Broad-spectrum Antiviral Research Articles

Allosteric Inhibition of Neutral Sphingomyelinase 2 (nSMase2) by DPTIP: From Antiflaviviral Activity to Deciphering Its Binding Site through In Silico Studies and Experimental Validation.

Flavivirus comprises globally emerging and re-emerging pathogens such as Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), among others. Although some vaccines are available, there is an unmet medical need as no effective antiviral treatment has been approved for flaviviral infections. The development of host-directed antivirals (HDAs) targeting host factors that are essential for viral replication cycle offers the opportunity for the development of broad-spectrum antivirals. In the case of flaviviruses, recent studies have revealed that neutral sphingomyelinase 2, (nSMase2), involved in lipid metabolism, plays a key role in WNV and ZIKV infection. As a proof of concept, we have determined the antiviral activity of the non-competitive nSMase2 inhibitor DPTIP against WNV and ZIKV virus. DPTIP showed potent antiviral activity with EC50 values of 0.26 µM and 1.56 µM for WNV and ZIKV, respectively. In order to unravel the allosteric binding site of DPTIP in nSMase2 and the details of the interaction, computational studies have been carried out. These studies have revealed that DPTIP could block the DK switch in nSMase2. Moreover, the analysis of the residues contributing to the binding identified His463 as a crucial residue. Interestingly, the inhibitory activity of DPTIP on the H463A mutant protein supported our hypothesis. Thus, an allosteric cavity in nSMase2 has been identified that can be exploited for the development of new inhibitors with anti-flaviviral activity.

A Short 5'triphosphate RNA nCoV-L Induces a Broad-Spectrum Antiviral Response by Activating RIG-I.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.

Activation of the Chemokine Receptor CCR1 and Preferential Recruitment of Gαi Suppress RSV Replication: Implications for Developing Novel Respiratory Syncytial Virus Treatment Strategies.

Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals, and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in nonimmune cells is rarely clear. In this study, we detected that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo or in vitro, viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction, or viral adhesion or entry inhibition. In contrast, it was related to the preferential recruitment and activation of the adaptor Gαi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation; explicitly, phosphorylated STAT3 (p-STAT3) was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of nonimmune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction. IMPORTANCE Our results revealed a novel mechanism by which RSV escapes innate immunity. That is, although it induces high CCL5 expression, RSV might attenuate the binding of CCL5 by downregulating the expression of CCR1 in respiratory epithelial cells to weaken the inhibitory effect of CCR1 on HSP90 activity and thereby facilitate RSV replication in nonimmune cells. This study provides a new target for the development of co-antiviral inhibitors against other components of the host and co-molecular chaperone/HSP90 and provides a scientific basis for the search for effective broad-spectrum antiviral drugs.

Broad Antiviral Effects of Echinacea purpurea against SARS-CoV-2 Variants of Concern and Potential Mechanism of Action.

SARS-CoV-2 variants of concern (VOCs) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Broad-spectrum antivirals could play an important role to control infections. The activity of Echinacea purpurea (Echinaforce® extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish), B1.525 (eta), and B.1.1.529.BA1 (omicron); (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild type SARS-CoV-2 (Hu-1) was analyzed. Molecular dynamics (MD) were applied to study the interaction of Echinacea's phytochemical markers with known pharmacological viral and host cell targets. EF extract broadly inhibited the propagation of all investigated SARS-CoV-2 VOCs as well as the entry of SARS-CoV-2 pseudoparticles at EC50's ranging from 3.62 to 12.03 µg/mL. The preventive addition of 25 µg/mL EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) and OC43. MD analyses showed constant binding affinities to VOC-typical S protein variants for alkylamides, caftaric acid, and feruloyl-tartaric acid in EF extract and interactions with serine protease TMPRSS-2. EF extract demonstrated stable virucidal activity across seven tested VOCs, likely due to the constant affinity of the contained phytochemical substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain the cell protective benefits of the extract by the inhibition of membrane fusion and cell entry. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.

Clinical and Biological Characteristics of a Griscelli Syndrome Type 2: About a Case Report

Introduction: Griscelli Syndrome (GS) is a rare autosomal recessive genetic disorder, characterized by a partial albinism that can be associated with early-onset severe psychomotor retardation, and normal immune status (type 1), or associated with an immune deficiency (type 2). GS type 2 results from mutations in the RAB27A gene, leading to immune deficiency. GS type 2 has a grave prognosis because of a rapidly fatal macrophage activated syndrome. Case Report: A 3-month-old infant having a family history of first-degree consanguinity in the parents and history of silvery gray hair in two uncles, Admitted to our department for management of a prolonged fever and an anemic syndrome. Hair on the scalp, eyebrows, and eyelashes were silvery gray and had a metallic sheen. The lymphocyte sub typing showed a severe combined immune deficiency. Histological examination of the hair showed enlarged, hyper pigmented basal melanocytes with sparse pigmentation of adjacent keratinocytes in favor of a Griscelli syndrome. The evolution was marked by clinical worsening, respiratory distress. On the biological level, a macrophage activation syndrome was retained in front of a hyperferritinemia, a hypertriglyceridemia and hepatic cytolysis. The patient received broad-spectrum antibiotic therapy, antivirals and antifungals, corticosteroid boluses 3 days in a row and an immunoglobulin infusion. Unfortunately, the patient died of septic shock. Conclusion: Type 2 GS is a rare genetic disease with a poor prognosis, which must be diagnosed early to allow better support. The only curative treatment is bone marrow transplantation.

Small molecule RAF265 as an antiviral therapy acts against HSV-1 by regulating cytoskeleton rearrangement and cellular translation machinery.

Host-targeting antivirals (HTAs) have received increasing attention for their potential as broad-spectrum antivirals that pose relatively low risk of developing drug resistance. The repurposing of pharmaceutical drugs for use as antivirals is emerging as a cost- and time- efficient approach to developing HTAs for the treatment of a variety of viral infections. In this study, we used a virus titer method to screen 30 small molecules for antiviral activity against Herpes simplex virus-1 (HSV-1). We found that the small molecule RAF265, an anticancer drug that has been shown to be a potent inhibitor of B-RAF V600E, reduced viral loads of HSV-1 by 4 orders of magnitude in Vero cells and reduced virus proliferation in vivo. RAF265mediated cytoskeleton rearrangement and targeted the host cell's translation machinery, which suggests that the antiviral activity of RAF265 may be attributed to a dual inhibition strategy. This study offers a starting point for further advances toward clinical development of antivirals against HSV-1.

Gossypol Broadly Inhibits Coronaviruses by Targeting RNA-Dependent RNA Polymerases.

Outbreaks of coronaviruses (CoVs), especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have posed serious threats to humans and animals, which urgently calls for effective broad-spectrum antivirals. RNA-dependent RNA polymerase (RdRp) plays an essential role in viral RNA synthesis and is an ideal pan-coronaviral therapeutic target. Herein, based on cryo-electron microscopy and biochemical approaches, gossypol (GOS) is identified from 881 natural products to directly block SARS-CoV-2 RdRp, thus inhibiting SARS-CoV-2 replication in both cellular and mouse infection models. GOS also acts as a potent inhibitor against the SARS-CoV-2 variant of concern (VOC) and exerts same inhibitory effects toward mutated RdRps of VOCs as the RdRp of the original SARS-CoV-2. Moreover, that the RdRp inhibitor GOS has broad-spectrum anti-coronavirus activity against alphacoronaviruses (porcine epidemic diarrhea virus and swine acute diarrhea syndrome coronavirus), betacoronaviruses (SARS-CoV-2), gammacoronaviruses (avian infectious bronchitis virus), and deltacoronaviruses (porcine deltacoronavirus) is showed. The findings demonstrate that GOS may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and other coronavirus outbreaks.

26 Kawasaki’s disease vs systemic juvenile idiopathic arthritis, a diagnostic dilemma

BackgroundBoth these conditions are separate inflammatory conditions whose clinical presentation overlap and thus make their distinction difficult. Each has a criterion that allows diagnosis but since features overlap, diagnoses may be missed.Recent research also shows that both disease processes have similar underlying pathogenesis and involve interleukin 1; which have now raised the question of whether both these disease processes are distinct entities or conditions of a similar spectrum.MethodsMedical records of from the inpatient admission at current and referring hospitals which included clinical history and examination, laboratory and radiological findings.Results13-year-old boy referred from Isiolo county with complains of unremitting fevers for 9 days with associated hip pains. Was initially treated for malaria, and started on broad spectrum antibiotics and antivirals. On further examination, was noted to have cracked lips, strawberry tongue, non-suppurative conjuctivitis and an anterior chest wall wheal like rash. Examination also revealed a left sided non tender submandibular lymph node. Initial laboratory investigations showed neutrophilia of 12.89, thrombocytosis of 431, CRP of 364 and normal joint radiographs. An ECHO done showed left ventricular enlargement with corresponding mild Mitral regurgitation. Blood cultures showed no organisms. Autoimmune markers and tropical fever markers were normal as well. He was then placed on prednisolone challenge and reported to be fever free for the first time in 12 days.Discussion and conclusionA persistent fever unremitting with parenteral antipyretics can have a multitude of differentials. In a tropical nation the likelihood of an infectious cause is more common than other aetiologies. Hence, it should always be investigated for thoroughly. In the case highlighted above, malaria, TB, osteomyelitis, septic arthritis and other infectious causes were investigated. Childhood malignancy can also present with the same. But of note is autoimmune conditions need to be considered. With history of arthritic like pain, quotidian pattern of fevers, evanescent salmon coloured chest wall rash and cervical lymphadenopathy, a diagnosis of Systemic Juvenile Arthritis needs to be considered. Often this may be overlooked or misinterpreted as Kawasaki’s; which is a close differential; or Still’s disease. There is a criterion for SJIA by the International League of Associations for Rheumatology, however, there can be an incomplete presentation as well. This adds on to the diagnostic challenge and hence, SJIA may remain undiagnosed for a long time.

Structural similarities between SARS-CoV2 3CLpro and other viral proteases suggest potential lead molecules for developing broad spectrum antivirals.

Considering the significant impact of the recent COVID-19 outbreak, development of broad-spectrum antivirals is a high priority goal to prevent future global pandemics. Antiviral development processes generally emphasize targeting a specific protein from a particular virus. However, some antiviral agents developed for specific viral protein targets may exhibit broad spectrum antiviral activity, or at least provide useful lead molecules for broad spectrum drug development. There is significant potential for repurposing a wide range of existing viral protease inhibitors to inhibit the SARS-CoV2 3C-like protease (3CLpro). If effective even as relatively weak inhibitors of 3CLpro, these molecules can provide a diverse and novel set of scaffolds for new drug discovery campaigns. In this study, we compared the sequence- and structure-based similarity of SARS-CoV2 3CLpro with proteases from other viruses, and identified 22 proteases with similar active-site structures. This structural similarity, characterized by secondary-structure topology diagrams, is evolutionarily divergent within taxonomically related viruses, but appears to result from evolutionary convergence of protease enzymes between virus families. Inhibitors of these proteases that are structurally similar to the SARS-CoV2 3CLpro protease were identified and assessed as potential inhibitors of SARS-CoV2 3CLpro protease by virtual docking. Several of these molecules have docking scores that are significantly better than known SARS-CoV2 3CLpro inhibitors, suggesting that these molecules are also potential inhibitors of the SARS-CoV2 3CLpro protease. Some have been previously reported to inhibit SARS-CoV2 3CLpro. The results also suggest that established inhibitors of SARS-CoV2 3CLpro may be considered as potential inhibitors of other viral 3C-like proteases.

Cyclic Phthalate Esters as Liver X Receptor Antagonists with Anti-hepatitis C Virus and Anti-severe Acute Respiratory Syndrome Coronavirus 2 Properties.

The liver X receptor is a nuclear hormone receptor that regulates lipid metabolism. Previously, we had demonstrated the antiviral properties of a liver X receptor antagonist associated with the hepatitis C virus and severe acute respiratory syndrome coronavirus 2. In this study, we screened a chemical library and identified two potential liver X receptor antagonists. Spectroscopic analysis revealed that the structures of both antagonists (compounds 1 and 2) were cyclic dimer and trimer of esters, respectively, that consisted of phthalate and 1,6-hexane diol. This study is the first to report the structure of the cyclic trimer of phthalate ester. Further experiments revealed that the compounds were impurities of solvents used for purification, although their source could not be traced. Both phthalate esters exhibited anti-hepatitis C virus activity, whereas the cyclic dimer showed anti-severe acute respiratory syndrome coronavirus 2 activity. Cyclic phthalate derivatives may constitute a novel class of liver X receptor antagonists and broad-spectrum antivirals.

RNA Viruses, Pandemics and Anticipatory Preparedness.

RNA viruses are likely to cause future pandemics and therefore we must create and organize a deep knowledge of these viruses to prevent and manage this risk. Assuming prevention will fail, at least once, we must be prepared to manage a future pandemic using all resources available. We emphasize the importance of having safe vaccine candidates and safe broad-spectrum antivirals ready for rapid clinical translation. Additionally, we must have similar tools to be ready for outbreaks of RNA viruses among animals and plants. Finally, similar coordination should be accomplished for other pathogens with pandemic potential.

IMB-0523 Inhibits Enterovirus 71 Replication by Activating Signal Transducer and Activator of Transcription 3 Signaling to Upregulate Interferon-Stimulated Genes Expression

Abstract Background Hand, foot, and mouth disease caused by enterovirus 71 (EV71) infection is prevalent in the Asia-Pacific region in recent years. Currently, no drug is available for the prevention and treatment of EV71 infection. IMB-0523, a N-phenylbenzamide derivative, inhibits hepatitis B virus replication by upregulating the expression of APOBEC3G. In the present study, the effect of IMB-0523 on EV71 replication and related mechanism were investigated. Methods The cytotoxicity of IMB-0523 was determined by cell counting kit. Quantitative real-time polymerase chain reaction and Western blot assay were used to detect the effect of IMB-0523 on EV71 replication and related mechanism. Cytopathic effect assay was used to investigate the effect of IMB-0523 on different EV71 strains, coxsackievirus A16, and coxsackieviruses of group B. Results The results showed that IMB-0523 could dose-dependently inhibit EV71 replication. Preliminary mechanism studies showed that IMB-0523 could activate STAT3 signaling to upregulate the expression of interferon-stimulated genes to play an antiviral role. In addition, IMB-0523 inhibited the replication of different EV71 strains, coxsackievirus A16, and coxsackieviruses of group B. Conclusions IMB-0523 inhibits EV71 replication by activating the STAT3 signaling pathway to upregulate interferon-stimulated gene expression. IMB-0523 has broad-spectrum antiviral potential and may be used as a lead compound for the development of broad spectrum antiviral drugs.

Pan-coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus infectious diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the causative agent of the currently ongoing coronavirus disease 2019 (COVID‐19) pandemic, has posed a serious threat to global public health. Recently, several SARS‐CoV‐2 variants of concern (VOCs) have emerged and caused numerous cases of reinfection in convalescent COVID‐19 patients, as well as breakthrough infections in vaccinated individuals. This calls for the development of broad‐spectrum antiviral drugs to combat SARS‐CoV‐2 and its VOCs. Pan‐coronavirus fusion inhibitors, targeting the conserved heptad repeat 1 (HR1) in spike protein S2 subunit, can broadly and potently inhibit infection of SARS‐CoV‐2 and its variants, as well as other human coronaviruses. In this review, we summarized the most recent development of pan‐coronavirus fusion inhibitors, such as EK1, EK1C4, and EKL1C, and highlighted their potential application in combating current COVID‐19 infection and reinfection, as well as future emerging coronavirus infectious diseases.

Remdesivir in treating hospitalized patients with COVID-19: A renewed review of clinical trials.

Since December 2019, COVID-19 has spread across the world almost through 2.5 years. As of 16 June 2022, the cumulative number of confirmed cases of COVID-19 worldwide has reached 542.62 million, and the death toll has risen to 6.33 million. With the increasing number of deaths, it is urgent to find effective treatment drugs. Remdesivir, an investigational broad-spectrum antiviral drug produced by Gilead has been shown to inhibit SARS-CoV-2, in vitro and in vivo. This review is aimed to analyze the feasibility of remdesivir in COVID-19 and put forward the shortcomings of present clinical studies. We systematically searched PubMed and Web of Science up until 24 May 2022, using several specific terms such as “remdesivir” or “GS-5734” and “COVID-19” or “SARS-CoV-2” and retrieved basic researches and clinical studies of remdesivir in COVID-19. In this review, we summarized and reviewed the mechanism of remdesivir in SARS-COV-2, clinical trials of using remdesivir in COVID-19, analyzed the efficacy and safety of remdesivir, and judged whether the drug was effective for the treatment of COVID-19. In different clinical trials, remdesivir showed a mixed result in the treatment of COVID-19. It seemed that remdesivir shortened the time to recovery and had an acceptable safety profile. However, more clinical trials are needed to test the efficacy and safety of remdesivir.

Effect of remdesivir on mortality rate and clinical status of COVID-19 patients: a systematic review with meta-analysis

Remdesivir (RDV) is a broad-spectrum antiviral drug, now approved by Regulatory Agencies for COVID-19 treatment. RDV is associated with improvements in clinical outcomes, but no conclusive studies have shown an effect in reducing mortality. This study aimed to carry out a systematic review with meta-analysis to investigate whether RDV can significantly modify the outcome of COVID-19 patients evaluating its effects on mortality, length of stay, time to clinical improvement and need for oxygen supplementation. No significant improvement in terms of survival in patients treated with standard therapy (ST)+RDV as compared to ST alone (P = 0.24) was found. The duration of oxygen support was significantly lower in patients treated with ST + RDV compared with ST alone (P = 0.03). Further investigations should be planned to assess the real impact of RDV in the management of COVID-19 patients.

COVID-19, Influenza, and Other Acute Respiratory Viral Infections: Etiology, Immunopathogenesis, Diagnosis, and Treatment. Part 2. Other Acute Respiratory Viral Infections.

The first part of this paper presented the current knowledge on two very significant respiratory diseases with high pandemic potential, COVID-19 and influenza. The second part reviews other pathogens that cause acute respiratory viral infections, ARVI, including parainfluenza viruses, adenoviruses, pneumoviruses and specifically respiratory syncytial virus, enteroviruses, rhinoviruses, bocaviruses, and seasonal coronaviruses. The review presents modern data on the structure and replication of viruses, epidemiology and immunopathogenesis of diseases, diagnostics, preventive vaccination, and antiviral drugs. Topical issues regarding ARVI vaccination and the search for new broad-spectrum antiviral drugs are discussed.

A potent neutralizing antibody provides protection against SARS-CoV-2 Omicron and Delta variants via nasal delivery

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still rapidly spreading worldwide. Many drugs and vaccines have been approved for clinical use show efficacy in the treatment and prevention of SARS-CoV-2 infections. However, the emergence of SARS-CoV-2 variants of concern (VOCs), such as Delta (B.1.617.2) and the recently emerged Omicron (B.1.1.529), has seriously challenged the application of current therapeutics. Therefore, there is still a pressing need for identification of new broad-spectrum antivirals. Here, we further characterized a human antibody (58G6), which we previously isolated from a patient, with a broadly authentic virus-neutralizing activity that inhibits the Delta and Omicron variants with half-maximal inhibitory concentrations (IC50) of 1.69 ng/ml and 54.31 ng/ml, respectively. 58G6 shows prophylactic and therapeutic efficacy in hamsters challenged with the Delta and Omicron variants through nasal delivery. Notably, a very low dosage (2 mg/kg daily) of 58G6 efficiently prevented Omicron variant replication in the lungs. These advantages may overcome the efficacy limitation of currently approved neutralizing antibodies that can be administered only by intravenous injection. In general, 58G6 is a promising prophylactic and therapeutic candidate against current circulating VOCs and even future emerging mutants. To the best of our knowledge, 58G6 is one of the most potent neutralizing antibodies against Omicron, with a broader spectrum than those approved for clinical use. 58G6 could be developed as a nebulized therapy, which would be more cost effective and user friendly and enhance the clinical outcome compared to that obtained with direct nasal delivery.

Broad-Spectrum Small-Molecule Inhibitors of the SARS-CoV-2 Spike-ACE2 Protein-Protein Interaction from a Chemical Space of Privileged Protein Binders.

Therapeutically useful small-molecule inhibitors (SMIs) of protein–protein interactions (PPIs) initiating the cell attachment and entry of viruses could provide novel alternative antivirals that act via mechanisms similar to that of neutralizing antibodies but retain the advantages of small-molecule drugs such as oral bioavailability and low likelihood of immunogenicity. From screening our library, which is focused around the chemical space of organic dyes to provide good protein binders, we have identified several promising SMIs of the SARS-CoV-2 spike—ACE2 interaction, which is needed for the attachment and cell entry of this coronavirus behind the COVID-19 pandemic. They included organic dyes, such as Congo red, direct violet 1, and Evans blue, which seem to be promiscuous PPI inhibitors, as well as novel drug-like compounds (e.g., DRI-C23041). Here, we show that in addition to the original SARS-CoV-2 strain, these SMIs also inhibit this PPI for variants of concern including delta (B.1.617.2) and omicron (B.1.1.529) as well as HCoV-NL63 with low- or even sub-micromolar activity. They also concentration-dependently inhibited SARS-CoV-2-S expressing pseudovirus entry into hACE2-expressing cells with low micromolar activity (IC50 < 10 μM) both for the original strain and the delta variant. DRI-C23041 showed good therapeutic (selectivity) index, i.e., separation between activity and cytotoxicity (TI > 100). Specificities and activities require further optimization; nevertheless, these results provide a promising starting point toward novel broad-spectrum small-molecule antivirals that act via blocking the interaction between the spike proteins of coronaviruses and their ACE2 receptor initiating cellular entry.

Small-molecule screening of ribonuclease L binders for RNA degradation

Small molecules targeting the ubiquitous latent ribonuclease (RNase L), which has limited sequence specificity toward single-stranded RNA substrates, hold great potential to be developed as broad-spectrum antiviral drugs by modulating the RNase L-mediated innate immune responses. The recent development of proximity-inducing bifunctional molecules, as described in the strategy of ribonuclease targeting chimeras, demonstrated that small-molecule RNase L activators can function as the essential RNase L-recruiting component to design bifunctional molecules for targeted RNA degradation. However, only a single screening study on small-molecule RNase L activators with poor potency has been reported to date. Herein, we established a FRET assay and conducted a screening of 240,000 small molecules to identify new RNase L activators with improved potency. The extremely low hit rate of less than 0.03% demonstrated the challenging nature of RNase L activation by small molecules available from current screening collections. A few hit compounds induced enhanced thermal stability of RNase L upon binding, although validation assays did not lead to the identification of compounds with significantly improved RNase L activating potency. The sulfonamide compound 17 induced a thermal shift of ~ 0.9 °C upon binding to RNase L, induced significant apoptosis in cancer cells, and showed single-digit micromolar inhibitory activity against cancer cell proliferation. This study paves the way for future structural optimization for the development of small-molecule RNase L binders.