Thomas W. Hudyma

Ligand-induced changes in hepatitis C virus NS5B polymerase structure.

Hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B) is required for viral replication. Crystal structures of the NS5B apoprotein show that the finger and thumb domains interact to encircle the active site, and that inhibitors defined by P495 resistance that bind to the thumb-finger interface displace the Δ1 finger loop and disrupt this structure. Since crystal structures may not reveal all of the conformations of a protein in solution we have developed an alternative method, using limited trypsin protease digestion, to investigate the impact of inhibitors as well as substrates on the movement of the Δ1 loop. This assay can be used to study NS5B under conditions that support enzymatic activity. In the absence of inhibitors, no specific region of NS5B was hypersensitive to trypsin, and no specific intermediate cleavage products were formed. Binding of P495-site inhibitors to NS5B induced specific trypsin hypersensitivity at lysine residues 50 and 51. Previously characterized inhibitors and mutant polymerases were used to link this specific trypsin hypersensitivity to movement of the Δ1 loop. Trypsin hypersensitivity identical to the inhibitor pattern was also induced by the binding of the RNA template. The addition of primer to the NS5B-template complex eliminated the hypersensitivity. The data are consistent with displacement of the Δ1 finger loop from the thumb by the binding of template, and reversal by the addition of primer or NTP. Our results complement inhibitor-enzyme co-crystal studies, and the assay provides a rapid and sensitive method to study dynamic changes in HCV NS5B polymerase conformation under conditions that support functional activity.

Syntheses and initial evaluation of a series of indolo-fused heterocyclic inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus.

Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety resulted in conformationally constrained heterocycles that possess multiple additional vectors for further exploration. The binding mode and pharmacokinetic (PK) properties of select examples, including: 13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo[2,1-d][1,4]benzodiazepine-10-carboxylic acid (7) (IC(50)=0.07 μM, %F=18), are reported.

Synthesis and release of doxorubicin from a cephalosporin based prodrug by a β-lactamase-immunoconjugate

Abstract A cephalosporin based prodrug of doxorubicin has been synthesized which effciently releases doxorubicin in the presence of an immunoconjugate consisting of a β-lactamase-MAb.

Synthesis and SAR studies of novel heteroaryl fused tetracyclic indole-diamide compounds: Potent allosteric inhibitors of the hepatitis C virus NS5B polymerase

Presented here are initial structure-activity relationship (SAR) studies on a series of novel heteroaryl fused tetracyclic indole-based inhibitors of the hepatitis C viral polymerase, NS5B. The introduction of alternative heterocyclic moieties into the indolo-fused inhibitor class significantly expands the reported SAR and resulted in the identification of pyridino analogs, typified by compounds 44 and 45 that displayed excellent potency against the NS5B polymerase of both HCV 1a and HCV 1b genotypes.

Synthesis and structure-activity relationship of C-3 quaternary ammonium cephalosporins exhibiting anti-MRSA activities

Abstract A series of cephalosporins bearing C-3 quaternary ammonium groups were prepared and evaluated for their anti-MRSA activity. They exhibit good to excellent in vitro activity (MICs = 1−8 μg/mL) against MRSA.

Synthesis and anti-MRSA Activity of Novel Cephalosporin Derivatives

Abstract Cephalosporin derivatives containing a unique combination of lipophilic C-7 sidechains and polar C-3 thiopyridinium groups were synthesized and found to exhibit potent anti-MRSA activity in vitro and in vivo. The optimum C-7 sidechains utilized were 2,5-dichlorophenylthioacetamido and 2,6-dichloropyrid-4-ylthioacetamido. The C-3 thiopyridinium rings were substituted at nitrogen with amino acid and pyruvic acid groups that were designed to confer aqueous solubility as required for IV formulation. This paper describes the characteristics of these novel cephalosporins and highlights synthetic methods developed to allow their practical, large-scale syntheses.