Path to another drug against COVID-19 The rapid development of vaccines has been crucial in battling the ongoing COVID-19 pandemic. However, access challenges remain, breakthrough infections occur, and emerging variants present increased risk. Developing antiviral therapeutics is therefore a high priority for the treatment of COVID-19. Some drug candidates in clinical trials act against the viral RNA-dependent RNA polymerase, but there are other viral enzymes that have been considered good targets for inhibition by drugs. Owen et al . report the discovery and characterization of a drug against the main protease involved in the cleavage of polyproteins involved in viral replication. The drug, PF-07321332, can be administered orally, has good selectivity and safety profiles, and protects against infection in a mouse model. In a phase 1 clinical trial, the drug reached concentrations expected to inhibit the virus based on in vitro studies. It also inhibited other coronaviruses, including severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, and could be in the armory against future viral threats. —VV

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3␤. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus͞hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases. Although many protein kinases phosphorylate tau at ADrelevant epitopes in vitro (reviewed in ref.2), only two have been copurified with microtubules, GSK3␤ and cdk5 (3, 4). To our knowledge, only these two kinases will phosphorylate tau in a cellular environment (e.g., refs. 5 and 6). We chose to focus on cdk5 because it is active predominantly in neurons whereas GSK3␤ plays a role in energy metabolism and is active in all cells. cdk5 is a member of the cyclin-dependent protein kinase gene family. Rather than cyclins, cdk5 associates with the positive allosteric regulators p35 (7), amino-terminal proteolytic fragments of p35 (e.g., p25; ref. 8), and p39 (9). These proteins share minimal amino acid sequence homology to cyclins, but the mechanism of activation of cdk5 by p25͞35 may be similar to the activation of cdk2 by cyclin A (10). p25͞35 is expressed predominantly in neurons, implying that most cdk5 activity is concentrated in neuronal structures (7,8). cdk5 plays a pivotal role in neuronal development as evidenced by the abnormal corticogenesis and perinatal lethality of cdk5 knockout mice (11) and the disturbances in neuronal migration and early death in p35 knockout mice (12). A number of potential cdk5 substrates have been identified and most are consistent with a putative role in neurite outgrowth and plasma membrane dynamics. These include cytoskeletal proteins such as tau and neurofilament (e.g., refs. 13 and 14) and synaptic vesicle proteins (15, 16). To clarify the potential role of cdk5 in neurodegenerative diseases in vivo, we overexpressed human p25 in the brains of transgenic mice to determine whether increased cdk5 activity would lead to hyperphosphorylation of tau and neurofilament and͞or cytoskeletal disturbances. Materials and MethodsAnimal Handling. All experimentation was performed under...

The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency, in a phase I clinical trial in healthy human participants. Clinical Trial Registration ID #: NCT04756531

High-permeability-low-molecular-weight acids/zwitterions [i.e., extended clearance classification system class 1A (ECCS 1A) drugs] are considered to be cleared by metabolism with a minimal role of membrane transporters in their hepatic clearance. However, a marked disconnect in the in vitro-in vivo (IVIV) translation of hepatic clearance is often noted for these drugs. Metabolic rates measured using human liver microsomes and primary hepatocytes tend to underpredict. Here, we evaluated the role of organic anion transporter 2 (OAT2)-mediated hepatic uptake in the clearance of ECCS 1A drugs. For a set of 25 ECCS 1A drugs, in vitro transport activity was assessed using transporter-transfected cells and primary human hepatocytes. All but two drugs showed substrate affinity to OAT2, whereas four (bromfenac, entacapone, fluorescein, and nateglinide) also showed OATP1B1 activity in transfected cells. Most of these drugs (21 of 25) showed active uptake by plated human hepatocytes, with rifamycin SV (pan-transporter inhibitor) reducing the uptake by about 25%-95%. Metabolic turnover was estimated for 19 drugs after a few showed no measurable substrate depletion in liver microsomal incubations. IVIV extrapolation using in vitro data was evaluated to project human hepatic clearance of OAT2-alone substrates considering 1) uptake transport only, 2) metabolism only, and 3) transporter-enzyme interplay (extended clearance model). The transporter-enzyme interplay approach achieved improved prediction accuracy (average fold error = 1.9 and bias = 0.93) compared with the other two approaches. In conclusion, this study provides functional evidence for the role of OAT2-mediated hepatic uptake in determining the pharmacokinetics of several clinically important ECCS 1A drugs.

Understanding the quantitative implications of P-glycoprotein and breast cancer resistance protein efflux is a key hurdle in the design of effective, centrally acting or centrally restricted therapeutics. Previously, a comprehensive physiologically based pharmacokinetic model was developed to describe the in vivo unbound brain-to-plasma concentration ratio as a function of efflux activity measured in vitro. In the present work, the predictive utility of this framework was examined through application to in vitro and in vivo data generated on 133 unique compounds across three preclinical species. Two approaches were examined for the scaling of efflux activity to in vivo, namely relative expression as determined by independent proteomics measurements and relative activity as determined via fitting the in vivo neuropharmacokinetic data. The results with both approaches indicate that in vitro efflux data can be used to accurately predict the degree of brain penetration across species within the context of the proposed physiologically based pharmacokinetic framework.