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Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (...

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19.In BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.HighlightsLY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY-CoV555, a SARS-CoV-2 spike protein–specific antibody, neutralizes SARS-CoV-2 and protects the airways of nonhuman primates against infection., A monoclonal for SARS-CoV-2 Among the most promising therapeutic options for individuals with coronavirus disease 2019 (COVID-19) are monoclonal antibodies (mAbs). In this study, Jones et al. identified, characterized, and tested one such mAb, LY-CoV555, in vitro and in vivo. They found that LY-CoV555 bound to the severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) spike protein and prevented its interaction with angiotensin-converting enzyme 2. Prophylactic treatment with LY-CoV555 protected the upper and lower respiratory tracts of nonhuman primates from becoming infected with SARS-CoV-2. Together, these data support the clinical use of LY-CoV555 for treating patients with COVID-19., Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour−1 kg−1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY2812223 [(1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid] was identified via structure-activity studies arising from the potent metabotropic glutamate mGlu2/3 receptor agonist LY354740 [(+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid] as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5′-O-(3-[35S]thio)triphosphate (GTPγS) functional binding assay, a method for measuring Gi-coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line–based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain.

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu2/3 antagonists. Exploration of this structure–activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu2) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu2 receptor protein. The resulting cocrystal structure revealed the specific ligand–protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu2 antagonist pharm...

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2 (-/-) mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug-drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

Background and Purpose CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity modifying protein 1 (RAMP1) and either calcitonin receptor-like receptor (CLR; forms canonical CGRP receptor) or calcitonin receptor (CT receptor; forms AMY1 receptor). The goal of this study was to test whether transgenic mice globally expressing human RAMP1 have increased CGRP receptor activity and whether the receptors are sensitive to human selective antagonist telcagepant. Experimental Approach cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and non-transgenic littermates. Functional activity and inhibition were compared with clonal cell lines expressing combinations of CLR or CT receptors with RAMP1. Key Results Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC50s. In contrast, cultured trigeminal ganglia from global hRAMP1 mice had a 40-fold leftward shift of the EC50, with similar Rmax values as littermates. In both hRAMP1 cultures, telcagepant blocked CGRP-induced cAMP production, but was not effective in non-transgenic cultures. IC50 values were closer to those observed for CT receptor/hRAMP1 than CLR/hRAMP1 in clonal cell lines. Conclusions and Implications Overexpression of hRAMP1 increases CGRP signalling by changing the maximal response or ligand sensitivity, depending on tissue type. Furthermore, telcagepant inhibited transgenic hRAMP1 CGRP receptors, but the degree of inhibition suggests that the transgenic mice are only partially humanized or both canonical CGRP and AMY1 receptors are functional in trigeminal ganglia neurons and vascular smooth muscle.

The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs.

The base of knowledge concerning RNA structure and function has been expanding rapidly in recent years. Simultaneously, an increasing awareness of the pivotal role RNA plays in viral diseases has prompted many researchers to apply new technologies in high-throughput screening and molecular modelling to the design of antiviral drugs that target RNA. While the two RNA viruses with the greatest unmet medical need, HIV and HCV, have been most actively pursued, the approaches discussed in this review are relevant to all virus infections. Both traditional small-molecule and large-molecule therapeutics, such as antisense, ribozymes and interfering dsRNAs have been described, and several molecules are under development for commercialization. The purpose of this review is to summarize the current state of the art in this field and to postulate new directions in the future.

Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3′ cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3′ single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3′-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

We have developed a simple in vitro translation method to analyze compounds that inhibit the rhinovirus 3C protease in peptide substrate assays but demonstrate no antiviral activity. This complementary assay, which provides both qualitative and quantitative results, detects the inhibition of the 3CD protease in the native polyprotein form.

Two allosteric modulators of the group I metabotropic glutamate receptors (mGluR1 and mGluR5) were evaluated as positron emission tomography (PET) radioligands for mGluR1.LY2428703, a full mGluR1 antagonist (IC(50) 8.9 nM) and partial mGluR5 antagonist (IC(50) 118 nM), and LSN2606428, a full mGluR1 and mGluR5 antagonist (IC(50) 35.3 nM and 10.2 nM, respectively) were successfully labeled with (11)C and evaluated as radioligands for mGluR1. The pharmacology of LY2428703 was comprehensively assessed in vitro and in vivo, and its biodistribution was investigated by liquid chromatography-mass spectrometry/mass spectrometry, and by PET imaging in the rat. In contrast, LSN2606428 was only evaluated in vitro; further evaluation was stopped due to its unfavorable pharmacological properties and binding affinity.(11)C-LY2428703 showed promising characteristics, including: (1) high potency for binding to human mGluR1 (IC(50) 8.9 nM) with no significant affinity for other human mGlu receptors (mGluR2 through mGluR8); (2) binding to brain displaceable by administration of an mGluR1 antagonist; (3) only one major radiometabolite in both plasma and brain, with a negligible brain concentration (with 3.5 % of the total radioactivity in cerebellum) and no receptor affinity; (4) a large specific and displaceable signal in the mGluR1-rich cerebellum with no significant in vivo affinity for mGluR5, as shown by PET studies in rats; and (5) lack of substrate behavior for efflux transporters at the blood-brain barrier, as shown by PET studies conducted in wild-type and knockout mice.(11)C-LY2428703, a new PET radioligand for mGluR1 quantification, displayed promising characteristics both in vitro and in vivo in rodents.

WIN compounds inhibit attachment of human rhinovirus 14 by binding to a hydrophobic pocket within the capsid and inducing conformational changes in the canyon floor, the region that binds the cellular receptor. To study the basis of drug resistance, we isolated and characterized a family of human rhinovirus 14 mutants resistant to WIN 52035-2. Thermostabilization data and single-cycle growth curves provided evidence for two classes of resistant mutants. One class, here called exclusion mutants, showed a marked decrease in drug-binding affinity and was characterized by substitution to bulkier amino acid side chains at two sites lining the hydrophobic pocket. The other class, called compensation mutants, displayed single-amino-acid substitutions in the drug-deformable regions of the canyon; these mutants were able to attach to cells despite the presence of bound drug. A delay in the rise period of the growth curves of compensation mutants indicated a second locus of drug action. WIN 52035-2 was found to inhibit the first step of uncoating, release of VP4. Attempts to identify this site of drug action by using single-step growth curves were obscured by abortive elution of a major fraction of cell-attached virus. The drug had no effect on the rate of this process but did affect the spectrum of particles produced.

Approximately 300,000 compounds from selected libraries were screened against a subdomain of a hepatitis C viral (HCV) RNA using a high throughput flow injection mass spectrometry (FIA-MS) method with automated data storage and analysis. Samples contained 2 μM RNA target and 10 μM of each of up to ten ligands. Preliminary studies to optimize operational parameters used the binding of aminoglycosides to the A44 subdomain of bacterial RNA. Binding (confirmed by titration) and sensitivity were maximized within the constraints of the library and throughput. The mobile phase of 5 mM ammonium acetate in 50% isopropanol maintained the noncovalent complexes and provided good detection by electrospray mass spectrometry. Additionally, this composition maximized general solubility of the various classes of compounds including the oligonucleotide and organic library molecules. Cation adduction was insignificant in this screen although some solute and target dependent acetate adduction was observed. The ion trap mass spectrometer provided sufficient mass resolution to identify complexes of RNA with known components of the library. Converted mass spectral data (netCDF) were subjected to two types of statistical evaluation based on binding. The first algorithm identified noncovalent complexes that correlated with the molecular weights of the injected compounds. The second yielded the largest peak in the noncovalent complex region of the spectrum; this spectrum may or may not correlate with expected well components. Sixty-three compounds were confirmed to bind by more stringent secondary testing. Titrations, which were carried out with selected binding compounds, yielded a range of dissociation constants. Biological activity was observed for eleven confirmed binders.

This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.

We have been exploring the mechanism of action of 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin), an antiviral drug that inhibits the replication of poliovirus in culture. By varying the time of drug addition to infected cells, we found that the drug acts at a stage which is late in the replication cycle and subsequent to the step inhibited by guanidine. Furthermore, we detected normal levels of full-length plus-strand virion RNA in hydantoin-treated cultures. A new assembly intermediate in addition to the expected assembly intermediates was detected in drug-treated cultures. This intermediate has properties consistent with that of a packaging intermediate. Drug-resistant mutants were readily isolated. Sequence analysis of three independent drug-resistant mutants identified amino acid substitutions in the 2C coding region. Reconstruction by site-directed mutagenesis confirmed that these single mutations were sufficient to confer drug resistance. Taken together, these data suggest that the poliovirus 2C region is involved in virus encapsidation and that hydantoin inhibits this stage of replication.

X-ray diffraction data were collected from human rhinovirus 14 crystals a few minutes after exposure to acid vapor and prior to excessive crystalline disorder. Conformational changes occurred (i) in the GH loop of viral protein (VP) 1, (ii) at the ion binding site on the outer surface of the pentamer center, and (iii) in VP3 and VP4 on the virion's interior in the vicinity of the fivefold axis. Amino acid substitutions in mutants resistant to low pH, or to drugs that inhibit uncoating, were concentrated in the vicinity of the GH loop. It is proposed that the acid-induced changes reflect processes that trigger uncoating.

Spontaneous mutants of human rhinovirus 14 resistant to WIN 52084, an antiviral compound that inhibits attachment to cells, were isolated by selecting plaques that developed when wild-type virus was plated in the presence of high (2 micrograms/ml) or low (0.1 to 0.4 micrograms/ml) concentrations of the compound. Two classes of drug resistance were observed: a high-resistance (HR) class with a frequency of about 4 x 10(-5), and a low-resistance (LR) class with a 10- to 30-fold-higher frequency. The RNA genomes of 56 HR mutants and 13 LR mutants were sequenced in regions encoding the drug-binding site. The HR mutations mapped to only 2 of the 16 amino acid residues that form the walls of the drug-binding pocket. The side chains of these two residues point directly into the pocket and were invariably replaced by bulkier groups. These findings, and patterns of resistance to related WIN compounds, support the concept that HR mutations may hinder the entry or seating of drug within the binding pocket. In contrast, all of the LR mutations mapped to portions of the polypeptide chain near the canyon floor that move when the drug is inserted. Because several LR mutations partially reverse the attachment-inhibiting effect of WIN compounds, these mutants provide useful tools for studying the regions of the capsid structure involved in attachment. This paper shows that the method of escape mutant analysis, previously used to identify antibody binding sites on human rhinovirus 14, is also applicable to analysis of antiviral drug activity.

The binding to human rhinovirus 14 of a series of eight antiviral agents that inhibit picornaviral uncoating after entry into host cells has been characterized crystallographically. All of these bind into the same hydrophobic pocket within the viral protein VP1 beta-barrel structure, although the orientation and position of each compound within the pocket was found to differ. The compounds cause the protein shell to be less flexible, thereby inhibiting disassembly. Although the antiviral potency of these compounds varies by 120-fold, they all induce the same conformational changes on the virion. The interactions of these compounds with the viral capsid are consistent with their observed antiviral activities against human rhinovirus 14 drug-resistant mutants and other rhinovirus serotypes. Crystallographic studies of one of these mutants confirm the partial sequencing data and support the finding that this is a single mutation that occurs within the binding pocket.

Organ explants of porcine ileum were cultured in different media for up to 48 h. Tissue preservation was evaluated by light microscopy and by transmission and scanning electron microscopy. Cellular structure was well maintained after incubation for 48 h in CMRL-1066 supplemented with insulin and cortisone. Explants of absorptive or lymphoid tissue from young or adult pigs were incubated with either coxsackievirus B5 (which is infectious for swine) or human poliovirus type 1 (which served as a control) for 24 h at 37 degrees C. Progeny virus was detected by plaque assay. Replication was most evident in the absorptive tissue explants from young pigs. In tissues from adults, replication occurred equally well in absorptive and lymphoid tissues. Infection in explants was inefficient, and the yield of progeny virus was low.