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Supplementary Figures 1-6, Table 1 from Intracellular β-Tubulin/Chaperonin Containing TCP1-β Complex Serves as a Novel Chemotherapeutic Target against Drug-Resistant Tumors

The coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a worldwide pandemic. The 3C-like protease (3CL

Metastasis, the movement of cancer cells from one site to another, is responsible for the highest number of cancer deaths, especially in lung cancer patients. In this study, we first identified a prognostic marker of lung adenocarcinoma, TCP-1 β subunit (chaperonin-containing TCP-1β; CCT-β). We showed a compound that disrupted the interaction of CCT-β with β-tubulin killed a highly metastatic non-small cell lung cancer cell line CL1-5 through inducing Endoplasmic reticulum stress and caspases activation. Moreover, at the dosage of EC20, the compound inhibited migration and invasion of the lung cancer cells by suppressing matrix metalloproteinase (MMP)-2/9 and epithelial–mesenchymal transition (EMT)-related proteins through downregulating mitogen-activated protein kinases (MAPKs), Akt/β-catenin and integrin–focal adhesion kinase signaling pathways. Unlike the anticancer drugs, such as Taxol, that target the adenosine triphosphate site of β-tubulin, this study reveals a therapeutic target, β-tubulin/CCT-β complex, for metastatic human lung adenocarcinoma. The study demonstrated CCT-β as a prognostic marker. Targeting β-tubulin/CCT-β complex caused apoptosis and inhibited invasion/migration of CCT-β overexpressed, highly metastatic lung adenocarcinoma.

Plant lignocellulosic biomass can be enzymatically degraded and converted into biofuel. We have studied a bifunctional cellulase/xylanase from thermophilic Clostridium thermocellum (CtCel5E), which can digest two major plant cell wall polysaccharides, cellulose (a polymer of glucose) and xylan (a major hemicellulose composed of xylose), into disaccharides cellobiose and xylobiose, respectively. In the study presented here, the bifunctional CtCel5E was fused with a β-glucosidase from Clostridium cellulovorans (CcBglA) to convert cellulose into glucose with improved activities toward carboxymethyl cellulose (CMC), regenerated amorphous cellulose (RAC), and alkali-treated rice straw as compared to the mixture of two individual enzymes by eliminating accumulation of cellobiose intermediate. The engineered CtCel5E-CcBglA was secreted from yeast to degrade alkali-treated rice straw into glucose that sustained cell growth. Our study provides a strategy of consolidating enzymes for more efficient biomass processing in vitro and by yeast cells.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV., Simeprevir, an HCV drug, suppresses SARS-CoV-2 replication by inhibiting two viral proteins and modulating host immune responses, thus providing novel insights in anti-CoV drug design.

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease., Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CLpro and PLpro assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CLpro inhibitors and 36 drugs as PLpro inhibitors active at 10 μM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC50) below or close to 10 μM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 μM and an antiviral activity with EC50 of 49.89 μM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 μM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 μM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

Chaperonin-containing TCP-1 (CCT) is a chaperonin composed of eight subunits that participates in intracellular protein folding. Here, we showed that increased levels of subunits of CCT, particularly CCT-&beta, were significantly correlated with lower survival rates for cancer patients. Endogenously high expression of CCT-&beta, was found in cancer cell lines, such as the triple-negative breast cancer cell line MDA-MB-231 and the highly metastatic non-small-cell lung cancer cell line CL1-5. Knocking down CCT-&beta, in these cancer cells led to decreased levels of anti-apoptotic proteins, such as XIAP, as well as inhibited phosphorylation of Ser473-AKT and GSK3, resulting in decrease of the nucleus-entering form of &beta, catenin, these changes reduced the chemoresistance and migration/invasion of the cells. Conversely, overexpression of CCT-&beta, recovered the chemoresistance and cell migration/invasion by promoting the AKT-GSK3&beta, &beta, catenin and XIAP-Survivin pathways. Coimmunoprecipitation data revealed that the CCT complex might directly bind and stabilize XIAP and &beta, catenin. This study not only elucidates the roles of CCT in chemoresistance and metastasis, which are two major obstacles for current cancer therapy, but also provides a possible therapeutic strategy against cancers with overexpressed CCT-&beta

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients.One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

Hanatoxin (HaTx) from spider venom works as an inhibitor of Kv2.1 channels, most likely by interacting with the voltage sensor (VS). However, the way in which this water-soluble peptide modifies the gating remains poorly understood as the VS is deeply embedded within the bilayer, although it would change its position depending on the membrane potential. To determine whether HaTx can indeed bind to the VS, the depth at which HaTx penetrates into the POPC membranes was measured with neutron reflectivity. Our results successfully demonstrate that HaTx penetrates into the membrane hydrocarbon core (∼9 Å from the membrane surface), not lying on the membrane-water interface as reported for another voltage sensor toxin (VSTx). This difference in penetration depth suggests that the two toxins fix the voltage sensors at different positions with respect to the membrane normal, thereby explaining their different inhibitory effects on the channels. In particular, results from MD simulations constrained by our penetration data clearly demonstrate an appropriate orientation for HaTx to interact with the membranes, which is in line with the biochemical information derived from stopped-flow analysis through delineation of the toxin-VS binding interface.

Background Caspase-3 acts as a major executioner protein in proteolytic degradation during apoptosis. The role of Caspase-3 in gastric cancer remains controversial. Methods A total of 366 gastric cancer patients who received curative surgery were enrolled. Caspase-3 expression in gastric tumors was examined by immunohistochemical staining. Correlations between Caspase-3 expression and the survival rates and between Caspase-3 expression and the clinicopathological parameters of the gastric cancer patients were analyzed. Results The 5-year overall survival rates of gastric cancer patients with and without Caspase-3 expression were 51.2% and 37.3%, respectively ( P = 0.030). The 5-year disease-free survival rates of gastric cancer patients with and without Caspase-3 expression were 49.2% and 34.6%, respectively ( P = 0.029). Analyses of the clinicopathological features showed that larger tumor size ( P = 0.030), more advanced Borrmann type ( P = 0.012), more aggressive stromal reaction ( P = 0.001), higher classification using Ming's infiltrating histology type ( P = 0.018), more lymph node involvement ( P = 0.019), and more lymphovascular involvement ( P = 0.045) were significantly correlated with a lack of Caspase-3 expression. The multivariate analysis showed that age ( P = 0.001), Borrmann classification ( P = 0.032), stromal reaction type ( P = 0.018), TNM pathological T category ( P = 0.002), TNM pathological N category ( P P = 0.041) were significantly correlated with the overall survival of gastric cancer patients. Conclusion Caspase-3 expression in gastric cancer patients is related to favorable clinicopathological features and a positive prognosis after curative surgery. Caspase-3 may act as a tumor suppressor in human gastric cancer.

Graphical abstract, Highlights • A fluorogenic detection of L-Cysteine was investigated using rGO/Au nanohybrids. • The rGO/Au nanohybrids was prepared by one-pot hydrothermal method. • A lower LOD (0.51 nm) was achieved. • Practicality of the sensor was evaluated in live cancer cells., A highly sensitive and selective fluorogenic sensing of L-Cysteine (L-Cys) was implemented based on gelatin stabilized gold nanoparticles decorated reduced graphene oxide (rGO/Au) nanohybrid. The rGO/Au nanohybrid was prepared by the one-pot hydrothermal method and well characterized by different physiochemical techniques. The nanohybrid exhibits a weak fluorescence of rGO due to the energy transfer from the rGO to Au NPs. The rGO/Au nanohybrid shows enhanced fluorescence activity due to the restoration of quenched fluorescence of rGO/Au nanohybrid in presence of L-Cys. The rGO/Au nanohybrid exhibits much lower detection limit of 0.51 nM for L-Cys with higher selectivity. The fluorescence sensing mechanism arose from the fluorescence recovery due to the stronger interaction between Au NPs and L-Cys, and consequently, the energy transfer was prevented between rGO and Au NPs. The practicability of rGO/Au sensor was implemented by invitro bioimaging measurements in Colo-205 (colorectal adenocarcinoma) and MKN-45 (gastric carcinoma) cancer live cells with excellent biocompatibility.

Graphical abstract, The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 μM against SARS-CoV-2 and CC50 of 453.5 μM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3Cpro), 3C-like protease (3CLpro) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC50 values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC50 values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses.

A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths via consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). cis-Prenyltransferases, which catalyze cis-double bond formation during IPP condensation, usually synthesize long-chain products as lipid carriers to mediate peptidoglycan biosynthesis in prokaryotes and protein glycosylation in eukaryotes. Unlike only one or two cis-prenyltransferases in bacteria, yeast, and animals, plants have several cis-prenyltransferases and their functions are less understood. As reported here, a cis-prenyltransferase from Lilium longiflorum anther, named LLA66, was expressed in Saccharomyces cerevisiae and characterized to produce C40/C45 products without the capability to restore the growth defect from Rer2-deletion, although it was phylogenetically categorized as a long-chain enzyme. Our studies suggest that evolutional mutations may occur in the plant cis-prenyltransferase to convert it into a shorter-chain enzyme.

Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.

The main protease of SARS‐associated coronavirus (SARS‐CoV), also called 3C‐like protease (3CLpro), is vital for the viral replication. It cleaves the replicase polyproteins at 11 sites and is a promising drug target. Several groups of inhibitors have been identified through high‐throughput screening and rational drug design. In addition to the pharmaceutical applications, a mutant 3CLpro (T25G) with an expanded S1′ space has been demonstrated to tolerate larger residues at P1′, facilitating the cleavage behind the recognition sequence. This review summarizes current developments in anti‐SARS agents targeting 3CLpro and the application of the mutant protease as a tag‐cleavage endopeptidase., Severe acute respiratory syndrome (SARS)‐associated coronavirus (CoV) has disappeared, but might re‐emerge in the future, together with other new and existing CoV. The 3CL protease of SARS‐CoV, a key enzyme for viral replication, is a promising drug target for inhibitor molecules in order to combat CoVs. This review summarizes current developments and research on inhibitors against proteases and the engineering of the mutant protease as an endopeptidase. WILEY-VCH

Background An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition. Method A Tmloop inserted CtCel5E and its related mutants were produced to investigate the role of Tmloop in catalysis. Crystal structure of CtCel5E-TmloopF267A followed by site-direct mutagenesis reveals the mechanism. RtCelB, a homolog with Tmloop was identified to have mannanase activity. Result Tmloop incorporation enables CtCel5E to gain mannanase activity. Tyr270, His277, and Trp282 in the Tmloop are indispensable for CtCel5E-Tmloop catalysis, and weakening hydrophobic environment near the Tmloop enhances enzyme kcat. Using our newly identified loop motif to search for structurally conserved homologs in other subfamilies of GH5, we identified RtCelB. This homolog, originally annotated as a cellulase also possesses mannanase and xylanase activities. Conclusion Our studies show that Tmloop enhances GH5 enzyme promiscuity and plays a role in catalysis. General significance The study identified a loop of GH5 for mannan recognition and catalysis. Weakening the hydrophobic environment near the loop can also enhance the enzyme catalytic rate. Our findings provide a new insight on mannan recognition and activity enhancement of GH5.

We have previously demonstrated the ability of I-Trp to disrupt the protein–protein interaction of β-tubulin with chaperonin-containing TCP-1β (CCT-β). This caused more severe apoptosis in multidrug-resistant MES-SA/Dx5, compared to MES-SA, due to its higher CCT-β overexpression. In this study, we screened a panel of cancer cell lines, finding CCT-β overexpression in the triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell lines Colo205 and HCT116, and a gastric cancer cell line MKN-45. Thus, I-Trp killed these cancers with sub- to low-μM EC50, whereas it was non-toxic to MCF-10A. We then synthesized analogs of I-Trp and evaluated their cytotoxicity. Furthermore, apoptotic mechanism investigations revealed the activation of both protein ubiquitination/degradation and ER-associated protein degradation pathways. These pathways proceeded through activation of MAPKs at the onset of CCT-β : β-tubulin complex disruption. We thus establish an effective strategy to treat CCT-β overexpressed cancers by disrupting the CCT-β : β-tubulin complex.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C40 long-chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans-prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo-form as well as in complexes with IPP and a FPP thio-analog, FsPP, at resolutions of 2.2-2.6 A, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active-site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand-bound crystal structures of a long-chain trans-prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation.

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure. These structures together with the results of site-directed mutagenesis suggest an unusual mechanism of action involving two Tyr residues. Given the similarity in local and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c), the possibility exists for the development of inhibitors that target not only virulence but also cell wall biosynthesis, based in part on the structures reported here.

A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths via consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). cis-Prenyltransferases, which catalyze cis-double bond formation during IPP condensation, usually synthesize long-chain products as lipid carriers to mediate peptidoglycan biosynthesis in prokaryotes and protein glycosylation in eukaryotes. Unlike only one or two cis-prenyltransferases in bacteria, yeast, and animals, plants have several cis-prenyltransferases and their functions are less understood. As reported here, a cis-prenyltransferase from Lilium longiflorum anther, named LLA66, was expressed in Saccharomyces cerevisiae and characterized to produce C40/C45 products without the capability to restore the growth defect from Rer2-deletion, although it was phylogenetically categorized as a long-chain enzyme. Our studies suggest that evolutional mutations may occur in the plant cis-prenyltransferase to convert it into a shorter-chain enzyme.

A novel coronavirus (CoV), unlike previous typical human coronaviruses (HCoVs), was identified as causative agent for severe acute respiratory syndrome (SARS). SARS first surfaced as a pandemic in late 2002 and originated in southern China. SARS-CoV rapidly spread to30 countries by 2003, infecting nearly 8,000 people and causing around 800 fatalities. After 10 years of silence, a 2012 report alarmed researchers about the emergence of a new strain of CoV causing SARS-like disease.To combat SARS, scientists applied for patents on various therapeutic agents, including small-molecule inhibitors targeting the essential proteases, helicase and other proteins of the virus, natural products, approved drugs, molecules binding to the virus, neutralizing antibodies, vaccines, anti-sense RNA, siRNA and ribozyme against SARS-CoV. In this article, the patents published from 2008 to the present for the new therapeutics that could potentially be used in the prophylaxis and treatment of SARS are reviewed.The therapeutic interventions or prophylaxis discussed in this review seems to offer promising solutions to tackle SARS. Rather than being complacent about the results, we should envisage how to transform them into drug candidates that may be useful in combating SARS and related viral infections in the future.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3Cpro), 3C-like protease (3CLpro) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC50 values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC50 values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses., Graphical abstract Image 1, Highlights • Aldehyde-containing peptidomimetics were identified to be potent inhibitors against MERS-CoV 3CLpro. • The active inhibitor showed sub-μM EC50 in killing MERS-CoV. • Compounds were also effective against other α and β-CoVs of both human and feline origin. • We identified broad-spectrum antiviral agents effective against both coronaviruses and picornaviruses.

Graphical abstract, Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CLpro of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R1 or R4 destabilizes the oxyanion hole in the 3CLpro. Interestingly, 3f, 3g and 3m could inhibit both NA and 3CLpro and serve as a starting point to develop broad-spectrum antiviral agents.

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.

Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C(40) octaprenyl diphosphate, which constitutes the side chain of ubiquinone or menaquinone. To understand the roles of active site amino acids in substrate binding and catalysis, we conducted site-directed mutagenesis studies with Escherichia coli OPPS. In conclusion, D85 is the most important residue in the first DDXXD motif for both FPP and IPP binding through an H-bond network involving R93 and R94, respectively, whereas R94, K45, R48, and H77 are responsible for IPP binding by providing H-bonds and ionic interactions. K170 and T171 may stabilize the farnesyl carbocation intermediate to facilitate the reaction, whereas R93 and K225 may stabilize the catalytic base (MgPP(i)) for H(R) proton abstraction after IPP condensation. K225 and K235 in a flexible loop may interact with FPP when the enzyme becomes a closed conformation, which is therefore crucial for catalysis. Q208 is near the hydrophobic part of IPP and is important for IPP binding and catalysis.

A group of prenyltransferases produce linear lipids by catalyzing consecutive condensation reactions of farnesyl diphosphate (FPP) with specific numbers of isopentenyl diphosphate (IPP), a common building block of isoprenoid compounds. Depending on the stereochemistry of the double bonds formed during IPP condensation, these prenyltransferases are categorized as cis- and trans-types. Undecaprenyl diphosphate synthase (UPPS) that catalyzes chain elongation of FPP by consecutive condensation reactions with eight IPP, to form C₅₅ lipid carrier for bacterial cell wall biosynthesis, serves as a model for understanding cis-prenyltransferases. In this review, the current knowledge in UPPS kinetics, mechanisms, structures, and inhibitors is summarized.

Development of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) agents is pivotal to prevent the reemergence of the life-threatening disease, SARS. In this study, more than 200 extracts from Chinese medicinal herbs were evaluated for anti-SARS-CoV activities using a cell-based assay that measured SARS-CoV-induced cytopathogenic effect (CPE) in vitro on Vero E6 cells. Six herbal extracts, one each from Gentianae Radix (龍膽 lóng dǎn; the dried rhizome of Gentiana scabra), Dioscoreae Rhizoma (山藥 shān yào; the tuber of Dioscorea batatas), Cassiae Semen (決明子 jué míng zǐ; the dried seed of Cassia tora) and Loranthi Ramus (桑寄生 sāng jì shēng; the dried stem, with leaf of Taxillus chinensis) (designated as GSH, DBM, CTH and TCH, respectively), and two from Rhizoma Cibotii (狗脊 gǒu jǐ; the dried rhizome of Cibotium barometz) (designated as CBE and CBM), were found to be potent inhibitors of SARS-CoV at concentrations between 25 and 200 μg/ml. The concentrations of the six extracts needed to inhibit 50% of Vero E6 cell proliferation (CC50) and 50% of viral replication (EC50) were determined. The resulting selective index values (SI = CC50/EC50) of the most effective extracts CBE, GSH, DBM, CTH and TCH were > 59.4, > 57.5, > 62.1, > 59.4, and > 92.9, respectively. Among these extracts, CBM and DBM also showed significant inhibition of SARS-CoV 3CL protease activity with IC50 values of 39 μg/ml and 44 μg/ml, respectively. Our findings suggest that these six herbal extracts may have potential as candidates for future development of anti-SARS therapeutics. Abbreviations SARS, severe acute respiratory syndrome CoV, coronavirus CPE, cytopathogenic effect TCM, traditional Chinese medicine

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) caused infection of ~8000 people and death of ~800 patients around the world during the 2003 outbreak. In addition, picornaviruses such as enterovirus, coxsackievirus and rhinovirus also can cause life-threatening diseases. Replication of picornaviruses and coronaviruses requires 3Cpro (3C protease) and 3CLpro (3C-like protease) respectively, which are structurally analogous with chymotrypsin-fold, but the former is a monomer and the latter is dimeric due to an extra third domain for dimerization. Subtle structural differences in the S2 and S3 pockets of these proteases make inhibitors selective, but some dual inhibitors have been discovered. Our findings as summarized in the present review provide new potential anti-coronavirus and anti-picornavirus therapeutic agents and a clue to convert 3CLpro inhibitors into 3Cpro inhibitors and vice versa.

A β-glucosidase from Clostridium cellulovorans (CcBG) was fused with one of three different types of cellulases from Clostridium thermocellum , including a cellulosomal endoglucanase CelD (CtCD), a cellulosomal exoglucanase CBHA (CtCA) and a non-cellulosomal endoglucanase Cel9I (CtC9I). Six bifunctional enzymes were constructed with either β-glucosidase or cellulase in the upstream. CtCD–CcBG showed the favorable specific activities on phosphoric acid swollen cellulose (PASC), an amorphous cellulose, with more glucose production (2 folds) and less cellobiose accumulation (3 folds) when compared with mixture of the single enzymes. Moreover, CtCD–CcBG had significantly improved thermal stability with a melting temperature ( T m ) of 10.9 °C higher than that of CcBG (54.5 °C) based on the CD unfolding experiments. This bifunctional enzyme is thus useful in industrial application to convert cellulose to glucose.

β-Glucosidases (EC 3.2.1.21) cleave β-glucosidic linkages in disaccharide or glucose-substituted molecules and play important roles in fundamental biological processes. β-Glucosidases have been widely used in agricultural, biotechnological, industrial and medical applications. In this study, a high yield expression (70–250 mg/l) in Escherichia coli of the three functional β-glucosidase genes was obtained from the bacterium Clostridium cellulovorans (CcBglA), the fungus Trichoderma reesei (TrBgl2), and the termite Neotermes koshunensis (NkBgl) with the crystal structures of CcBglA, TrBgl2 and NkBgl, determined at 1.9 A, 1.63 A and 1.34 A resolution, respectively. The overall structures of these enzymes are similar to those belonging to the β-retaining glycosyl hydrolase family 1, which have a classical (α/β)8-TIM barrel fold. Each contains a slot-like active site cleft and a more variable outer opening, related to its function in processing different lengths of β-1,4-linked glucose derivatives. The two essential glutamate residues for hydrolysis are spatially conserved in the active site. In both TrBgl2 and NkBgl structures, a Tris molecule was found to bind at the active site, explaining the slight inhibition of hydrolase activity observed in Tris buffer. Manganese ions at 10 mM exerted an approximate 2-fold enzyme activity enhancement of all three β-glucosidases, with CcBglA catalyzing the most efficiently in hydrolysis reaction and tolerating Tris as well as some metal inhibition. In summary, our results for the structural and functional properties of these three β-glucosidases from various biological sources open important avenues of exploration for further practical applications.

Research highlights: {yields} The extremely slow trans-OPPS reaction using 2-Fluoro-FPP supports the sequential mechanism with the carbocation intermediate. {yields} The similar UPPS reaction rate under single turnover supports the concerted mechanism, without the carbocation intermediate. {yields} The secondary kinetic isotope effect also supports associate transition state for UPPS reaction, without the carbocation intermediate. -- Abstract: Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C{sub 55} product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-{sup 2}H{sub 2}]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 {+-} 0.022 measured for UPPS reaction using [1,1-{sup 2}H{sub 2}]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.

Graphical abstract A series of pyrazolone compounds as possible SARS-CoV 3CL protease inhibitors were designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide in which several showed potent inhibition against the 3CL protease. Interestingly, one of the inhibitors was also active against 3C protease from coxsackievirus B3. These inhibitors could be potentially developed into anti-coronaviral and anti-picornaviral agents., A series of pyrazolone compounds as possible SARS-CoV 3CL protease inhibitors were designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide in which several showed potent inhibition against the 3CL protease. Interestingly, one of the inhibitors was also active against 3C protease from coxsackievirus B3. These inhibitors could be potentially developed into anti-coronaviral and anti-picornaviral agents.