We next focused our design building into the
We next focused our design building into the ribose binding site, which had not been yet utilized in this effort. Compounds bearing a series of five-membered aromatic heterocycles (compound , ) showed reduced inhibition of the Gram-positive isozymes compared with compound , but a 20-fold improved potency for inhibition of a Gram-negative isozyme which was of more interest as such activity is more difficult and is where there is a larger medical need. Inhibition of bacterial growth was less potent and solubility was lower compared with compound . The loss in cellular activity could be attributed to weaker penetration into the silibinin or more efficient efflux by pump systems. The ligand efficiency (LE) for Sau was maintained through this effort, there was a loss in ligand lipophilicity Efficiency (LLE) which is not uncommon as inhibitors are built up. Additional analogs to better understand the impact of properties and cellular activity are in progress. Compounds were also profiled for additional Gram-negative antimicrobial activity in (wild type and pump mutant strains), , and . No activity was observed in the wild type stains, but activity was seen in the pump mutant strain for compound , similar to what was observed for .
Protein interactions for the active site with compound are illustrated below in two views, a, based on the 3D structure of the active site and then graphically as a MOE diagram. The inhibitor is in general ligand efficient and maintains multiple interactions with the active site: Leu117, Glu114, Lys291. In addition the aromatic core π-stacks with the Tyr 226 residue. The -butyl in this case efficiently fills the flexible lipophilic pocket and the pyrrole fills the entry to the ribose pocket. The ribose pocket also provides opportunities for additional hydrogen bond interactions and improvements to physical properties such as greater solubility and lower plasma binding.
The general synthetic route to analogs such as compound is shown below, . A condensation of the -butyl amidine with the ethylmalonate provides the right hand side of the core. The pyrimidine is then extended through simultaneous conversion of the phenolic groups to chlorides and the addition of an aldehyde. One of the chlorides is then converted to an aniline by treatment with 2M ammonia followed by Suzuki coupling to bring in the pyrrole. Condensation with cyanoacetamide in piperidine results in the protected product. The Boc group is then removed upon treatment with HCl in dioxane.
Introduction DNA non-homologous end-joining (NHEJ) is the major mechanism for the rejoining of DNA double-strand breaks (DSBs) in mammalian cells . Five proteins have been identified that function in NHEJ. The heterodimeric protein, Ku, binds to double-stranded DNA ends, recruits and activates the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs. Together this complex constitutes the DNA-dependent protein kinase (DNA-PK). DNA-PK then recruits Xrcc4 and DNA ligase IV, which tightly co-associate. DNA ligase IV carries out the final rejoining step of NHEJ. In addition to playing a major role in the rejoining of radiation and endogenously induced DSBs, NHEJ also effects the rejoining step during V(D)J recombination . Although Xrcc4 and DNA ligase IV are essential in mice, LIG4 syndrome has been identified as a human disorder conferred by hypomorphic mutations in DNA ligase IV , , , . The disorder is associated with clinical radiosensitivity and immunodeficiency, consistent with the known functions of DNA ligase IV. LIG4 syndrome patients also display developmental delay and microcephaly suggesting that DNA ligase IV plays an important role during development.