Discovery of GS-7682, a Novel 4'-Cyano-Modified -Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine -nucleoside GS-646089 () with broad antiviral activity against RSV (EC = 3-46 nM), human metapneumovirus (EC = 210 nM), human rhinovirus (EC = 54-61 nM), and enterovirus (EC = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [()-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation and .

Mechanism and spectrum of inhibition of a 4'-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682.

The larval attachment organ of the bowfin Amia ocellicauda Richardson, 1836 (Amiiformes: Amiidae) and its phylogenetic significance.

Larval attachment organs (LAOs) are unicellular or multicellular organs that enable the larvae of many actinopterygian fishes to adhere to a substrate before yolk-sac absorption and the free-swimming stage. Bowfins (Amiiformes) exhibit a sizable LAO on the snout, which was first described in the late 19th and early 20th centuries. In this study, we document the LAO of Amia ocellicauda (Richardson, 1836) using a combination of scanning electron microscopy (SEM) and light microscopy, and histochemistry.

External nasal gland morphology of Eurycea bislineata (Amphibia, Urodela, Plethodontidae).

Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands.

Genome size drives morphological evolution in organ-specific ways.

Morphogenesis is an emergent property of biochemical and cellular interactions during development. Genome size and the correlated trait of cell size can influence these interactions through effects on developmental rate and tissue geometry, ultimately driving the evolution of morphology. We tested whether variation in genome and body size is related to morphological variation in the heart and liver using nine species of the salamander genus Plethodon (genome sizes 29-67 gigabases).

Latitudinal gradients in sexual dimorphism: Alternative hypotheses for variation in male traits.

Biological patterns across latitudinal gradients elucidate a number of striking natural clines from which numerous processes can be further explored. The trade-off between reproduction and somatic maintenance and growth represents a suite of life-history traits with variable energy allocation and potential latitudinal patterns. Specifically, male sexually dimorphic traits in female choice systems represent one such reproductive investment constrained by resource acquisition and subsequent allocation.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine -Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified -nucleoside (RSV A2 EC = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (, GS-5734) that is >30-fold more potent than against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, -, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of following molar normalized IV dosing of compared to that of .

The larval attachment organ of the tropical gar Atractosteus tropicus Gill, 1863 (Lepisosteiformes: Lepisosteidae).

Larval attachment organs (LAOs) are unicellular or multicellular organs that allow larvae to adhere to a substrate before yolk-sac absorption and the free-swimming stage. This study documents the LAO of tropical gar, Atractosteus tropicus, using a combination of scanning electron microscopy and light microscopy. It is shown that the LAO of A.

Modification of genital kidney nephrons for sperm transport in a plethodontid salamander, Eurycea longicauda.

Salamanders possess kidneys with two distinct regions: a caudal pelvic portion and cranial genital portion. Nephrons of the pelvic region are responsible for urine formation and transport. Nephrons of the genital region transport sperm from testes to Wolffian ducts; however, nephrons of the genital region possess all the same functional regions found in pelvic kidney nephrons that are involved with urine formation and transport (renal corpuscles, proximal tubules, distal tubules, and collecting ducts).

Discovery of the pan-genotypic hepatitis C virus NS3/4A protease inhibitor voxilaprevir (GS-9857): A component of Vosevi.

Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3.

Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.

Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model.

The Lack of Nasolacrimal Ducts in Plethodontid Salamanders?

Nasolacrimal ducts are a terrestrial vertebrate adaptation and appear to have co-evolved with orbital glands. Although plethodontid salamanders possess orbital glands, a recent study concluded that plethodontid salamanders lack nasolacrimal ducts. Functionally, the absence of nasolacrimal ducts closes the route for orbital gland secretion passage into the nasal and vomeronasal organ cavities.

Histology of the Urogenital System in the American Bullfrog (Rana catesbeiana), with Emphasis on Male Reproductive Morphology.

Previous studies have revealed variations in the urogenital system morphology of amphibians. Recently, the urogenital system of salamanders was reviewed and terminology was synonymized across taxa. Discrepancies exist in the terminology describing the urogenital system of anurans, which prompted our group to develop a complete, detailed description of the urogenital system in an anuran species and provide nomenclature that is synonymous with those of other amphibian taxa.

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.

Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe.

GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases.

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV.

Phylogeny of Mental Glands, Revisited.

Mental glands and their associated delivery behaviors during courtship are unique to the plethodontid salamanders. Because previous interpretations of the evolution of these features were conducted using older phylogenetic hypotheses, we reanalyzed these traits with newer courtship descriptions and contemporary phylogenetic methods. Using Bayesian ancestral state reconstruction methods that have been developed since the first phylogenetic analyses were conducted in the mid-1990s, we reconstructed mental gland and courtship behavior evolution on a Bayesian phylogeny of the nuclear gene The most probable ancestral condition for plethodontids was resolved as presence of a mental gland.

Ultrastructural analysis of spermiogenesis in the Eastern Fence Lizard, Sceloporus undulatus (Squamata: Phrynosomatidae).

Studies on reptilian sperm morphology have shown that variation exists at various taxonomic levels but studies on the ontogeny of variation are rare. Sperm development follows a generalized bauplan that includes acrosome development, nuclear condensation and elongation, and flagellar development. However, minute differences can be observed such as the presence/absence of manchette microtubules, structural organization during nuclear condensation, and presence/absence of a nuclear lacuna.

Spermatogenic cycle of a plethodontid salamander, (Amphibia, Urodela).

Previous investigators have described the spermatogenic cycles of numerous species of plethodontid salamanders. Most studies describe a fairly stereotypical cycle with meiotic divisions of spermatogenesis commencing in the spring/summer. However, many studies lack details obtainable from histological examination and/or testicular squashes and, instead, provide only mensural data from the testes.

Discovery of β-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted β-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable.

Discovery of an oral respiratory syncytial virus (RSV) fusion inhibitor (GS-5806) and clinical proof of concept in a human RSV challenge study.

GS-5806 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a screening hit. The oral absorption properties were optimized by converting to the pyrazolo[1,5-a]-pyrimidine heterocycle, while potency, metabolic, and physicochemical properties were optimized by introducing the para-chloro and aminopyrrolidine groups. A mean EC50 = 0.

Histology and ultrastructure of the caudal courtship glands of the red-backed salamander, Plethodon cinereus (Amphibia: Plethodontidae).

Caudal courtship glands (CCGs) are sexually dimorphic glands described in the skin of the dorsal tail base of some male salamanders in the genera Desmognathus, Eurycea, and Plethodon in the family Plethodontidae. These glands are believed to deliver pheromones to females during courtship, when the female rests her chin on the dorsal tail base during the stereotypic tail straddling walk unique to plethodontids. Although CCGs have been studied histologically, no investigations of their ultrastructure have been made.

C7-derivatization of C3-alkylindoles including tryptophans and tryptamines.

A versatile strategy for C7-selective boronation of tryptophans, tryptamines, and 3-alkylindoles by way of a single-pot C2/C7-diboronation-C2-protodeboronation sequence is described. The combination of a mild iridium-catalyzed C2/C7-diboronation followed by an in situ palladium-catalyzed C2-protodeboronation allows efficient entry to valuable C7-boroindoles that enable further C7-derivatization. The versatility of the chemistry is highlighted by the gram-scale synthesis of C7-boronated N-Boc-L-tryptophan methyl ester and the rapid synthesis of C7-halo, C7-hydroxy, and C7-aryl tryptophan derivatives.