RAY1216 for COVID-19

AbstractSARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an α-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with aKi of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the α-ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mproactive site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

Abstract SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an α-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease:inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the α-ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials to test real-world therapeutic efficacy against COVID-19.

The main protease (Mpro) plays a pivotal role in the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is considered a highly conserved viral target. Disruption of the catalytic activity of Mpro produces a detrimental effect on the course of the infection, making this target one of the most attractive for the treatment of COVID-19. The current success of the SARS-CoV-2 Mpro inhibitor Nirmatrelvir, the first oral drug for the treatment of severe forms of COVID-19, has further focused the attention of researchers on this important viral target, making the search for new Mpro inhibitors a thriving and exciting field for the development of antiviral drugs active against SARS-CoV-2 and related coronaviruses.

In this study, we present the highly efficient and rapid synthesis of substituted dihydropyrimidinone derivatives through an ultrasound-accelerated approach. We utilize copper ferrite (CuFe2O4) magnetic nanoparticles as heterogeneous catalysts, employing the well-known Biginelli reaction, under solvent-free conditions. The impact of the solvent, catalyst amount, and catalyst type on the reaction performance is thoroughly investigated. Our method offers several notable advantages, including facile catalyst separation, catalyst reusability for up to three cycles with the minimal loss of activity, a straightforward procedure, mild reaction conditions, and impressive yields, ranging from 79% to 95%, within short reaction times of 20 to 40 min. Furthermore, in the context of fighting COVID-19, we explore the potential of substituted dihydropyrimidinone derivatives as inhibitors of three crucial SARS-CoV-2 proteins. These proteins, glycoproteins, and proteases play pivotal roles in the entry, replication, and spread of the virus. Peptides and antiviral drugs targeting these proteins hold great promise in the development of effective treatments. Through theoretical molecular docking studies, we compare the binding properties of the synthesized dihydropyrimidinone derivatives with the widely used hydroxychloroquine molecule as a reference. Our findings reveal that some of the tested molecules exhibit superior binding characteristics compared to hydroxychloroquine, while others demonstrate comparable results. These results highlight the potential of our synthesized derivatives as effective inhibitors in the fight against SARS-CoV-2.

This article systematically presents the current clinically significant therapeutic developments for the treatment of COVID-19 by providing an in-depth review of molecular mechanisms of action for SARS-CoV-2 antivirals and critically analyzing the potential targets that may allow the selection of resistant viral variants. Two main categories of agents can display antiviral activity: direct-acting antivirals, which act by inhibiting viral enzymes, and host-directed antivirals, which target host cell factors that are involved in steps of the viral life cycle. We discuss both these types of antivirals, highlighting the agents that have already been approved for treatment of COVID-19, and providing an overview of the main molecules that are currently in drug development. Direct-acting antivirals target viral enzymes that are essential in the viral life cycle. Three direct-acting antivirals are currently in use: two are nucleoside analogs that inhibit the RNA-dependent RNA polymerase of SARS-CoV-2, i.e., remdesivir and molnupiravir, and the third one, nirmatrelvir/ritonavir, is an inhibitor of SARS-CoV-2 main protease. The potential for induction of viral resistance is discussed for each of these antivirals, along with their clinical activity on each of the SARS-CoV-2 variants and sublineages that have been dominant over the course of the pandemic, i.e., Alpha, Delta, as well as Omicron and its sublineages BA.1, BA.2, BA.5, BQ.1 and XBB. Host-directed antivirals are currently in preclinical or clinical development; these agents target host cell enzymes that are involved in facilitating viral entry, replication, or virion release. By blocking these enzymes, viral replication can theoretically be effectively stopped. As no SARS-CoV-2 host-directed antiviral has been approved so far, further research is still needed and we present the host-directed antivirals that are currently in the pipeline. Another specific type of agents that have been used in the treatment of COVID-19 are neutralizing antibodies (NAbs). Their main binding site is the spike protein, and therefore their neutralization activity is influenced by mutations occurring in this region. We discuss the main changes in neutralization activity of NAbs for the most important dominant SARS-CoV-2 variants. Close monitoring of emerging variants and sublineages is still warranted, to better understand the impact of viral mutations on the clinical efficiency of antivirals and neutralizing antibodies developed for the treatment of COVID-19.