Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members. [Cancer Res 2007;67(24):11924-32]

Summary Secreted Hedgehog (Hh) ligands signal through the canonical receptor Patched (Ptch1). However, recent studies implicate three additional Hh-binding, cell surface proteins, Gas1, Cdo and Boc, as putative co-receptors for Hh ligands. A central question is to what degree these co-receptors function similarly and their collective requirement in Hh signal transduction. Here we provide evidence that Gas1, Cdo, and Boc, play overlapping and essential roles during Hh-mediated ventral neural patterning of the mammalian neural tube. Specifically, we demonstrate two important roles for these molecules: an early role in cell fate specification of multiple neural progenitors, and a later role in motor neuron progenitor maintenance. Most strikingly, genetic loss-of-function experiments indicate an obligatory requirement for Gas1, Cdo and Boc in Hh pathway activity in multiple tissues.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members.

The bis(heteroaryl)piperazine U-88204E is a potent inhibitor of HIV-1 reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the RNA- and DNA-dependent DNA polymerases of RT were carried out in order to determine whether the inhibitor interacts directly with the template:primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The experimental results were analyzed using steady-state or Briggs-Haldane kinetics, by assuming that the template:primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The results of the analysis show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template:primer and the dNTP binding sites. The potency of U-88204E on the RNA-directed DNA polymerase activity depends on the base composition of the template:primer. The Ki values for the poly(rC):(dG)10-directed reactions were at least 7 times lower than the ones for reactions directed by poly(rA):(dT)10. The inhibitor did not inhibit the RNase H function of HIV-1 RT nor did it impair the RNA-directed DNA polymerase activity of HIV-2 RT. These data thus demonstrate the unique specificity of U-88204E for HIV-1 RT.

Bisheteroarylpiperazines are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We describe a novel bisheteroarylpiperazine, U-90152 {1-(5-methanesulfonamido-IH-indol-2-yI-carbonyl)-4-[3-(1-methylethyl-amino)pyridinyllpiperazine}, which inhibited recombinant HIV-1 RT at a 50%Y inhibitory concentration (IC.) of 0.26 FLM (compared with IC50s of >440 ,uM for DNA polymerases at and B). U-90152 blocked the replication in peripheral blood lymphocytes of 25 primary HWV-i isolates, including variants that were highly resistant to 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine, with a mean 50%o effective dose of 0.066 + 0.137 pM. U-90152 had low cellular cytotoxicity, causing less than 8% reduction in peripheral blood lymphocyte viability at 100 ,uM. In experiments assessing inhibition of the spread of HIV-MB in cell cultures, U-90152 was much more effective than AZT. When approximately 500 HIV-1l1m-infected MT-4 cells were mixed 1:1,000 with uninfected cells, 3 ,uM AZT delayed the evidence of rapid viral growth for 7 days. In contrast, 3 ,uM U-90152 totally prevented the spread of HIV-i, and death and/or dilution of the original inoculum of infected cells prevented renewed viral growth after U-90152 was removed at day 24. The combination of U-90152 and AZT, each at 0.5 ,uM, also totally prevented viral spread. Finally, although the RT amino acid substitutions K103N (lysine 103 to asparagine) and Y181C (tyrosine 181 to cysteine), which confer cross-resistance to several nonnucleoside inhibitors, also decrease the potency of U-90152, this drug retains significant activity against these mutant RTs in vitro (IC50s, approximately 8 ,uM).