Search Results (40)

Despite current vaccines and therapeutics targeting SARS-CoV-2, the causative agent of the COVID-19 pandemic, cases remain high causing a burden on health care systems. Spike-protein mediated membrane fusion of SARS-CoV-2 is a critical step in viral entry. Herein, we describe entry inhibitors identified by first screening a library of about 160 compounds and then analogue synthesis. Specifically, compound 261 was found to inhibit SARS-CoV-2 infection in a tissue model with IC₅₀ of 0.3 µM. Using NMR, we found that 261 interacts with key residues in the aromatic-rich region of the spike protein directly next to the transmembrane domain. Molecular dynamic simulations of the 261 binding pocket in the spike protein was also mapped to the transmembrane domain, consistent with NMR findings. The amino acids in the binding site are conserved among different coronaviruses known to infect humans; therefore, inhibitors targeting this conserved binding site could be a useful addition to current therapeutics and may have pan-coronavirus antiviral activities. Full article

The prognosis of advanced (UICC IIb-IV) primary colorectal cancer (pCRC) remains poor. More effective targeted therapies are needed. Heat shock protein 90 alpha/beta (Hsp90α/β) expression was immunohistologically quantified in 89 pCRCs and multivariately correlated with survival. Pimitespib (Pim, TAS-116), a Hsp90α/β-specific inhibitor, was tested in pCRC cell lines and patient-derived cancer spheroids (PDCS) and referenced to the pan-Hsp90 inhibitor ganetespib (Gan, STA-9090) and standard-of-care therapies. A total of 26.97% pCRCs showed strong tumoral Hsp90α/β expression (Hsp90α/β > 40%), which correlated with reduced PFS (HR: 3.785, 95%CI: 1.578–9.078, p = 0.003) and OS (HR: 3.502, 95%CI: 1.292–9.494, p = 0.014). Co-expression of Hsp90α/β > 40% with its clients BRAF-V600E and Her2/neu aggravated the prognosis (BRAF-V600E mutated: PFS, p = 0.002; OS, p = 0.012; Her2/neu score3: PFS, p = 0.029). The prognostic cut-off Hsp90α/β > 40% was also a predictor for response to Pim-based therapy. Pim efficacy was increased in combination with 5-FU, 5-FU + oxaliplatin, and 5-FU + irinotecan (all p < 0.001). Pim induced sensitization to all chemotherapies in HT-29 (p < 0.001), Caco-2 (p < 0.01), and HCT116 (p < 0.05) cells. Pim combined with encorafenib in HT-29 and with trastuzumab in Caco-2 cells was most effective in dual-target inhibition approaches (HT-29: p < 0.005; Caco-2: p < 0.05). The anti-cancer effect and chemosensitization of Pim-based therapy were prospectively confirmed in PDCS directly generated from Hsp90α/β > 40% pCRCs. Protein profiling combined with functional drug testing stratifies Hsp90α/β > 40% pCRC patients diagnosed with UICC IIb-IV for effective Pim-based therapy. Full article

Human coronavirus OC43 (HCoV-OC43) is usually associated with common colds, but also related to severe disease in the frail. Its envelope glycoproteins spike (S) is responsible for host-cell attachment and membrane fusion. To understand the molecular basis of membrane fusion of HCoV-OC43, we solved the 3.34 Å crystal structure of the post-fusion state formed by two heptad repeat domains (HR1P and HR2P) of OC43-S. This fusion core comprises a parallel trimeric coiled coil of three HR1 helices with 61 Å at length, around which three HR2 helices are entwined in an antiparallel manner, as anticipated. Moreover, a pan-CoV fusion inhibitor EK1 derived from OC43-HR2P was also crystalized with OC43-HR1P in the resolution of 2.71 Å. Parallel comparisons rationalize the design of EK1, maintaining various hydrophobic and charged or hydrophilic interactions formed in the initial fusion core to stabilize the overall conformation. Together, our results not only reveal the critical intrahelical and interhelical interactions underlying the mechanism of action of OC43-S fusion, but also help our understanding on the mechanism of HCoV-OC43 inhibition by analogue HR2 mimic peptide. Full article

Furin, a serine protease enzyme located in the Golgi apparatus of animal cells, plays a crucial role in cleaving precursor proteins into their mature, active forms. It is ubiquitously expressed across various tissues, including the brain, lungs, gastrointestinal tract, liver, pancreas, and reproductive organs. Since its discovery in 1990, furin has been recognized as a significant therapeutic target, leading to the active development of furin inhibitors for potential use in antiviral, antibacterial, anticancer, and other therapeutic applications. This review provides a comprehensive overview of the progress in the development and characterization of furin inhibitors, encompassing peptides, linear and macrocyclic peptidomimetics, and non-peptide compounds, highlighting their potential in the treatment of both infectious and non-infectious diseases. Full article

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice). Full article

The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic coronaviral research. In this study, a comprehensive approach using molecular docking, in vitro assays, and molecular dynamics simulations was applied to identify potential inhibitors for SARS-CoV-2 papain-like protease (PL^pro), a key and underexplored viral enzyme target. A focused protease inhibitor library was initially created and molecular docking was performed using CmDock software (v0.2.0), resulting in the selection of hit compounds for in vitro testing on the isolated enzyme. Among them, compound 372 exhibited promising inhibitory properties against PL^pro, with an IC50 value of 82 ± 34 μM. The compound also displayed a new triazolopyrimidinyl scaffold not yet represented within protease inhibitors. Molecular dynamics simulations demonstrated the favorable binding properties of compound 372. Structural analysis highlighted its key interactions with PL^pro, and we stress its potential for further optimization. Moreover, besides compound 372 as a candidate for PL^pro inhibitor development, this study elaborates on the PL^pro binding site dynamics and provides a valuable contribution for further efforts in pan-coronaviral PL^pro inhibitor development. Full article

Human society is facing the threat of various viruses. Proteases are promising targets for the treatment of viral infections. In this study, we collected and profiled 170 protease sequences from 125 viruses that infect humans. Approximately 73 of them are viral 3-chymotrypsin-like proteases (3CL^pro), and 11 are pepsin-like aspartic proteases (PAPs). Their sequences, structures, and substrate characteristics were carefully analyzed to identify their conserved nature for proposing a pan-3CL^pro or pan-PAPs inhibitor design strategy. To achieve this, we used computational prediction and modeling methods to predict the binding complex structures for those 73 3CL^pro with 4 protease inhibitors of SARS-CoV-2 and 11 protease inhibitors of HCV. Similarly, the complex structures for the 11 viral PAPs with 9 protease inhibitors of HIV were also obtained. The binding affinities between these compounds and proteins were also evaluated to assess their pan-protease inhibition via MM-GBSA. Based on the drugs targeting viral 3CL^pro and PAPs, repositioning of the active compounds identified several potential uses for these drug molecules. As a result, Compounds 1–2, modified based on the structures of Ray1216 and Asunaprevir, indicate potential inhibition of DENV protease according to our computational simulation results. These studies offer ideas and insights for future research in the design of broad-spectrum antiviral drugs. Full article

The peptide-based pan-coronavirus fusion inhibitor EK1 is in phase III clinical trials, and it has, thus far, shown good clinical application prospects against SARS-CoV-2 and its variants. To further improve its in vivo long-acting property, we herein developed an Fc-binding strategy by conjugating EK1 with human immunoglobulin G Fc-binding peptide (IBP), which can exploit the long half-life advantage of IgG in vivo. The newly engineered peptide IBP-EK1 showed potent and broad-spectrum inhibitory activity against SARS-CoV-2 and its variants, including various Omicron sublineages and other human coronaviruses (HCoVs) with low cytotoxicity. In mouse models, IBP-EK1 possessed potent prophylactic and therapeutic efficacy against lethal HCoV-OC43 challenge, and it showed good safety profile and low immunogenicity. More importantly, IBP-EK1 exhibited a significantly extended in vivo half-life in rhesus monkeys of up to 37.7 h, which is about 20-fold longer than that reported for EK1. Strikingly, IBP-EK1 displayed strong in vitro or ex vivo synergistic anti-HCoV effect when combined with monoclonal neutralizing antibodies, including REGN10933 or S309, suggesting that IBP-conjugated EK1 can be further developed as a long-acting, broad-spectrum anti-HCoV agent, either alone or in combination with neutralizing antibodies, to combat the current COVID-19 pandemic or future outbreaks caused by emerging and re-emerging highly pathogenic HCoVs. Full article

Aprotinin (APR) was discovered in 1930. APR is an effective pan-protease inhibitor, a typical “magic shotgun”. Until 2007, APR was widely used as an antithrombotic and anti-inflammatory drug in cardiac and noncardiac surgeries for reduction of bleeding and thus limiting the need for blood transfusion. The ability of APR to inhibit proteolytic activation of some viruses leads to its use as an antiviral drug for the prevention and treatment of acute respiratory virus infections. However, due to incompetent interpretation of several clinical trials followed by incredible controversy in the literature, the usage of APR was nearly stopped for a decade worldwide. In 2015–2020, after re-analysis of these clinical trials’ data the restrictions in APR usage were lifted worldwide. This review discusses antiviral mechanisms of APR action and summarizes current knowledge and prospective regarding the use of APR treatment for diseases caused by RNA-containing viruses, including influenza and SARS-CoV-2 viruses, or as a part of combination antiviral treatment. Full article

We previously identified a lipopeptide, EK1C4, by linking cholesterol to EK1, a pan-CoV fusion inhibitory peptide via a polyethylene glycol (PEG) linker, which showed potent pan-CoV fusion inhibitory activity. However, PEG can elicit antibodies to PEG in vivo, which will attenuate its antiviral activity. Therefore, we designed and synthesized a dePEGylated lipopeptide, EKL1C, by replacing the PEG linker in EK1C4 with a short peptide. Similar to EK1C4, EKL1C displayed potent inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses. In this study, we found that EKL1C also exhibited broad-spectrum fusion inhibitory activity against human immunodeficiency virus type 1 (HIV-1) infection by interacting with the N-terminal heptad repeat 1 (HR1) of viral gp41 to block six-helix bundle (6-HB) formation. These results suggest that HR1 is a common target for the development of broad-spectrum viral fusion inhibitors and EKL1C has potential clinical application as a candidate therapeutic or preventive agent against infection by coronavirus, HIV-1, and possibly other class I enveloped viruses. Full article

SARS-CoV-2 ORF3a accessory protein was found to be involved in virus release, immunomodulation and exhibited a pro-apoptotic character. In order to unravel a potential ORF3a-induced apoptotic and inflammatory death mechanism, lung epithelial cells (A549) were transfected with in vitro synthesized ORF3a mRNA. The protein’s dynamic involvement as “stress factor” for the endoplasmic reticulum, causing the activation of PERK kinase and other UPR-involved proteins and therefore the upregulation of their signaling pathway executioners (ATF6, XBP-1s, PERK, phospho eIF2a, ATF4, CHOP, GADD34), has been clearly demonstrated. Furthermore, the overexpression of BAX and BH3-only pro-apoptotic protein PUMA, the upregulation of Bcl-2 family genes (BAX, BAK, BID, BAD), the reduced expression of Bcl-2 in mRNA and protein levels, and lastly, the cleavage of PARP-1 and caspase family members (caspase-3,-8 and -9) indicate that ORF3a displays its apoptotic character through the mitochondrial pathway of apoptosis. Moreover, the upregulation of NFκB, phosphorylation of p65 and IκΒα and the elevated expression of pro-inflammatory cytokines (IL-1b, IL-6, IL-8 and IL-18) in transfected cells with ORF3a mRNA indicate that this protein causes the inflammatory response through NFκB activation and therefore triggers lung injury. An intriguing finding of our study is that upon treatment of the ORF3a-transfected cells with GSK2606414, a selective PERK inhibitor, both complications (apoptosis and inflammatory response) were neutralized, and cell survival was favored, whereas treatment of transfected cells with z-VAD (a pan-caspase inhibitor) despite inhibiting cell death, could not ameliorate the inflammatory response of transfected A549 cells. Given the above, we point out that PERK kinase is a “master tactician” and its activation constitutes the main stimulus for the emergence of ORF3a apoptotic and inflammatory nature and therefore could serve as potential target for developing novel therapeutic approaches against COVID-19. Full article

The unprecedented pandemic of COVID-19, caused by a novel coronavirus, SARS-CoV-2, and its highly transmissible variants, led to massive human suffering, death, and economic devastation worldwide. Recently, antibody-evasive SARS-CoV-2 subvariants, BQ and XBB, have been reported. Therefore, the continued development of novel drugs with pan-coronavirus inhibition is critical to treat and prevent infection of COVID-19 and any new pandemics that may emerge. We report the discovery of several highly potent small-molecule inhibitors. One of which, NBCoV63, showed low nM potency against SARS-CoV-2 (IC₅₀: 55 nM), SARS-CoV-1 (IC₅₀: 59 nM), and MERS-CoV (IC₅₀: 75 nM) in pseudovirus-based assays with excellent selectivity indices (SI > 900), suggesting its pan-coronavirus inhibition. NBCoV63 showed equally effective antiviral potency against SARS-CoV-2 mutant (D614G) and several variants of concerns (VOCs) such as B.1.617.2 (Delta), B.1.1.529/BA.1 and BA.4/BA.5 (Omicron), and K417T/E484K/N501Y (Gamma). NBCoV63 also showed similar efficacy profiles to Remdesivir against authentic SARS-CoV-2 (Hong Kong strain) and two of its variants (Delta and Omicron), SARS-CoV-1, and MERS-CoV by plaque reduction in Calu-3 cells. Additionally, we show that NBCoV63 inhibits virus-mediated cell-to-cell fusion in a dose-dependent manner. Furthermore, the absorption, distribution, metabolism, and excretion (ADME) data of NBCoV63 demonstrated drug-like properties. Full article

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1’ and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV. Full article

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (M^pro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 M^pro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 M^pro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors. Full article

During the past three decades, humans have been confronted with different new coronavirus outbreaks. Since the end of the year 2019, COVID-19 threatens the world as a rapidly spreading infectious disease. For this work, we targeted the non-structural protein 16 (nsp16) as a key protein of SARS-CoV-2, SARS-CoV-1 and MERS-CoV to develop broad-spectrum inhibitors of nsp16. Computational methods were used to filter candidates from a natural product-based library of 224,205 compounds obtained from the ZINC database. The binding of the candidates to nsp16 was assessed using virtual screening with VINA LC, and molecular docking with AutoDock 4.2.6. The top 9 compounds were bound to the nsp16 protein of SARS-CoV-2, SARS-CoV-1, and MERS-CoV with the lowest binding energies (LBEs) in the range of −9.0 to −13.0 kcal with VINA LC. The AutoDock-based LBEs for nsp16 of SARS-CoV-2 ranged from −11.42 to −16.11 kcal/mol with predicted inhibition constants (pKi) from 0.002 to 4.51 nM, the natural substrate S-adenosyl methionine (SAM) was used as control. In silico results were verified by microscale thermophoresis as in vitro assay. The candidates were investigated further for their cytotoxicity in normal MRC-5 lung fibroblasts to determine their therapeutic indices. Here, the IC₅₀ values of all three compounds were >10 µM. In summary, we identified three novel SARS-CoV-2 inhibitors, two of which showed broad-spectrum activity to nsp16 in SARS-CoV-2, SARS-CoV-1, and MERS-CoV. All three compounds are coumarin derivatives that contain chromen-2-one in their scaffolds. Full article