermediates in a medicinal chemistry program for the discovery of anilide inhibitors against the SARS-CoV 3CL protease, were accessed in nine step routes wherein the majority of operations do not form strategic bonds. [13] In contrast, enantioselective decarboxylative acylation furnishes the same structures in a fraction of the steps previously required. From the standpoint of logic employed in a medicinal chemistry setting this method offers increased [a] Yields of isolated products are indicated in each case unless otherwise specified. ...
A new approach to the enantiocontrolled synthesis of α‐amino ketone derivatives is disclosed by employing a decarboxylative acylation strategy. Thus, when an acyl chloride and an α‐amido‐containing redox‐active ester are exposed to a nickel catalyst, chiral ligand, and metal reductant, α‐amido ketones are produced in good yield and high ee. The reaction exhibits broad substrate scope, can be easily scaled up, and is applied to dramatically simplify the synthesis of several known structures.
... Niclosamide and derivatives were reported as potent protease inhibitors of several viruses, including SARS-CoV (Li et al., 2017(Li et al., , 2020aShie et al., 2005;Xu et al., 2020d). Although niclosamide itself does not inhibit the 3CLpros of SARS-CoV and CoV-2 even at 50 μM concentration in our experiment, its derivatives were potent inhibitors against SARS-CoV (Shie et al., 2005). ...
... Niclosamide and derivatives were reported as potent protease inhibitors of several viruses, including SARS-CoV (Li et al., 2017(Li et al., , 2020aShie et al., 2005;Xu et al., 2020d). Although niclosamide itself does not inhibit the 3CLpros of SARS-CoV and CoV-2 even at 50 μM concentration in our experiment, its derivatives were potent inhibitors against SARS-CoV (Shie et al., 2005). In this project, we synthesized different derivatives of niclosamide and checked their inhibitory potential using FRET and cell-based assays. ...
SARS-CoV-2 has raised the alarm to search for effective therapy for this virus. To date several vaccines have been approved but few available drugs reported recently still need approval from FDA. Remdesivir was approved for emergency use only. In this report, the SARS-CoV-2 3CLpro was expressed and purified. By using a FRET-based enzymatic assay, we have screened a library consisting of more than 300 different niclosamide derivatives and identified three molecules JMX0286, JMX0301, and JMX0941 as potent allosteric inhibitors against SARS-CoV-2 3CLpro, with IC50 values similar to that of known covalent inhibitor boceprevir. In a cell-based antiviral assay, these inhibitors can inhibit the virus growth with EC50 in the range of 2–3 μM. The mechanism of action of JMX0286, JMX0301, and JMX0941 were characterized by enzyme kinetics, affinity binding and protein-based substrate digestion. Molecular docking, molecular dynamics (MD) simulations and hydration studies suggested that JMX0286, JMX0301, JMX0941 bind specifically to an allosteric pocket of the SARS-CoV-2 3CL protease. This study provides three potent compounds for further studies.
... The journey of different analogues towards development of SARS-CoV 3CLpro inhibitors is provided in Figure 3. Here, we have classified the SARS-CoV 3CLpro inhibitors into two categories: 1) Peptidomimetic inhibitors (PIs), and 2) Small-molecule inhibitors (SMIs). The PIs consist of different analogues, including: Keto-Glutamine analogues 23 , Anilide analogues 24 , Peptidomimetic α,β-unsaturated esters 25 , Glutamic acid and glutamine peptides 26 , Peptidomimetic (TG-0205221) 27 , Phthalhydrazide peptide analogues 28 , Cinanserin analogues 29 , Trifluoromethyl ketone containing peptides 30 , Trifluoromethyl, benzothiazolyl or thiazolyl ketone containing peptidomimetics 31 , Michael type of peptidomimetics 32 , Cysteine protease inhibitors 33 , Potent dipeptide type of peptidomimetics 34 , Novel dipeptide type of peptidomimetics 35 , Nitrile based peptidomimetics 36 , Tripeptide type of peptidomimetics 37 , Peptidomimetics containing cinnamoyl warhead 38 , Peptidomimetics containing decahydroisoquinolin moiety 39 , Ketone-based covalent inhibitors 14 , and α-Ketoamide derivatives 40 . The SMIs are classified into: isatin derivatives 41 , Chloropyridyl ester derivatives 42 , pyrazolone derivatives 43 , pyrimidine derivatives 44 , macrocyclic inhibitors 45 , 5-sulphonyl isatin derivatives 46 , pyrazolone derivatives 47 , serine derivatives 48 , phenylisoserine derivatives 49 , and Octahydroisochromene derivatives 50 . ...
... A multiple series of peptide anilides were developed by Shie and coworkers in 2005 based on antihelmintic drug (Niclosamide) 69 and evaluated the anti-SARS activity targeting the 3CLpro enzyme 24 . The anilide analogues were derived from 2-chloro-4-nitroaniline, L-phenylalanine, and 4-(dimethylamino)benzoic acid. ...
Severe Acute Respiratory Syndrome (SARS) is a severe febrile respiratory disease caused by the beta genus of human coronavirus, known as SARS-CoV. Last year, 2019-n-CoV (COVID-19) was a global threat for everyone caused by the outbreak of SARS-CoV-2. 3CLpro, chymotrypsin-like protease, is a major cysteine protease that substantially contributes throughout the viral life cycle of SARS-CoV and SARS-CoV-2. It is a prospective target for the development of SARS-CoV inhibitors by applying a repurposing strategy. This review focuses on a detailed overview of the chemical synthesis and computational chemistry perspectives of peptidomimetic inhibitors (PIs) and small-molecule inhibitors (SMIs) targeting viral proteinase discovered from 2004-2020. The PIs and SMIs are one of the primary therapeutic inventions for SARS-CoV. The journey of different analogues towards the evolution of SARS-CoV 3CLpro inhibitors and complete synthetic preparation of nineteen derivatives of PIs and ten derivatives of SMIs and their computational chemistry perspectives were reviewed. From each class of derivatives, we have identified and highlighted the most compelling PIs and SMIs for SARS-CoV 3CLpro. The protein-ligand interaction of 29 inhibitors were also studied that involved with the 3CLpro inhibition, and the frequent amino acid residues of the protease were also analyzed that are responsible for the interactions with the inhibitors. This work will provide an initiative to encourage further research for the development of effective and drug-like 3CLpro inhibitors against coronaviruses in the near future.
... With a target to improve SARS-CoV Mpro inhibitory potency of anilide derivatives Shie and co-workers reported a group of anilides as potent Mpro inhibitors [23]. Although these 2-chloro-4nitroanilides exhibited a decent Mpro inhibition, the ketomethylene isosters displayed comparatively lower potency in compare to the other derivatives. ...
... The dimethylaminophenyl moiety fitted inside the P68, M165, Q189 and Q192 created cleft while the phenyl ring of the benzyl moiety entered the S1 pocket of the SARS-CoV Mpro. In addition, the 2chloro-4-nitorphenyl function fitted inside the pocket formed by T25, H41, C44, A46 and T45 where the NO 2 group interacted with amino acid residue A46 [23]. Inspired by the human rhinovirus (HRV) 3Cpro inhibitor, A002, further convening their study on SARS-CoV Mpro inhibitors. ...
The fascinating similarity between the SARS-CoV and SARS-CoV-2, inspires scientific community to investigate deeper into the SARS-CoV proteases such as main protease (Mpro) and papain-like protease (PLpro) and their inhibitors for the discovery of SARS-CoV-2 protease inhibitors. Because of the similarity in the proteases of these two corona viruses, there is a greater chance for the previous SARS-CoV Mpro and PLpro inhibitors to provide effective results against SARS-CoV-2. In this context, the molecular fragments from the SARS-CoV protease inhibitors through the fragment-based drug design and discovery technique can be useful guidance for COVID-19 drug discovery. Here, we have focused on the structure-activity relationship studies of previous SARS-CoV protease inhibitors and discussed about crucial fragments generated from previous SARS-CoV protease inhibitors important for the lead optimization of SARS-CoV-2 protease inhibitors. This study surely offers different strategic options of lead optimization to the medicinal chemists to discover effective anti-viral agent against the devastating disease, COVID-19.
... Another study found that NCS didn't reveal clear inhibitory potency towards SARS-CoV(3CL protease) even up to 50 μM concentration. The study concluded that the drug may acts through different mechanism against the virus [31]. In Vero B4 cells, the evaluation of 10 μMNCS e against MERS-CoV demonstrated up to 1000-fold inhibition of MERS-CoV replication at 48 h p.i [32]. ...
Since the world health organization declared the pandemic of covid 19, many drugs have been tested and re-evaluated to find effective treatment for the novel corona virus infection. Niclosamide and Levamisole which are FDA approved anthelmintic drugs have been evaluated by many researchers and agencies to repurpose of these drugs as additional options for existing treatment strategy used for patients with Covid-19. Hence we are trying in this review to introduce most reports that evaluated the use of Niclosamide and Levamisole for treatment of patients infected with Covid 19. We concluded that the encouraging studies regarding the repurpose of the two drugs may highlighting for further studies that can widening the options for existing treatment strategy used for patients with COVID-19.
... Various molecular studies regarding SARS-CoV genomic analysis confirmed that SARS-CoV 3CL-protease is an important enzyme for processing viral polyprotein and is regarded as a potential drug target [69]. However, NCL showed no noticeable effect on SARS-CoV 3CL-protease even at a higher concentration [25]. Furthermore, NCL inhibits MERS-CoV replication by inhibiting E3-ligase S-phase kinase associated protein 2 (SKP2), which is involved in the degradation of proteasomes [26,70]. ...
Aim/purpose
Niclosamide (NCL) is an anthelminthic drug, which is widely used to treat various diseases due to its pleiotropic anti-inflammatory and antiviral activities. NCL modulates of uncoupling oxidative phosphorylation and different signaling pathways in human biological processes. The wide-spectrum antiviral effect of NCL makes it a possible candidate for recent pandemic SARS-CoV-2 infection and may reduce Covid-19 severity. Therefore, the aim of the present study was to review and clarify the potential role of NCL in Covid-19.
Methods
This study reviewed and highlighted the protective role of NCL therapy in Covid-19. A related literature search in PubMed, Scopus, Web of Science, Google Scholar, and Science Direct was done.
Results
NCL has noteworthy anti-inflammatory and antiviral effects. The primary antiviral mechanism of NCL is through neutralization of endosomal PH and inhibition of viral protein maturation. NCL acts as a proton carrier, inhibits homeostasis of endosomal PH, which limiting of viral proliferation and release. The anti-inflammatory effects of NCL are mediated by suppression of inflammatory signaling pathways and release of pro-inflammatory cytokines. However, the major limitation in using NCL is low aqueous solubility, which reduces oral bioavailability and therapeutic serum concentration that reducing the in vivo effect of NCL against SARS-CoV-2.
Conclusions
NCL has anti-inflammatory and immune regulatory effects by modulating the release of pro-inflammatory cytokines, inhibition of NF-κB /NLRP3 inflammasome and mTOR signaling pathway. NCL has an anti-SARS-CoV-2 effect via interruption of viral life-cycle and/or induction of cytopathic effect. Prospective clinical studies and clinical trials are mandatory to confirm the potential role of NCL in patients with Covid-19 concerning the severity and clinical outcomes.
... The electrophilic warhead of that inhibitor is based on nitroanilide structure where the thiol group of M pro attacks electrophilic sp 2 carbon of aromatic ring (Figure 20, 186). [89] Protease inhibitors incorporating spring-loaded rigs of aziridines and epoxides built-in peptide structure were described by Martina and co-workers ( Figure 20, 187 and 188). [90] Other potent inhibitors of CoV M pro incorporating electrophilic warhead are aldehydes [91] (Figure 20, 189) and halomethylketones [92] (Figure 20, 190) able for hemiacetal bonding. ...
A significant number of antiviral agents used in clinical practice are amino acids, short peptides, or peptidomimetics. Among them, several HIV protease inhibitors (e. g. lopinavir, atazanavir), HCV protease inhibitors (e. g. grazoprevir, glecaprevir), and HCV NS5A protein inhibitors have contributed to a significant decrease in mortality from AIDS and hepatitis. However, there is an ongoing need for the discovery of new antiviral agents and the development of existing drugs; amino acids, both proteinogenic and non‐proteinogenic in nature, serve as convenient building blocks for this purpose. The synthesis of non‐proteinogenic amino acid components of antiviral agents could be challenging due to the need for enantiomerically or diastereomerically pure products. Herein, we present a concise review of antiviral agents whose structures are based on amino acids of both natural and unnatural origin. Special attention is paid to the synthetic aspects of non‐proteinogenic amino acid components of those agents.
... One dataset containing approximately 40 actives was used for each of the three inhibitor-enzyme complexes selected in this work. To do this, we randomly selected forty actives for each target from a small library that was built with ∼140 known Mpro inhibitors of SARS-CoV and SARS-CoV-2, compiled from literature [27][28][29][30][35][36][37][38][39][40][41][42][43][44], considering either reported data concerning their IC 50 values or existence of respective PDB files (see Supplementary Table 1). Decoy generation was executed from the webserver Database of Useful Decoys Enhanced (DUD-E) [45], which furnished 50 decoys per active compound. ...
Background: The new coronavirus pandemic has had a significant impact worldwide, and therapeutic treatment for this viral infection is being strongly pursued. Efforts have been undertaken by medicinal chemists to discover molecules or known drugs that may be effective in COVID-19 treatment – in particular, targeting the main protease (Mpro) of the virus. Materials & methods: We have employed an innovative strategy – application of ligand- and structure-based virtual screening – using a special compilation of an approved and diverse set of SARS-CoV-2 crystallographic complexes that was recently published. Results and conclusion: We identified seven drugs with different original indications that might act as potential Mpro inhibitors and may be preferable to other drugs that have been repurposed. These drugs will be experimentally tested to confirm their potential Mpro inhibition and thus their effectiveness against COVID-19.
... Besides the peptidomimetic inhibitors mentioned above, small-molecule inhibitors targeting SARS-CoV M pro have also been extensively explored. Wong and coworkers produced a series of nitroaniline derivatives, among which compound 29 (Fig. 16) was discovered and functions as a competitive, noncovalent inhibitor targeting SARS-CoV M pro ; it displays potent inhibitory efficacy (K i and IC 50 values of 0.03 and 0.06 μM, respectively) [120]. Currently, the increasing exploitation of metal-conjugated compounds as broad-spectrum antiviral agents has been widely investigated [121][122][123][124]. ...
Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still a pandemic around the world. Currently, specific antiviral drugs to control the epidemic remain deficient. Understanding the details of SARS-CoV-2 structural biology is extremely important for development of antiviral agents that will enable regulation of its life cycle. This review focuses on the structural biology and medicinal chemistry of various key proteins (Spike, ACE2, TMPRSS2, RdRp and Mpro) in the life cycle of SARS-CoV-2, as well as their inhibitors/drug candidates. Representative broad-spectrum antiviral drugs, especially those against the homologous virus SARS-CoV, are summarized with the expectation they will drive the development of effective, broad-spectrum inhibitors against coronaviruses. We are hopeful that this review will be a useful aid for discovery of novel, potent anti-SARS-CoV-2 drugs with excellent therapeutic results in the near future.
... [40] The recent experimental binding study of 4-(dimethylamino) benzoic acid suggests about its effective protease inhibitory action. [41] Similar computational research work that involves 3D structure prediction followed by docking methods has been successfully developed and implemented by many researchers for discovery of potential inhibitors against other diseases. Recently, many advance version of computational prediction methods are being used for secondary and tertiary structure of proteins lead to prediction of the function . ...
Background: Currently, the helicase enzyme of novel severe acute respiratory syndrome coronavirus 2019 has been proposed as a
potential drug target. This work envisages predicting the three dimensional (3D) structure of helicase (nonstructural protein 13) and
screen for the novel inhibitor molecules. Methods: For this purpose, the sequence information of helicase enzyme was obtained
from NCBI, and 3D model was predicted using I TASSER server followed by model validation. The helicase enzyme sequence
was then used to search for the potential inhibitors in the Drug Bank database. The search resulted eight numbers of probable
drug molecules against the receptor. To confirm the binding affinity of the drug molecules, further molecular docking study was
conducted using AutoDock Vina software. Results: From the docking result, it was obtained that, among all eight numbers, only
the molecule remdesivir shows more binding affinity to the nucleoside triphosphate binding site of helicase enzyme and further
confirmed by analysis of amino acid interaction profile. Conclusion: In the present study, it was predicted that, the drug molecule
remdesivir can be suitably used as a helicase inhibitor in case of novel severe acute respiratory syndrome coronavirus 2019.
Keywords: Binding affinity, docking, drug mol
... SARS-CoV 3CL protease is identified with a vital role in the processing of replicase polyprotein, and could be a major target for development of anti-SARS agents [119,120]. In one study on concentrations up to 50 μM, no clear inhibitory effect was found against SARS-CoV 3CL protease, therefore, they may be other explanations for its effects [51]. ...
Coronavirus disease 2019 (COVID-19) is an infective disease generated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given the pandemic urgency and lack of an effective cure for this disease, drug repurposing could open the way for finding a solution. Lots of investigations are ongoing to test the compounds already identified as antivirals. On the other hand, induction of type I interferons are found to play an important role in the generation of immune responses against SARS-CoV-2. Therefore, it was opined that the antivirals capable of triggering the interferons and their signaling pathway, could rationally be beneficial for treating COVID-19. On this basis, using a database of antivirals, called drugvirus, some antiviral agents were derived, followed by searches on their relevance to interferon induction. The examined list included drugs from different categories such as antibiotics, immunosuppressants, anti-cancers, non-steroidal anti-inflammatory drugs (NSAID), calcium channel blocker compounds, and some others. The results as briefed here, could help in finding potential drug candidates for COVID-19 treatment. However, their advantages and risks should be taken into account through precise studies, considering a systemic approach. Even though the adverse effects of some of these drugs may overweight their benefits, considering their mechanisms and structures may give a clue for designing novel drugs in the future. Furthermore, the antiviral effect and IFN-modifying mechanisms possessed by some of these drugs might lead to a synergistic effect against SARS-CoV-2, which deserve to be evaluated in further investigations.
... Shie et al. [96] developed a series of peptide based anilide derivatives and out of all these, Niclosamide type anilides were found to be potent inhibitors of 3CL pro . Literature reveals that Niclosamide, 55 itself acts as a 3CL pro inhibitor with IC 50 value > 50 mM [97]. ...
Originated in China, coronavirus disease 2019 (COVID-19)- the highly contagious and fatal respiratory disease caused by SARS-CoV-2 has already infected more than 29 million people worldwide with a mortality rate of 3.15% (according to World Health Organization’s (WHO’s) report, September 2020) and the number is exponentially increasing with no remedy whatsoever discovered till date. But it is not the first time this infectious viral disease has appeared, in 2002 SARS-CoV infected more than 8000 individuals of which 9.6% patients died and in 2012 approximately 35% of MERS-CoV infected patients have died. Literature reports indicate that a chymotripsin-like cystein protease (3CLpro) is responsible for the replication of the virus inside the host cell. Therefore, design and synthesis of 3CLpro inhibitor molecules play a great impact in drug development against this COVID-19 pandemic. In this review, we are discussing the anti-SARS effect of some small molecule 3CLpro inhibitors with their various binding modes of interactions to the target protein.
... (8) 肽硝基苯胺类抑制剂 Shie等 [86] 筛选了含有多种苯胺的肽类化合物对于 化合物40的选择性也有明显的增强, 在100 μM高浓度 下对HIV蛋白酶没有明显抑制作用 [88] . ...
Global pandemic caused by coronavirus remains a huge threat to public health. No efficacious drugs and vaccines are available so far. Therefore, it is urgent to design and develop anti-coronavirus clinical drugs. The 3C-like protease is a promising key drug target, as it plays a pivotal role in viral replication and is highly conserved in all coronaviruses. This review summarizes the major advances in the structure, catalytic mechanism, substrate specificity, auto-processing mechanism and inhibitor discovery for the 3C-like protease of SARS-CoV, MERS-CoV and SARS-CoV-2, classifies known inhibitors and analyzes their possible inhibition mechanisms. Potential future directions of 3C-like protease inhibitor discovery with novel mechanisms are also discussed.
... The viral antigen production was destroyed at niclosamide concentration of 1.56 μM which was revealed by immunoblot analysis (Wu et al., 2004). Niclosamide unlike other nitro anilide based peptides does not get cleaved by SARS-CoV 3CL protease enzyme but shows a slight inhibitory action against the enzyme (Shie et al., 2005). Another cell-based assay of cytopathogenic effect done in Vero E6 cells infected with the SARS virus showed a high level of inhibition mediated by niclosamide at concentrations between 3.3 and 20 μM. ...
Coronaviruses are pleomorphic, enveloped, or spherical viruses, which have a size ranging from 80 to 120 nm. These viruses act on receptors that cause the triggering of fusion. Coronaviruses were first described after cultivation from patients with common colds by Tyrell and Bynoe in 1966. There are various subtypes of coronavirus, 7 out of these can cause infection in human beings. The Alpha subtype is responsible for mild infection showing symptoms or infection without any prevailing symptoms. On the other hand, the beta subtype is responsible for very serious diseases leading to fatality. The lineage of this novel SARS-CoV-2 falls under the beta lineage of the beta coronavirus which has been observed to have a relation to the MERS and SARS coronavirus. In the Huanan market selling seafood, the transition of this novel virus in humans from animals has occurred. It has the potential to be the cause of widespread fatality amongst the people of the globe. On August 16, 2020, the World Health Organisation had reported 2,1294,845 cases which are confirmed to date out of which 413,372 deaths have occurred. Currently, no targeted antiviral vaccines or drugs to fight against COVID-19 infection have been approved for use in humans. This pandemic is fast emerging and drug repurposing is the only ray of hope which can ensure quick availability. Vaccine development is progressing each day with various platforms such as DNA, Live Attenuated Virus, Non-Replicating Viral Vector, Protein Subunit, and RNA, being utilized for the development. COVID-19 attacks the immune system of the host & this can result in a cytokine storm. As a result, various herbal agents both acting as antivirals and immunomodulatory can also be used. Convalescent Plasma Therapy and Mesenchymal Stem Cell therapy are also being explored as a plausible therapeutic. There remains a considerable unmet need for therapeutics to be addressed. The development and availability of accessible and efficient therapy are essential for the treatment of patients. This review discusses the epidemiology, pathogenesis, the tale of origin, and transmission of COVID-19 or Sars-Cov2 virus and gives evidence of potential therapeutic agents that can be explored to cast away this pandemic.
... Shie et al. studied a diverse library of synthesized aniline against SARS-CoV-1 3CL protease. 2chloro-4-nitroaniline, l-phenylalanine, and 4-(dimethylamino) benzoic acid showed the highest inhibition potential with Ki ¼ 0.03 mmM binding with CYS 145 containing thiol group in the active site (Shie et al., 2005). Lalani and Poh studied the in-vitro antiviral potency of flavonoids and their derivatives against enteroviral protease from the Enterovirus EV-A71 strain. ...
The study aims to evaluate the potency of two hundred natural antiviral phytocompounds against the active site of the Severe Acquired Respiratory Syndrome - Coronavirus − 2 (SARS-CoV-2) Main-Protease (Mpro) using AutoDock 4.2.6. The three- dimensional crystal structure of the Mpro (PDB Id: 6LU7) was retrieved from the Protein Data Bank (PDB), the active site was predicted using MetaPocket 2.0. Food and Drug Administration (FDA) approved viral protease inhibitors were used as standards for comparison of results. The compounds theaflavin-3-3’-digallate, rutin, hypericin, robustaflavone, and (-)-solenolide A with respective binding energy of −12.41 (Ki = 794.96 pM); −11.33 (Ki = 4.98 nM); −11.17 (Ki = 6.54 nM); −10.92 (Ki = 9.85 nM); and −10.82 kcal/mol (Ki = 11.88 nM) were ranked top as Coronavirus Disease − 2019 (COVID-19) Mpro inhibitors. The interacting amino acid residues were visualized using Discovery Studio 3.5 to elucidate the 2-dimensional and 3-dimensional interactions. The study was validated by i) re-docking the N3-peptide inhibitor-Mpro and superimposing them onto co-crystallized complex and ii) docking decoy ligands to Mpro. The ligands that showed low binding energy were further predicted for and pharmacokinetic properties and Lipinski’s rule of 5 and the results are tabulated and discussed. Molecular dynamics simulations were performed for 50 ns for those compounds using the Desmond package, Schrödinger to assess the conformational stability and fluctuations of protein-ligand complexes during the simulation. Thus, the natural compounds could act as a lead for the COVID-19 regimen after in-vitro and in- vivo clinical trials.
Communicated by Ramaswamy H. Sarma
... Among the 32 tested compounds, the peptide anilide JMF1507 (30) showed the most potent inhibition, with an IC 50 value of 0.06 μM and a K i value of 0.03 μM. 102 Another highly potent compound that is a peptidomimetic inhibitor (31) was reported to have a SARS-CoV 3CL pro inhibitory activity of 0.20 μM. 103 3.2.2. ...
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 20 million people infected worldwide with an average mortality rate of 3.6%. This virus poses major challenges to public health, as it not only is highly contagious but also can be transmitted by asymptomatic infected individuals. COVID-19 is clinically difficult to manage due to a lack of specific antiviral drugs or vaccines. In this article, Chinese therapy strategies for treating COVID-19 patients, including current applications of traditional Chinese medicine (TCM), are comprehensively reviewed. Furthermore, 72 small molecules from natural products and TCM with reported antiviral activity against human coronaviruses (CoVs) are identified from published literature, and their potential applications in combating SARS-CoV-2 are discussed. Among these, the clinical efficacies of some accessible drugs such as remdesivir (RDV) and favipiravir (FPV) for COVID-19 are emphatically summarized. We hope this review provides a foundation for managing the worsening pandemic and developing antivirals against SARS-CoV-2.
... However, the steric effect caused by "Cl" atoms at ortho-position disturbs the coplanarity of 2-chloro-4-nitrophenyl ring with the amido group, thereby, discouraging the process of hydrolysis. The antiviral potential of these compounds, 10-13(Figure 4)suggests the therapeutic potential of anilides, and amide functionality for the rational development of SARS-CoV-2 targeting drugs(Shie et al., 2005). ...
... Flavonoids (10)(11)(12)(13)(14) are plant-derived natural products with diverse reported biological activities, and they have been shown to be also able to inhibit the 3CLpro (Figure 4). 17,18 The broad-spectrum and established use of plant-based medicines to combat infectious diseases in TCM is the basis of several currently ongoing clinical trials in China. ...
SARS-CoV-2 (previously 2019-nCoV or Wuhan coronavirus) caused an unprecedented fast-spreading worldwide pandemic. Although currently with a rather low mortality rate, the virus spread rapidly over the world using the modern world’s traffic highways. The coronavirus (CoV) family members were responsible for several deadly outbreaks and epidemics during the last decade. Not only governments but also the scientific community reacted promptly to the outbreak, and information is shared quickly. For example, the genetic fingerprint was shared, and the 3D structure of key proteins was rapidly solved, which can be used for the discovery of potential treatments. An overview is given on the current knowledge of the spread, disease course, and molecular biology of SARS-CoV-2. We discuss potential treatment developments in the context of recent outbreaks, drug repurposing, and development timelines.
... Compound 38 also showed selectivity towards SARS-CoV 3CLpro, displaying significantly lower activity against trypsin, chymotrypsin and papain (IC 50 values of 110, 200 and 220 μM, respectively). [86] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 In 2011, Akaji and co-workers developed a series of SARS 3CLpro inhibitors containing a terminal aldehyde. An initial lead sequence, namely Ac-Ser-Ala-Val-Leu-His-H (42, IC 50 = 5.7 μM, Figure 17) was employed for further optimization. ...
The COVID‐19 pandemic caused by SARS‐CoV‐2 infection is spreading at an alarming rate and has created an unprecedented health emergency around the globe. There is no effective vaccine or approved drug treatment against COVID‐19 and other pathogenic coronaviruses. The development of antiviral agents is an urgent priority. Biochemical events critical to the coronavirus replication cycle provided a number of attractive targets for drug development. These include, spike protein for binding to host cell‐surface receptors, proteolytic enzymes that are essential for processing polyproteins into mature viruses, and RNA‐dependent RNA polymerase for RNA replication. There has been a lot of ground work for drug discovery and development against these targets. Also, high‐throughput screening efforts have led to the identification of diverse lead structures, including natural product‐derived molecules. This review highlights past and present drug discovery and medicinal‐chemistry approaches against SARS‐CoV, MERS‐CoV and COVID‐19 targets. The review hopes to stimulate further research and will be a useful guide to the development of effective therapies against COVID‐19 and other pathogenic coronaviruses.
... Furthermore, recent study has shown that compounds containing electrophilic arylketone moiety were designed and synthesized as new SARS-Cov 3CL protease inhibitors [70]. The anilide derived from 2-chloro-4-nitroaniline, l-phenylalanine and 4-(dimethylamino)benzoic acid was found to be a competitive inhibitor of the SARS-CoV 3CL protease with K(i) = 0.03 uM by using a fluorogenic tetradecapeptide substrate [70,71]. It is demonstrated that trioxa-adamantane-triols (TATs) (BN, IBNCA, VANBA, euBN), trivially termed bananins, were identified to be effective inhibitors of SARS-CoV NSP10/nsp13 RNA/DNA helicase/NTPase protein ATPase enzymatic function. ...
An outbreak of novel coronavirus-related pneumonia COVID-19, that was identified in December 2019, has expanded rapidly, with cases now confirmed in more than 211 countries or areas. This constant transmission of a novel coronavirus and its ability to spread from human to human have prompted scientists to develop new approaches for treatment of COVID-19. A recent study has shown that remdesivir and chloroquine effectively inhibit the replication and infection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, 2019-nCov) in vitro. In the United States, one case of COVID-19 was successfully treated with compassionate use of remdesivir in January of 2020. In addition, a clinically proven protease inhibitor, camostat mesylate, has been demonstrated to inhibit Calu-3 infection with SARS-CoV-2 and prevent SARS-2-spike protein (S protein)-mediated entry into primary human lung cells. Here, we systemically discuss the pharmacological therapeutics targeting RNA-dependent RNA polymerase (RdRp), proteinase and S protein for treatment of SARS-CoV-2 infection. This review should shed light on the fundamental rationale behind inhibition of SARS-CoV-2 enzymes RdRp as new therapeutic approaches for management of patients with COVID-19. In addition, we will discuss the viability and challenges in targeting RdRp and proteinase, and application of natural product quinoline and its analog chloroquine for treatment of coronavirus infection. Finally, determining the structural-functional relationships of the S protein of SARS-CoV-2 will provide new insights into inhibition of interactions between S protein and angiotensin-converting enzyme 2 (ACE2) and enable us to develop novel therapeutic approaches for novel coronavirus SARS-CoV-2.
... Among the various inhibitors that have been reported, the majority was found to target at His-41, His-163, His-164, Cys-145, Glu-166, Met-49, and Met-165 residues via aromatic π-π stackings and hydrogen bonds. 2,13,[19][20][21][22][23][24][25][26][27][28][29] Possible COVID-19 treatment could be provided through the development of the COVID-19 3CLpro inhibitors, which can be hugely encouraged from the understanding of the inhibition mechanism of SARS-CoV 3CLpro, due to the structural similarity between the two target proteins. ...
The rapid spread of the COVID-19 outbreak is now a global threat with over a million diagnosed cases and more than 70 thousand deaths. Specific treatments and effective drugs regarding such disease are in urgent need. To contribute to the drug discovery against COVID-19, we performed computational study to understand the inhibition mechanism of the COVID-19 3c-like protease, and search for possible drug candidates from approved or experimental drugs through drug repurposing screening against the DrugBank database. Two novel computational methods were applied in this study. We applied the "Consecutive Histogram Monte Carlo" (CHMC) sampling method for understanding the inhibition mechanism from studying the 2-D binding free energy landscape. We also applied the "Movable Type" (MT) free energy method for the lead compound screening by evaluating the binding free energies of the COVID-19 3c-like protease-inhibitor complexes. Lead compounds from the DrugBank database were first filtered using ligand similarity comparison to 19 published SARS 3c-like protease inhibitors. 70 selected compounds were then evaluated for protein-ligand binding affinities using the MT free energy method. 4 drug candidates with strong binding affinities and reasonable protein-ligand binding modes were selected from this study, i.e. Enalkiren (DB03395), Rupintrivir (DB05102), Saralasin (DB06763) and TRV-120027 (DB12199).
... Among the various inhibitors that have been reported, the majority was found to target at His-41, His-163, His-164, Cys-145, Glu-166, Met-49, and Met-165 residues via aromatic π-π stackings and hydrogen bonds. 2,13,[19][20][21][22][23][24][25][26][27][28][29] Possible COVID-19 treatment could be provided through the development of the COVID-19 3CLpro inhibitors, which can be hugely encouraged from the understanding of the inhibition mechanism of SARS-CoV 3CLpro, due to the structural similarity between the two target proteins. ...
p>The rapid spread of the COVID-19 outbreak is now a global threat with over a million diagnosed cases and more than 70 thousand deaths. Specific treatments and effective drugs regarding such disease are in urgent need. To contribute to the drug discovery against COVID-19, we performed computational study to understand the inhibition mechanism of the COVID-19 3c-like protease, and search for possible drug candidates from approved or experimental drugs through drug repurposing screening against the DrugBank database. Two novel computational methods were applied in this study. We applied the “Consecutive Histogram Monte Carlo” (CHMC) sampling method for understanding the inhibition mechanism from studying the 2-D binding free energy landscape. We also applied the “Movable Type” (MT) free energy method for the lead compound screening by evaluating the binding free energies of the COVID-19 3c-like protease – inhibitor complexes. Lead compounds from the DrugBank database were first filtered using ligand similarity comparison to 19 published SARS 3c-like protease inhibitors. 70 selected compounds were then evaluated for protein-ligand binding affinities using the MT free energy method. 4 drug candidates with strong binding affinities and reasonable protein-ligand binding modes were selected from this study, i.e. Enalkiren (DB03395), Rupintrivir (DB05102), Saralasin (DB06763) and TRV-120027 (DB12199). </p
... In contrast to classical electrophilic building blocks like Michael-acceptor systems [9][10][11][12][13][14][15][16][17], nitriles [18][19][20][21], aldehydes, ketones [22][23][24], three-membered heterocycles [25][26][27][28] or a iodoacetic acid moiety [29,30], (hetero)aromatic electrophiles, which can react via nucleophilic addition (yielding πor σ-complexes) or substitution reactions (S N Ar) have only rarely been employed [31][32][33][34][35]. Such aromatic moieties have also seldom been investigated as non-covalently binding parts of the recognition units of enzyme inhibitors [36,37]. ...
Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the SNAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (Ki = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the SNAr reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation.
... In some extreme cases k4 = 0, and hence, these irreversible inhibitors covalently bind the target until the target disappears. 7,8 Considering the inhibitors of SARS 3CL pro may be also bio-active to SARS-CoV-2, we have created a molecular library including all the reported SARS-3CL pro inhibitors (284 molecules) 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 and we will also add new validated molecular structures into this library with our research progresses. To date, there are no clinically approved vaccines or drugs specifically targeting SARS-CoV-2. ...
The focused drug repurposing of known approved drugs (such as lopinavir/ritonavir) has been reported failed for curing SARS-CoV-2 infected patients. It is urgent to generate new chemical entities against this virus. As a key enzyme in the life-cycle of coronavirus, the 3C-like main protease (3CLpro or Mpro) is the most attractive for antiviral drug design. Based on a recently solved structure (PDB ID: 6LU7), we developed a novel advanced deep Q-learning network with the fragment-based drug design (ADQN-FBDD) for generating potential lead compounds targeting SARS-CoV-2 3CLpro. We obtained a series of derivatives from those lead compounds by our structure-based optimization policy (SBOP). All the 47 lead compounds directly from our AI-model and related derivatives based on SBOP are accessible in our molecular library at https://github.com/tbwxmu/2019-nCov. These compounds can be used as potential candidates for researchers in their development of drugs against SARS-CoV-2.
... Recent years have witnessed so much research for the discovery of antiviral drugs but the drugs are not effective against SARS. Certain 3ClPro inhibitors such as HIV protease inhibitor Tl-3, a thiophenecarboxylate, zinc conjugates [4] and anilides [5] were discovered but effective inhibition in the nano range against SARS was not reported. Certain stable benzotriazole esters derivatives were also discovered is late years. ...
The aim of this study is to identify a potential antiviral protease inhibitor for Sars CoV using In-silico docking studies with the Molegro Virtual Docker (MVD). The structures of benzotriazole esters were drawn using the Chem sketch software and protein PDB ID: 1uk4 was obtained from the protein data bank. Various electron withdrawing and donating groups were substituted in the active position of benzotriazole esters and docked against Sars CoV 3Cl pro. Automatic and manual methods were selected and Flexible docking was performed and results were observed. It was found that, among the docked ligands compound 38 (best with automatic docking method) and compound 27 (best with manual docking method) was said to possess the highest ranking score. The selected lead compounds were compared with the standard drug Maraviroc with parameters such as docking score and binding capacity with the target. The lead compounds were found to possess similar binding properties as that of the standard drug Maraviroc.
... 3C-like protease Benzotriazole esters CE-5 [284] Papain-like protease Thiopurines 6-thioguanine, 6-mercaptopurine [113] Helicase Triazole SSYA10-001 [162] RNA-dependent RNA polymerase Nucleotide prodrug GS-5734 [159] RNA-dependent RNA polymerase Nucleoside analog BCX4430 [160] Membrane-bound RNA synthesis Small molecule inhibitor K22 [285] Furin inhibitior Small molecule inhibitor Decanoyl-RVKR [286] SARS-CoV Viral/cellular target Drug class Drug 3C-like protease Benzotriazole esters CE-5 [284] 3C-like protease Anilides Peptide nitroanilides [287] 3C-like protease C 2 -symmetric inhibitors containing diol cores TL-3 [288] 3C-like protease Pyrazole analogs Pyrazolones [289] 3C-like protease Serine inhibitor Trifluoromethyl ketones [290] 3C-like protease Serotonin receptor antagonist Cinanserin [291] 3C-like protease Zinc-conjugated inhibitor JMF 1586 [292] Papain-like protease Thiopurines 6-thioguanine, 6-mercaptopurine [113] Helicase Triazole SSYA10-001 [162] Helicase Bananin derivatives Vanillinbananin, Idobananin [163] NTPase/Helicase Aryl diketoacids Dihydroxychromone and hydroxychromone derivatives ...
No specific antivirals are currently available for two emerging infectious diseases, Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS). A literature search was performed covering pathogenesis, clinical features and therapeutics, clinically developed drugs for repurposing and novel drug targets. This review presents current knowledge on the epidemiology, pathogenesis and clinical features of the SARS and MERS coronaviruses. The rationale for and outcomes with treatments used for SARS and MERS is discussed. The main focus of the review is on drug development and the potential that drugs approved for other indications provide for repurposing. The drugs we discuss belong to a wide range of different drug classes, such as cancer therapeutics, antipsychotics, and antimalarials. In addition to their activity against MERS and SARS coronaviruses, many of these approved drugs have broad-spectrum potential and have already been in clinical use for treating other viral infections. A wealth of knowledge is available for these drugs. However, the information in this review is not meant to guide clinical decisions, and any therapeutic described here should only be used in context of a clinical trial. Potential targets for novel antivirals and antibodies are discussed as well as lessons learned from treatment development for other RNA viruses. The article concludes with a discussion of the gaps in our knowledge and areas for future research on emerging coronaviruses.
... Therefore, compounds with 3-pyrrolidinone functions (Compounds 5-8) are preferable for the higher inhibitory activity. Shie et al. (2005a) reported a series of potent anilide inhibitors against SARS-3CL pro (Fig. 11.4;Table 11.6), for which the QSAR model obtained was as shown by Eq. (11.5). This model showed the importance of dipole moment along the X-axis (D X ), MW, PSA, and volume (Vol) for controlling the enzyme inhibition. ...
Severe acute respiratory syndrome (SARS), caused by SARS-coronavirus (SARS-CoV), is a dreadful infection worldwide having economic and medical importance and a global threat for health. It was turned into an epidemic in South China followed by a chain of infections across three generations. A number of pathogeneses in human may occur due to the virus. This infection has not been taken into account before the SARS outbreak, and still it is a neglected one. Therefore, there is an urgent need to develop small molecule antivirals to combat the SARS-CoV. No vaccines are available till date though a number of SARS-CoV 3C-like and 3C protease inhibitors were reported. In this chapter, quantitative structure–activity relationship technique is used for development of anti-SARS and anti-HRV drugs and outcome discussed in details. This approach may be a useful strategy to design novel and potential anti-SARS drugs to combat these dreadful viral diseases.
A new approach to the enantiocontrolled synthesis of α‐amino ketone derivatives is disclosed by employing a decar‐boxylative acylation strategy. Thus, when an acyl chloride and an α‐amido‐containing redox‐active ester are exposed to a nickel catalyst, chiral ligand, and metal reductant, α‐amido ketones are produced in good yield and high ee. The reaction exhibits broad substrate scope, can be easily scaled up, and is applied to dramatically simplify the synthesis of several known structures.
In order to address the world-wide health challenge caused by the COVID-19 pandemic, the 3CL protease/SARS-CoV-2 main protease (SARS-CoV-2-Mpro) coded by its nsp5 gene became one of the biochemical targets for the design of antiviral drugs. In less than 3 years of research, 4 inhibitors of SARS-CoV-2-Mpro have actually been authorized for COVID-19 treatment (nirmatrelvir, ensitrelvir, leritrelvir and simnotrelvir) and more such as EDP-235, FB-2001 and STI-1558/Olgotrelvir or five undisclosed compounds (CDI-988, ASC11, ALG-097558, QLS1128 and H-10517) are undergoing clinical trials. This review is an attempt to picture this quite unprecedented medicinal chemistry feat and provide insights on how these cysteine protease inhibitors were discovered. Since many series of covalent SARS-CoV-2-Mpro inhibitors owe some of their origins to previous work on other proteases, we first provided a description of various inhibitors of cysteine-bearing human caspase-1 or cathepsin K, as well as inhibitors of serine proteases such as human dipeptidyl peptidase-4 or the hepatitis C protein complex NS3/4A. This is then followed by a description of the results of the approaches adopted (repurposing, structure-based and high throughput screening) to discover coronavirus main protease inhibitors.
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) identified in 2003 infected ∼8000 people in 26 countries with 800 deaths, which was soon contained and eradicated by syndromic surveillance and enhanced quarantine. A closely related coronavirus SARS-CoV-2, the causative agent of COVID-19 identified in 2019, has been dramatically more contagious and catastrophic. It has infected and caused various flu-like symptoms of billions of people in >200 countries, including >6 million people died of or with the virus. Despite the availability of several vaccines and antiviral drugs against SARS-CoV-2, finding new therapeutics is needed because of viral evolution and a possible emerging coronavirus in the future. The main protease (Mpro) of these coronaviruses plays important roles in their life cycle and is essential for the viral replication. This article represents a comprehensive review of the function, structure and inhibition of SARS-CoV and -CoV-2 Mpro, including structure-activity relationships, protein-inhibitor interactions and clinical trial status.
In this article, emulsomes (EMLs) were fabricated to encapsulate the N-(5-nitrothiazol-2-yl)-carboxamido derivatives (3a–3g) in an attempt to improve their biological availability and antiviral activity. Next, both cytotoxicity and anti-SARS-CoV-2 activities of the examined compounds loaded EMLs (F3a–g) were assessed in Vero E6 cells via MTT assay to calculate the CC50 and inhibitory concentration 50 (IC50) values. The most potent 3e-loaded EMLs (F3e) elicited a selectivity index of 18 with an IC50 value of 0.73 μg/mL. Moreover, F3e was selected for further elucidation of a possible mode of action where the results showed that it exhibited a combination of virucidal (>90%), viral adsorption (>80%), and viral replication (>60%) inhibition. Besides, molecular docking and MD simulations towards the SARS-CoV-2 Mpro were performed. Finally, a structure–activity relationship (SAR) study focussed on studying the influence of altering the size, type, and flexibility of the α-substituent to the carboxamide in addition to compound contraction on SARS-CoV-2 activity.
Highlights
Emulsomes (EMLs) were fabricated to encapsulate the N-(5-nitrothiazol-2-yl)-carboxamido derivatives (3a–3g).
The most potent 3e-loaded EMLs (F3e) showed an IC50 value of 0.73 μg/mL against SARS-CoV-2.
F3e exhibited a combination of virucidal (>90%), viral adsorption (>80%), and viral replication (>60%) inhibition.
Molecular docking, molecular dynamics (MD) simulations, and MM-GBSA calculations were performed.
Structure–activity relationship (SAR) study was discussed to study the influence of altering the size, type, and flexibility of the α-substituent to the carboxamide on the anti-SARS-CoV-2 activity.
The prevalence of coronavirus disease 2019 (COVID-19) is the third registered spillover of an animal coronavirus to humans from the early 21st century. Coronaviruses are important human and animal pathogens. The 2019 novel coronavirus (2019-nCoV) rapidly spreads, resulting in an epidemic throughout China, followed by an increasing number of cases in other countries throughout the world. Recently, a wide range of inhibitors have been introduced for treatment of COVID-19, and also promising vaccines are in late phase of development. Here, we aim to present an overview of recent findings of the biological and clinical aspects of SARS-CoV-2 infection, along with possible treatments and future vaccines.
Since the initial outbreak in December 2019, the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 3 million deaths worldwide. There is an urgent need for developing a potential therapy against SARS-CoV-2. Exploring the potentials of phytochemicals towards inhibition of SARS-CoV-2 proteins remains of significant scientific interest. The therapeutic values of phytochemicals in the treatment of diseases, such as viral infections, are known for a long time. In this review, we present a brief overview of the past experimental and computational efforts on evaluating phytochemicals against SARS coronaviruses, an earlier coronavirus strain. We discuss natural metabolites of different structural and chemical scaffolds, including polyphenols, flavonoids, and phytosterols, which can be promising compounds for screening against the currently evolving SARS-CoV-2 virus.
The drug repurposing of known approved drugs (e.g., lopinavir/ritonavir) has failed to treat SARS-CoV-2-infected patients. Therefore, it is important to generate new chemical entities against this virus. As a critical enzyme in the lifecycle of the coronavirus, the 3C-like main protease (3CLpro or Mpro) is the most attractive target for antiviral drug design. Based on a recently solved structure (PDB ID: 6LU7), we developed a novel advanced deep Q-learning network with a fragment-based drug design (ADQN–FBDD) for generating potential lead compounds targeting SARS-CoV-2 3CLpro. We obtained a series of derivatives from the lead compounds based on our structure-based optimization policy (SBOP). All of the 47 lead compounds obtained directly with our AI model and related derivatives based on the SBOP are accessible in our molecular library. These compounds can be used as potential candidates by researchers to develop drugs against SARS-CoV-2.
Severe acute respiratory syndrome coronavirus‐2 is the etiological agent of the ongoing pandemic of coronavirus disease‐2019, a multi‐organ disease that has triggered an unprecedented global health and economic crisis. The virally encoded 3C‐like protease (3CLpro), which is named after picornaviral 3C protease (3Cpro) due to their similarities in substrate recognition and enzymatic activity, is essential for viral replication and has been considered as the primary drug target. However, information regarding the cellular substrates of 3CLpro and its interaction with the host remains scarce, though recent work has begun to shape our understanding more clearly. Here we summarized and compared the mechanisms by which picornaviruses and coronaviruses have evolved to evade innate immune surveillance, with a focus on the established role of 3Cpro in this process. Through this comparison, we hope to highlight the potential action and mechanisms that are conserved and shared between 3Cpro and 3CLpro. In this review, we also briefly discussed current advances in the development of broad‐spectrum antivirals targeting both 3Cpro and 3CLpro.
Background:
A novel coronavirus disease, 2019-nCoV (COVID-19), reported first in Wuhan, the capital of Hubei, China in late December 2019 and subsequently reached pandemic level affecting around 213 countries. As of 24th May 2020, the total number of positive cases confirmed is 5,446,514 and 344,754 death reports worldwide. COVID-19 infection causes pneumonia-like severe respiratory infection and acute lung failure. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA beta coronavirus that is a confirmed causative agent of COVID-19. SARS-CoV-2 may use angiotensin-converting enzyme 2 (ACE2), unlike the receptor utilized by SARS-CoV (emerged in 2002) to infect humans. People with a history of hypertension, chronic obstructive pulmonary disease, diabetes, cardiovascular disease are more susceptible to SARS-CoV-2.
Objective:
The purpose behind this review is to help the society to distinguish and deal with SARS-CoV-2, and make available a reference for forthcoming studies.
Methods:
Recently, a diagnostic primer sets on the SARS-CoV-2 genome have been identified. The receptor-binding domain of SARS-COV-2 highlighted the mode by which beta-CoV recognizes ACE2. Various diagnostic tools are available to differentiate and identify SARS-CoV-2 infection as RT-PCR, antigen detection assay, and antibody detection assay. Different strategies have been employed to control the SARS-CoV-2, considering various drug targets like the main protease (3CLPro), papain-like protease (PLpro), helicase (NSP13), RNA dependent RNA polymerase (RdRp), and viral envelope (E) protein.
Conclusion:
In the present review, we have updated details of transmission, pathogenesis, genome structure, diagnostic criteria, clinical characteristics, therapeutics, and vaccine development of the SARS-CoV-2 infection, which may be significant in the control and response to the COVID-19 outbreak.
Coronaviruses (CoVs) are enveloped positive-stranded RNA viruses with spike (S) protein projections that allow the virus to enter and infect host cells. The S protein is a key virulence factor determining viral pathogenesis, host tropism, and disease pathogenesis. There are currently diverse corona viruses that are known to cause disease in humans.
The occurrence of Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), as fatal human CoV diseases, has induced significant interest in the medical field. The novel coronavirus disease (COVID-19) is an infectious disease caused by a novel strain of coronavirus (SAR-CoV-2). The SARS-CoV2 outbreak has been evolved in Wuhan, China, in December 2019, and identified as a pandemic in March 2020, resulting in 53.24 M cases and 1.20M deaths worldwide.
SARS-CoV-2 main proteinase (MPro), a key protease of CoV-2, mediates viral replication and transcription. SARS-CoV-2 MPro has been emerged as an attractive target for SARS-CoV-2 drug design and development. Diverse scaffolds have been released targeting SARS-CoV-2 MPro. In this review, we culminate the latest published information about SARS-CoV-2 main proteinase (MPro) and reported inhibitors.
A brief history of the human coronaviruses (HCoVs) tells us that they are inherently zoonotic in origin. The
molecular analysis of these HCoVs revealed a large genome of approximately 30 kb that encodes for a fixed
subset of structural proteins, non-structural proteins, and varied accessory proteins. Additionally, the HCoV genomes have a prerequisite ability to undergo mutation and recombination that fosters advantageous gene gain and gene losses. A culmination of all these factors has led to the evolution of HCoVs that can cause mild infections as well as the global pandemic COVID-19.
Using simplified phylogenetic trees, we elucidate the probable primary zoonotic origin and the animal reservoirs for the HCoV family. This chapter aims to highlight the evolution of the common cold triggering HCoV cluster (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1), as well as the severe epidemic and pandemic causing HCoV cluster (SARS-CoV-1, SARS-CoV-2, and MERS-CoV). The final section of this chapter is dedicated to exploring clinical therapies against the above CoVs. As such the CoV replication pathways are exploited, and a select few repurposed drug candidates, novel agents, and their combination are organized based on the steps they inhibit in this pathway.
Coronaviruses (CoVs) infect both humans and animals. In humans, CoVs can cause respiratory, kidney, heart, brain, and intestinal infections that can range from mild to lethal. Since the start of the 21st century, three β-coronaviruses have crossed the species barrier to infect humans: severe-acute respiratory syndrome (SARS)-CoV-1, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2 (2019-nCoV). These viruses are dangerous and can easily be transmitted from human to human. Therefore, the development of an-ticoronaviral therapies is urgently needed. However, to date, no approved vaccines or drugs against CoV infections are available. In this review, we focus on the medicinal chemistry efforts toward the development of antiviral agents against SARS-CoV-1, MERS-CoV, SARS-CoV-2, targeting biochemical events important for viral replication and its life cycle. These targets include the spike glycoprotein and its host-receptors for viral entry, proteases that are essential for cleaving Med Res Rev. 2020;1-64. wileyonlinelibrary.com/journal/med | 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Corona is an avian and mammalian Ribovirus which generally evade host immune mechanism hampering respiratory tract of the host. Structurally it has an envelope of protein enclosing a single-stranded positively coiled RNA genome. Coronavirus uses around 4-5 different classes of protein for its replication in the host cell. Due to the high mutation rate of the viral genome development of a vaccine against corona has been a difficult task. In the current scenario, the world is facing a problem with CoViD-19 as the biggest pandemic. Several combinations of drugs like Hydroxychloroquine, Plaquine, Chloroquine, etc. targeting viral protein have been utilized for controlling viral infection. The possibility of insect brain proteins was checked against the selected non-structural proteins of CoViD-19 that take an active role in the replication of the virus in the host. Molecular docking methodology plays an important role in predicting interaction between the insect proteins with non-structural protein (nsp) of coronavirus. The results predict good bonding affinity with possible interaction as hydrogen bond and salt bridge between antibacterial protein and nsp of CoViD-19 indicating as afuture alternative medications
Since known approved drugs like liponavir and ritonavir failed to cure SARS-CoV-2 infected patients, it is high time to generate new chemical entities against this virus. 3CL main protease acts as key enzyme for the growth of a virus which acts as biocatalyst and helps to break protein and ultimately in the growth of coronavirus. Based on a recently solved structure (PDB ID: 6LU7), we developed a novel advanced deep Q-learning network with the fragment-based drug design (ADQN-FBDD) along with variational autoencoder with KL annealing and circular annealing for generating potential lead compounds targeting SARS-CoV-2 3CL pro. Structure-based optimization policy (SBOP) is used in reinforcement learning. The reason for using variational autoencoders is that it does not deviate much from actual inhibitors, but since VAE suffers from KL diminishing we have used KL annealing and circular annealing to address this issue. Researchers can use this compound as potential drugs against SARS-CoV-2.
The recent outbreak of coronavirus disease 2019 (COVID-19) highlights an urgent need for therapeutics. Through a series of drug repurposing screening campaigns, niclosamide, an FDA-approved anthelminthic drug, was found to be effective against various viral infections with nanomolar to micromolar potency such as SARS-CoV, MERS-CoV, ZIKV, HCV and human adenovirus, indicating its potential as an antiviral agent. In this brief article, we summarize the broad antiviral activities of niclosamide and highlight its potential clinical use for treatment of COVID-19.
NEW peptide series of 2-(2-oxo-2H benzo[h] chromen-4-yl) acetyl derivatives 3-5, 15-20 and their corresponding methyl esters 6-14 were synthesized. Structures of the synthesized products were characterized by correct elemental analysis, IR, Mass and H-NMR spectroscopy. DNA binding activities were performed. Some synthesized compounds showed high binding affinity against DNA. Virtual screening using molecular docking studies of the synthesized compounds were performed, the molecular docking results indicate that, most of the synthesized compounds are more suitable inhibitors against carbonic anhydrase isozyme (CA) than reference drug.
Sixty-one SARS coronavirus genomic sequences derived from the early,
middle, and late phases of the severe acute respiratory syndrome (SARS)
epidemic were analyzed together with two viral sequences from palm
civets. Genotypes characteristic of each phase were discovered, and the
earliest genotypes were similar to the animal SARS-like coronaviruses.
Major deletions were observed in the Orf8 region of the genome, both at
the start and the end of the epidemic. The neutral mutation rate of the
viral genome was constant but the amino acid substitution rate of the
coding sequences slowed during the course of the epidemic. The spike
protein showed the strongest initial responses to positive selection
pressures, followed by subsequent purifying selection and eventual
stabilization.
In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak.
We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens.
None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population.
A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.
The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent.
Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques.
A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated.
The novel coronavirus might have a role in causing SARS.
We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and we constructed a homology model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Molecular modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.
A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiological agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, we report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to our knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.
Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.
SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be critical for catalysis by mutating to Ala, while the Cys145 to Ser mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pK(a)'s for His41 and Cys145 in the wild-type enzyme were estimated to be 6.38 and 8.34, while the pK(a)'s for His41 and Ser145 in the C145S mutant were estimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D(2)O for general base catalysis, that is, reacts slower in D(2)O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pK(a) values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.
SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the alpha-helical domain. Differential scanning calorimetry indicates that the alpha-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.
Antiviral agents are urgently needed to fight severe acute respiratory syndrome (SARS). We showed that niclosamide, an existing
antihelminthic drug, was able to inhibit replication of a newly discovered coronavirus, SARS-CoV; viral antigen synthesis
was totally abolished at a niclosamide concentration of 1.56 μM, as revealed by immunoblot analysis. Thus, niclosamide represents
a promising drug candidate for the effective treatment of SARS-CoV infection.
Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel human coronavirus. Currently, no effective antiviral agents exist against this type of virus. A cell-based assay, with SARS virus and Vero E6 cells, was developed to screen existing drugs, natural products, and synthetic compounds to identify effective anti-SARS agents. Of >10,000 agents tested, approximately 50 compounds were found active at 10 microM; among these compounds, two are existing drugs (Reserpine 13 and Aescin 5) and several are in clinical development. These 50 active compounds were tested again, and compounds 2-6, 10, and 13 showed active at 3 microM. The 50% inhibitory concentrations for the inhibition of viral replication (EC(50)) and host growth (CC(50)) were then measured and the selectivity index (SI = CC(50)/EC(50)) was determined. The EC(50), based on ELISA, and SI for Reserpine, Aescim, and Valinomycin are 3.4 microM (SI = 7.3), 6.0 microM (SI = 2.5), and 0.85 microM (SI = 80), respectively. Additional studies were carried out to further understand the mode of action of some active compounds, including ELISA, Western blot analysis, immunofluorescence and flow cytometry assays, and inhibition against the 3CL protease and viral entry. Of particular interest are the two anti-HIV agents, one as an entry blocker and the other as a 3CL protease inhibitor (K(i) = 0.6 microM).
The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.
A novel and robust automated docking method that predicts the bound conformations of flexible ligands to macromolecular targets has been developed and tested, in combination with a new scoring function that estimates the free energy change upon binding. Interestingly, this method applies a Lamarckian model of genetics, in which environmental adaptations of an individual's phenotype are reverse transcribed into its genotype and become heritable traits (sic). We consider three search methods, Monte Carlo simulated annealing, a traditional genetic algorithm, and the Lamarckian genetic algorithm, and compare their performance in dockings of seven protein–ligand test systems having known three-dimensional structure. We show that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckian genetic algorithm is the most efficient, reliable, and successful of the three. The empirical free energy function was calibrated using a set of 30 structurally known protein–ligand complexes with experimentally determined binding constants. Linear regression analysis of the observed binding constants in terms of a wide variety of structure-derived molecular properties was performed. The final model had a residual standard error of 9.11 kJ mol⁻¹ (2.177 kcal mol⁻¹) and was chosen as the new energy function. The new search methods and empirical free energy function are available in AUTODOCK, version 3.0. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 1639–1662, 1998
A novel and robust automated docking method that predicts the bound conformations of flexible ligands to macromolecular targets has been developed and tested, in combination with a new scoring function that estimates the free energy change upon binding. Interestingly, this method applies a Lamarckian model of genetics, in which environmental adaptations of an individual's phenotype are reverse transcribed into its genotype and become . heritable traits sic . We consider three search methods, Monte Carlo simulated annealing, a traditional genetic algorithm, and the Lamarckian genetic algorithm, and compare their performance in dockings of seven protein)ligand test systems having known three-dimensional structure. We show that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckian genetic algorithm is the most efficient, reliable, and successful of the three. The empirical free energy function was calibrated using a set of 30 structurally known protein)ligand complexes with experimentally determined binding constants. Linear regression analysis of the observed binding constants in terms of a wide variety of structure-derived molecular properties was performed. The final model had a residual standard y1 y1 .
The cleavage mechanism of severe acute respiratory syndrome (SARS) coronavirus main proteinase (Mpro or 3CLpro) for the octapeptide AVLQSGFR is studied using molecular mechanics (MM) and quantum mechanics (QM). The catalytic dyad His-41 and Cys-145 in the active pocket between domain I and II seem to polarize the π-electron density of the peptide bond between Gln and Ser in the octapeptide, leading to an increase of positive charge on C(CO) of Gln and negative charge on N(NH) of Ser. The possibility of enhancing the chemical bond between Gln and Ser based on the “distorted key” theory [Anal. Biochem. 233 (1996) 1] is examined. The scissile peptide bond between Gln and Ser is found to be solidified through “hybrid peptide bond” by changing the carbonyl group CO of Gln to CH2 or CF2. This leads to a break of the π-bond system for the peptide bond, making the octapeptide (AVLQSGFR) a “distorted key” and a potential starting system for the design of anti SARS drugs.
An efficient synthetic route to a key intermediate for the preparation of the rhinovirus protease inhibitor AG7088 has been developed employing a key asymmetric dianionic cyanomethylation of N-Boc-l-(+)-glutamic acid dimethyl ester. This methodology enables the preparation of this compound in kilogram quantities with an overall yield of 30%.
Eine effiziente und einfache Methode im Mikrotiterplattenformat zur Identifizierung potenter und selektiver Inhibitoren von α-Fucosidasen wurde entwickelt. Sie beruht auf der Amidbildung einer Fuconojirimycin-Kernstruktur mit Carbonsäuren und anschließendem Inhibierungs-Assay ohne Isolierung des Produkts. Gezeigt sind die Strukturen der beiden potentesten Inhibitoren.
A novel and robust automated docking method that predicts the bound conformations of flexible ligands to macromolecular targets has been developed and tested, in combination with a new scoring function that estimates the free energy change upon binding. Interestingly, this method applies a Lamarckian model of genetics, in which environmental adaptations of an individual's phenotype are reverse transcribed into its genotype and become . heritable traits sic . We consider three search methods, Monte Carlo simulated annealing, a traditional genetic algorithm, and the Lamarckian genetic algorithm, and compare their performance in dockings of seven protein)ligand test systems having known three-dimensional structure. We show that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckian genetic algorithm is the most efficient, reliable, and successful of the three. The empirical free energy function was calibrated using a set of 30 structurally known protein)ligand complexes with experimentally determined binding constants. Linear regression analysis of the observed binding constants in terms of a wide variety of structure-derived molecular properties was performed. The final model had a residual standard y1 y1. error of 9.11 kJ mol 2.177 kcal mol and was chosen as the new energy
The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines.
We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04).
We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing.
Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations.
In order to stimulate the development of drugs against severe acute respiratory syndrome (SARS), based on the atomic coordinates of the SARS coronavirus main proteinase determined recently [Science 13 (May) (2003) (online)], studies of docking KZ7088 (a derivative of AG7088) and the AVLQSGFR octapeptide to the enzyme were conducted. It has been observed that both the above compounds interact with the active site of the SARS enzyme through six hydrogen bonds. Also, a clear definition of the binding pocket for KZ7088 has been presented. These findings may provide a solid basis for subsite analysis and mutagenesis relative to rational design of highly selective inhibitors for therapeutic application. Meanwhile, the idea of how to develop inhibitors of the SARS enzyme based on the knowledge of its own peptide substrates (the so-called "distorted key" approach) was also briefly elucidated.
An efficient and simple method has been developed to identify potent and selective inhibitors against α-fucosidases based on the amide-forming reaction in a microtiter plate of a fuconojirimycin core with various carboxylic acids, followed by a direct inhibition assay without product isolation. The structures of the two most potent inhibitors are shown.
SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.
3C-like (3CL) protease is essential for the life cycle of severe acute respiratory syndrome-coronavirus (SARS-CoV) and therefore represents a key anti-viral target. A compound library consisting of 960 commercially available drugs and biologically active substances was screened for inhibition of SARS-CoV 3CL protease. Potent inhibition was achieved using the mercury-containing compounds thimerosal and phenylmercuric acetate, as well as hexachlorophene. As well, 1-10 microM of each compound inhibited viral replication in Vero E6 cell culture. Detailed mechanism studies using a fluorescence-based protease assay demonstrated that the three compounds acted as competitive inhibitors (Ki=0.7, 2.4, and 13.7 microM for phenylmercuric acetate, thimerosal, and hexachlorophene, respectively). A panel of metal ions including Zn2+ and its conjugates were then evaluated for their anti-3CL protease activities. Inhibition was more pronounced using a zinc-conjugated compound (1-hydroxypyridine-2-thione zinc; Ki=0.17 microM) than using the ion alone (Ki=1.1 microM).
Glycosidase inhibitors have shown great medicinal and pharmaceutical values as exemplified by the therapeutic treatment of influenza virus and non-insulin-dependent diabetes. We herein report the discovery of picomolar slow tight-binding inhibitors 2-5 against the alpha-fucosidase from Corynebacterium sp. by a rapid screening for an optimal aglycon attached to 1-aminomethyl fuconojirimycin (1). The time-dependent inhibition displays the progressive tightening of enzyme-inhibitor complex from a low nanomolar K(i) to picomolar K(i)* value. Particularly compound 2 with a K(i)* of 0.46 pM represents the most potent glycosidase inhibitor to date. The effect of compound 3 on the intrinsic fluorescence of alpha-fucosidase is both time- and concentration-dependent in a saturation-type manner, which is consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (E.I), followed by the slower formation of a tightly bound enzyme-inhibitor complex (E.I*). The binding affinity increases 3.5 x 10(4)-fold from 1 (K(i) = 16.3 nM) to 2 (K(i)* = 0.46 pM). This work clearly demonstrates the effectiveness of our combinatorial approach leading to the rapid discovery of potent inhibitors.
The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected more than 8,000 people across 29 countries and caused more than 900 fatalities. Based on the concept of chemical genetics, we screened 50,240 structurally diverse small molecules from which we identified 104 compounds with anti-SARS-CoV activity. Of these 104 compounds, 2 target the SARS-CoV main protease (M(pro)), 7 target helicase (Hel), and 18 target spike (S) protein-angiotensin-converting enzyme 2 (ACE2)-mediated viral entry. The EC(50) of the majority of the 104 compounds determined by SARS-CoV plaque reduction assay were found to be at low micromolar range. Three selected compounds, MP576, HE602, and VE607, validated to be inhibitors of SARS-CoV M(pro), Hel, and viral entry, respectively, exhibited potent antiviral activity (EC(50) < 10 microM) and comparable inhibitory activities in target-specific in vitro assays.
The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50 = 0.5-7 microM) toward SARS-CoV 3CLpro.
The 3C-like proteinase (3CL(pro)) of severe acute respiratory syndrome (SARS) coronavirus is a key target for structure-based drug design against this viral infection. The enzyme recognizes peptide substrates with a glutamine residue at the P1 site. A series of keto-glutamine analogues with a phthalhydrazido group at the alpha-position were synthesized and tested as reversible inhibitiors against SARS 3CL(pro). Attachment of tripeptide (Ac-Val-Thr-Leu) to these glutamine-based "warheads" generated significantly better inhibitors (4a-c, 8a-d) with IC(50) values ranging from 0.60 to 70 microM.
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