Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5'-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase.

There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5'-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5'-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5'-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5'-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104 This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

Asymmetric synthesis of host-directed inhibitors of myxoviruses.

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC(50) values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

S(N)2-type nucleophilic opening of beta-thiolactones (thietan-2-ones) as a source of thioacids for coupling reactions.

Beta-thiolactones monosubstituted in the 3-position by alkyl and carbamoyl groups undergo nucleophilic ring opening by arenethiolates through a process involving an S(N)2-type attack at the 4-position leading to 3-arylthiopropionates substituted in the 2-position. These thiocarboxylates can be trapped in situ by Mukaiyama's reagent or Sanger's reagent through a nucleophilic aromatic substitution process leading to highly activated thioesters that are then allowed to react further with primary or secondary amines leading, overall, to one-pot, three-component syntheses of 3-arylthiopropionamides carrying various substituents in the 2-position. Alternatively, the trapping combination of an electron deficient aryl halide and an amine may be replaced by a 2,4-dinitrobenzenesulfonamide, resulting in the formation of the same products overall with the incorporation of the latent amine in the sulfonamide into the final amide product. In another embodiment, the thiocarboxylate intermediate is allowed to react with a sulfonyl azide, resulting overall in N-arenesulfonyl 3-arylthiopropionamide derivatives.

Solid-phase synthesis of peptidyl thioacids employing a 9-fluorenylmethyl thioester-based linker in conjunction with Boc chemistry.

A method for the synthesis of peptidyl thioacids is described on the basis of the use of the N-[9-(thiomethyl)-9H-fluoren-2-yl]succinamic acid and cross-linked aminomethyl polystyrene resin. The method employs standard Boc chemistry SPPS techniques in conjunction with 9-fluorenylmethyloxycarbonyl protection of side-chain alcohols and amines and 9-fluorenylmethyl protection of side-chain acids and thiols. Cleavage from the resin is accomplished with piperidine, which also serves to remove the side-chain protection and avoids the HF conditions usually associated with the resin cleavage stage of Boc chemistry SPPS. The so-obtained thioacids are converted to simple thioesters in high yield by standard alkylation according to well-established methods.

Amino acid and peptide synthesis and functionalization by the reaction of thioacids with 2,4-dinitrobenzenesulfonamides.

Readily prepared amino thioacids react at room temperature in DMF in the presence of cesium carbonate with 2,4-dinitrobenzenesulfonamides to give amides. When the sulfonamide is derived from an amino acid the method results in peptide bond formation, whereas the use of carbohydrate derived sulfonamides gives neoglycoconjugates.

Synthesis and Metabolic Studies of Host Directed Inhibitors for Anti Viral Therapy.

Targeting host cell factors required for virus replication provides an alternative to targeting pathogen components and represents a promising approach to develop broad-spectrum antiviral therapeutics. High-throughput screening (HTS) identified two classes of inhibitors (2 and 3) with broad-spectrum antiviral activity against ortho- and paramyxoviruses including influenza A virus (IAV), measles virus (MeV), respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (HPIV3). Hit-to-lead optimization delivered inhibitor, 28a, with EC50 values of 0.88 and 0.81 µM against IAV strain WSN and MeV strain Edmonston, respectively. It was also found that compound 28a delivers good stability in human liver S9 fractions with a half-life of 165 minutes. These data establish 28a as a promising lead for antiviral therapy through a host-directed mechanism.