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. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

Soft-tissue profile growth in patients with repaired complete unilateral cleft lip and palate: A cephalometric comparison with normal controls at ages 7, 11, and 18 years.

INTRODUCTION: In this retrospective longitudinal study, we aimed to study differences in the soft-tissue profiles in growing children with clefts in comparison with controls through the period of facial growth from 7 to 18 years. METHODS: Lateral cephalometric measurements made at 7 years (T1), 11.1 years (T2), and 17.9 years (T3) of age of 70 white children (35 boys, 35 girls) with complete unilateral cleft lip and palate (UCLP) who received primary lip and palate repair surgeries at The Hospital for Sick Children, Toronto, were compared with those of a control group of similar ages, sexes, and racial backgrounds, and having skeletal Class I facial growth, selected from the Burlington Growth Study. None of the included subjects had received any surgeries other than the primary lip and palate repairs, and none had undergone nasal septum surgery or nasal molding during infancy. Between-group comparisons were made at each time point using generalized linear models adjusted for age and sex effects. Longitudinal comparisons across all time points were conducted using the mixed model approach, adjusting for these effects and their interactions with time. RESULTS: Bimaxillary retrognathism, progressive maxillary retrognathism, and increasing lower anterior face height with downward and backward growth rotation of the mandible in the UCLP group were seen. Unlike the hard-tissue face height ratio, their soft-tissue face height ratio was not affected. The upper lips in the UCLP group were shorter by 1.81 mm at T2 (P <0.001) and by 1.16 mm at T3 (P = 0.018), whereas their lower lips were 2.21 mm longer at T3 (P = 0.003). A reduced upper lip to lower lip length ratio at T2 and T3 (P <0.001) resulted. Their upper lips were relatively retruded by 1.44 mm at T1, 1.66 mm at T2, and 1.86 mm at T3 (all, P <0.001), and their lower lips were relatively protruded by 1.07 mm at T1 (P = 0.003), 1.40 mm at T2 (P <0.001), and 1.62 mm at T3 (P <0.001). Nose depths in the UCLP group were shallower by at least 1 mm from T1 to T3, and columellar length was shorter by almost 2 mm (all, P <0.001). Their columellae and nose tips rotated downward with growth, with the most significant rotations experienced from T2 to T3, and progressive reductions in their soft-tissue profile convexity were seen from T1 to T3 (P <0.001). CONCLUSIONS: Key attributes of the imbalance in the soft-tissue profile in children with repaired UCLP were identified in the lip and nose regions. Although many profile differences were visible as early as 7 years of age, they became more apparent by 11 years of age and increased in severity thereafter. The short upper lip combined with a long lower lip resulted in the characteristic lip length imbalance, whereas the progressively retruding upper lip and protruding lower lip led to developing a step relationship in the sagittal lip profile during the adolescent growth period. Their columellae and nose tips rotated downward during this time.

Closed genomes of four multidrug resistance Salmonella enterica serotype Infantis isolated in Costa Rica.

Here, we report the complete genome of four S. enterica Infantis isolated in Costa Rica from human, poultry rinse, and raw chicken meat from 2017 to 2019. All genomes belonged to ST32 and carried a 310-kb plasmid with many antimicrobial resistance genes including the bla CTX-M65 gene.

Rapid differentiation of nucleotide phosphoramidate diastereomers by electrospray ionization tandem mass spectrometry.

RATIONALE: Nucleotide phosphoramidates are prodrugs which effectively deliver the active nucleotide to target tissues. It was shown that the individual phosphoramidate diastereomers have different antiviral activity, although the active nucleotide is the same. Therefore, a fast and simple analytical method is needed to characterize the individual diastereomeric phosphoramidate prodrugs. METHODS: Stock solutions of diastereomeric nucleotide phosphoramidate prodrugs, i.e., 5'-phosphate derivatives of the ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleotide, were made in 25% acetonitrile to achieve a final concentration of 10 µg/mL. The samples were studied using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: The MS/MS spectra of diastereomeric pairs showed substantial differences in the relative abundances of a characteristic ion in negative mode, which is proposed to be a cyclic phosphoramidate ion. Results were confirmed by the MS/MS spectrum of an analog without the NH proton and deuterium exchange experiment. Furthermore, the diastereomer-specific fragmentation behavior in negative ESI-MS was used to characterize a series of nucleotide phosphoramidates with different amino acid and aromatic substituents. CONCLUSIONS: An HPLC/MS/MS method was developed for the differentiation of the diastereomers of phosphoramidate prodrugs. In negative mode MS/MS spectra, the cyclic phosphoramidate ions yielded unambiguous distinction. This method presented a rapid and simple way for the characterization of nucleotide phosphoramidates.

ß-D-2'-α-F-2'-ß-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: a structure-activity relationship study.

The 3',5'-cyclic phosphate prodrug 9-[ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100 µm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.

A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma.

A rapid and stereospecific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the separation and determination of PSI-7851 diastereomers in human K2EDTA plasma has been developed. The analytical method involves direct protein precipitation with acetonitrile, followed by separation of the diastereomers on a Luna C18 column, positive mode electrospray ionization and selected reaction monitoring mode mass spectrometry detection. The mobile phase composition and pH were investigated for the resolution of the two diastereomers of PSI-7851. The optimized method showed good resolution (R(s) = 4.8) within short analysis time (approximately 8 min). The assay range was 5-2500 ng/mL for both diastereomers using a 1/x² weighted linear regression analysis for standard curve fitting. Replicate sample analysis indicated that intra- and inter-day accuracy and precision were within ±15.0%. The recovery of diastereomers from human plasma was greater than 85% and no significant matrix effect was observed. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies.

A Chemical Approach to Introduce 2,6-Diaminopurine and 2-Aminoadenine Conjugates into Oligonucleotides without Need for Protecting Groups.

We report a simple, postsynthetic strategy for synthesis of oligonucleotides containing 2,6-diaminopurine nucleotides and 2-aminoadenine conjugates using 2-fluoro-6-amino-adenosine. The strategy allows introduction of 2,6-diaminopurine and other 2-amino group-containing ligands. The strongly electronegative 2-fluoro deactivates 6-NH2 obviating the need for any protecting group on adenine, and simple aromatic nucleophilic substitution of fluorine makes reaction with aqueous NH3 or R-NH2 feasible at the 2-position.

Synthesis of diastereomerically pure nucleotide phosphoramidates.

Prodrugs of therapeutic nucleoside monophosphates masked as phosphoramidate derivatives have become an increasingly important class of antiviral drugs in pharmaceutical research for delivering nucleotides in vitro and in vivo. Conventionally, phosphoramidate derivatives are prepared as a mixture of two diastereomers. We report a class of stable phosphoramidating reagents containing an amino acid ester and two phenolic groups, one unsubstituted and the other with electron-withdrawing substituents. The reagents can be isolated as single diastereomers and reacted with the 5'-hydroxyl group of nucleosides through selective nucleophilic displacement of the substituted phenol to prepare single diastereomer phosphoramidate products. This method has been used to prepare the HCV clinical candidate PSI-7977 in high yield and high diastereomeric purity.

Stereoselective synthesis of PSI-352938: a ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methyl-3',5'-cyclic phosphate nucleotide prodrug for the treatment of HCV.

PSI-352938 is a novel 2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug currently under investigation for the treatment of hepatitis C virus (HCV) infection. PSI-352938 demonstrated superior characteristics in vitro that include broad genotype coverage, superior resistance profile, and high levels of active triphosphate in vivo in the liver compared to our first and second generation nucleoside inhibitors of this class. Consequently, PSI-352938 was selected for further development and an efficient and scalable synthesis was sought to support clinical development. We report an improved, diastereoselective synthesis of a key 1'-ß-nucleoside intermediate 13 via S(N)2 displacement of 1-α-bromo ribofuranose sugar 16 with the potassium salt of 6-chloro-2-amino purine and an efficient method to prepare cis-Rp cyclic phosphate (PSI-352938) in a highly stereoselective manner without any chromatographic purification. The 1-α-bromo sugar 16 was stereospecifically prepared from the corresponding 1-ß-lactol in high yield under mild bromination conditions using CBr(4)/PPh(3) (Appel reaction). The desired cis-Rp 3',5'-cyclic phosphate construction was accomplished using isopropyl phosphorodichloridate readily obtained from POCl(3) and isopropyl alcohol. The base combination of Et(3)N/NMI was identified as a key factor for producing PSI-352938 as the major (>95%) diastereomer (cis-Rp) in high yield after the final cyclization step. The current route described in this article was successfully used to produce PSI-352938 on multikilogram scale.

PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine monophosphate, is a potent and pan-genotype inhibitor of hepatitis C virus replication.

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

Synthesis and anti-HCV activity of 3',4'-oxetane nucleosides.

Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor, RG7128 has already established proof-of-concept in the clinic and is currently in phase IIb clinical trials. As part of our continuing efforts to discover novel anti-HCV agents, 3',4'-oxetane cytidine and adenosine nucleosides were prepared as inhibitors of HCV RNA replication. These nucleosides were shown not to be inhibitors of HCV as determined in a whole cell subgenomic replicon assay. However, 2'-mono/diflouro analogs, 4, 5, and 6 were readily phosphorylated to their monophosphate metabolites by deoxycytidine kinase and their triphosphate derivatives were shown to be inhibitors of HCV NS5B polymerase in vitro. Lack of anti-HCV activity in the replicon assay may be due to the inability of the monophosphates to be converted to their corresponding diphosphates.

2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs as inhibitors of HCV NS5B polymerase: discovery of PSI-352938.

A series of novel 2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs were synthesized and evaluated for their in vitro anti-HCV activity and safety. These prodrugs demonstrated a 10-100-fold greater potency than the parent nucleoside in a cell-based replicon assay due to higher cellular triphosphate levels. Our structure-activity relationship (SAR) studies provided compounds that gave high levels of active triphosphate in rat liver when administered orally to rats. These studies ultimately led to the selection of the clinical development candidate 24a (PSI-352938).

An efficient and diastereoselective synthesis of PSI-6130: a clinically efficacious inhibitor of HCV NS5B polymerase.

R7128 is the prodrug of 2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130), a potent and selective inhibitor of HCV NS5B polymerase. Currently, R7128 is in clinical trials for the treatment of HCV infection. To support clinical development efforts, we needed an efficient and scalable synthesis of PSI-6130. We describe an improved, diastereoselective synthetic route starting with protected d-glyceraldehyde. No chiral reagents or catalysts were used to produce the three new contiguous stereocenters. Introduction of fluorine at the C-2 tertiary carbon was accomplished in a highly regio- and stereoselective manner through nucleophilic substitution on a cyclic sulfate. Scale-limiting chromatographic purifications were eliminated through the use of crystalline intermediates.

BASC: an integrated bioinformatics system for Brassica research.

The BASC system provides tools for the integrated mining and browsing of genetic, genomic and phenotypic data. This public resource hosts information on Brassica species supporting the Multinational Brassica Genome Sequencing Project, and is based upon five distinct modules, ESTDB, Microarray, MarkerQTL, CMap and EnsEMBL. ESTDB hosts expressed gene sequences and related annotation derived from comparison with GenBank, UniRef and the genome sequence of Arabidopsis. The Microarray module hosts gene expression information related to genes annotated within ESTDB. MarkerQTL is the most complex module and integrates information on genetic markers, maps, individuals, genotypes and traits. Two further modules include an Arabidopsis EnsEMBL genome viewer and the CMap comparative genetic map viewer for the visualization and integration of genetic and genomic data. The database is accessible at http://bioinformatics.pbcbasc.latrobe.edu.au.

Comparing in vitro and in vivo activity of 2'-O-[2-(methylamino)-2-oxoethyl]- and 2'-O-methoxyethyl-modified antisense oligonucleotides.

A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.

An efficient and scalable synthesis of 2,6-diaminopurine riboside.

An efficient process to synthesize 2,6-diaminopurine riboside in high yield and quality is described. This inexpensive approach was scaled up to multi-hundred kilogram quantities for use in oligonucleotide therapeutics.

Permanent maxillary central incisor and first molar rotations in the mixed dentition in repaired complete unilateral cleft lip and palate and their relationship with absence of teeth in their vicinity.

OBJECTIVES: To describe qualitatively and quantitatively the directions and magnitudes of rotations of permanent maxillary central incisors and first molars in the mixed dentition in repaired complete unilateral cleft lip and palate (UCLP) and study their associations with absence of teeth in their vicinity. MATERIALS AND METHODS: Dental casts and orthodontic records taken prior to orthodontic preparation for alveolar bone grafting of 74 children with repaired UCLP (53 male, 21 female; aged 8.9 ± 1.0 years) were studied. Directions and magnitudes of permanent maxillary central incisor and first molar rotations were recorded. Tooth absence was confirmed from longitudinal radiographic records. Incisor and molar rotations were analyzed in relation to the absence of teeth in their vicinity. RESULTS: Distolabial rotation of the permanent maxillary central incisor was noted in 77.14% on the cleft side, while distopalatal rotation was noted in 82.19% on the noncleft side. Incisor rotation was greater when a permanent tooth was present distal to the cleft side central incisor, in the greater segment. The permanent maxillary first molar showed mesiopalatal rotation, which was greater on the cleft side and when there was absence of one or more teeth in the buccal segment. CONCLUSIONS: Presence and absence of teeth were associated with the severity of incisor and molar rotations in UCLP. Crowding of anterior teeth in the greater segment was associated with a greater magnitude of rotation of the cleft side permanent central incisor. Absence of one or more buccal segment teeth was associated with greater magnitude of rotation of the molar.

Incisor and molar overjet, arch contraction, and molar relationship in the mixed dentition in repaired complete unilateral cleft lip and palate: A qualitative and quantitative appraisal.

OBJECTIVE: To compare the mixed dentition incisor and molar overjet, severity of contraction of the dental arch, and the sagittal molar relationship on the cleft side vs the noncleft side in children with repaired complete unilateral cleft of the lip and palate (UCLP). MATERIALS AND METHODS: Orthodontic records taken prior to orthodontic preparation for alveolar bone grafting were screened to select study casts from patients with nonsyndromic repaired complete UCLP who did not have mandibular skeletal or dental asymmetry. The study sample comprised dental casts from 74 children aged 8.9 ± 1 years. Standardized digital photographs were acquired at 1:1 magnification. A coordinate system was developed using digital image-processing software (Photoshop CS4 and Adobe Illustrator). Incisor and molar overjet, Angle's classification, and arch contraction were recorded. Descriptive statistics, paired t-tests, and kappa statistics were used to compare the cleft and noncleft sides. RESULTS: A negative overjet of -1 to -5 mm was often present at the incisors, with greater frequency and magnitude on the cleft side. Class II molar relation was more frequent on the cleft side (61.1%) than on the noncleft side (47.2%). Significantly greater contraction of the cleft side deciduous canine and deciduous first molar was noted, while the difference was very minor at the first permanent molar. CONCLUSIONS: Cleft side maxillary arch contraction was most severe in the deciduous canine and first deciduous molar region and progressively less severe in the posterior region of the arch. A greater frequency and severity of negative overjet and Class II molar relationship was seen on the cleft side.

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 EC50 = 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. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Efficient large-scale synthesis of 5'-O-dimethoxytrityl-N4-benzoyl-5-methyl-2'-deoxycytidine.

An efficient process to synthesize 5'-O-dimethoxytrityl-N4-benzoyl-5-methyl-2'-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multikilogram quantities for routine use in oligonucleotide therapeutics.