David G. Hangauer

Prevention of noise-induced hearing loss with Src-PTK inhibitors

Studies from our lab show that noise exposure initiates cell death by multiple pathways [Nicotera, T.M., Hu, B.H., Henderson, D., 2003. The caspase pathway in noise-induced apoptosis of the chinchilla cochlea. J. Assoc. Res. Otolaryngol. 4, 466-477] therefore, protection against noise may be most effective with a multifaceted approach. The Src protein tyrosine kinase (PTK) signaling cascade may be involved in both metabolic and mechanically induced initiation of apoptosis in sensory cells of the cochlea. The current study compares three Src-PTK inhibitors, KX1-004, KX1-005 and KX1-174 as potential protective drugs for NIHL. Chinchillas were used as subjects. A 30 microl drop of one of the Src inhibitors was placed on the round window membrane of the anesthetized chinchilla; the vehicle (DMSO and buffered saline) alone was placed on the other ear. After the drug application, the middle ear was sutured and the subjects were exposed to noise. Hearing was measured before and several times after the noise exposure and treatment using evoked responses. At 20 days post-exposure, the animals were anesthetized their cochleae extracted and cochleograms were constructed. All three Src inhibitors provided protection from a 4 h, 4 kHz octave band noise at 106 dB. The most effective drug, KX1-004 was further evaluated by repeating the exposure with different doses, as well as, substituting an impulse noise exposure. For all conditions, the results suggest a role for Src-PTK activation in noise-induced hearing loss (NIHL), and that therapeutic intervention with a Src-PTK inhibitor may offer a novel approach in the treatment of NIHL.

The design, synthesis and activity of non-ATP competitive inhibitors of pp60c-src tyrosine kinase. Part 1 : Hydroxynaphthalene derivatives

A series of hydroxynaphthalene pp60(c-src) non-peptide inhibitors was designed, using the crystal structure of the insulin receptor tyrosine kinase as a qualitative model, to target the peptide substrate binding site. Representative inhibitors were shown to bind non-competitively with respect to ATP.

KX-01, a novel Src kinase inhibitor directed toward the peptide substrate site, synergizes with tamoxifen in estrogen receptor α positive breast cancer

KX-01 is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward Src kinase. The present study was designed to evaluate the effects of KX-01 as a single agent and in combination with tamoxifen (TAM) on cell growth and apoptosis of ERα positive breast cancer in vitro and in vivo. Flow cytometry demonstrated that KX-01 induced cell cycle arrest in G2/M phase. Immunofluorescent staining for mitotic phase markers and TUNEL staining indicated that cells had arrested in the mitotic phase and mitotic arrested cells were undergoing apoptosis. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2, and Cdc25C that is required for progression past the G2/M checkpoint. Apoptosis resulted from activation of caspases 6, 7, 8, and 9. Combinational index analysis revealed that combinations of KX-01 with TAM resulted in synergistic growth inhibition of breast cancer cell lines. KX-01 combined with TAM resulted in decreased ERα phosphorylation at Src-regulated phosphorylation sites serines 118 and 167 that were associated with reduced ERα transcriptional activity. Orally administered KX-01 resulted in a dose dependent growth inhibition of MCF-7 tumor xenografts, and in combination with TAM exhibited synergistic growth inhibition. Immunohistochemical analysis revealed that combinational treatment reduced angiogenesis, and ERα signaling in tumors compared to either drug alone that may underlie the synergistic tumor growth inhibition. Combinations of KX-01 with endocrine therapy present a promising new strategy for clinical management of ERα positive breast cancer.

The design and synthesis of substituted biphenyl libraries

A novel scaffold system for the generation of diversity libraries has been designed which allows for rapid modification not only of functional groups, but their spatial arrangements as well. The biphenyl scaffold allows for display of three or four diverse functional groups in a wide variety of spatial arrangements depending on the substitution pattern selected. The libraries are generated by a combination of solution and solid-phase chemistries and are cleaved off the solid-support for screening.

Targeting Src and tubulin in mucinous ovarian carcinoma

Purpose: To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma. Experimental Design: The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models. Results: In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2–M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01–sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules. Conclusion: Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma. Clin Cancer Res; 19(23); 6532–43. ©2013 AACR.

Protection from impulse noise-induced hearing loss with novel Src-protein tyrosine kinase inhibitors

Apoptosis is a significant mechanism of cochlear hair cell loss from noise. Molecules that inhibit apoptotic intracellular signaling reduce cochlear damage and hearing loss from noise. The current study is an extension of a previous study of the protective value of Src-protein tyrosine kinase inhibitors against noise (Harris et al., 2005). The current study tested three Src-inhibitors: the indole-based KX1-141, the biaryl-based KX2-329, and the ATP-competitive KX2-328. Each of the three drugs was delivered into the chinchillas cochleae by allowing the solutions to diffuse across the round window membrane thirty minutes prior to exposure to impulse noise. Hearing thresholds were measured using auditory evoked responses from electrodes in the inferior colliculi. Ears treated with KX2-329 showed significantly lower threshold shifts and outer hair cell losses than the control group. The cochleae treated with KX1-141 and KX2-328 did not show statistically significant protection from the impulse noise. The finding of protection with KX2-329 demonstrates that a biaryl-based Src inhibitor has protective capacity against noise-induced hearing loss that is as good as that demonstrated by KX1-004, a Src inhibitor drug that has been studied extensively as an otoprotectant against noise, and suggests that KX2-329 could be useful for protection against noise.

C-10 epimerization of hydroazulen-7-onesby vinylogus activation

Abstract Incorporation of α-(phenylthio)-α,β-unsaturation into a cycloheptanone ring permits epimerization, without deconjugation, of an otherwise unactivated chiral center at the γ-position.

Abstract 2535: KX-01, a novel Src kinase inhibitor directed towards the peptide substrate site, induces robust apoptosis and synergizes with tamoxifen and chemotherapy in breast cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnNew therapeutic regimens are needed to increase efficacy and reduce resistance of endocrine therapy and chemotherapy in breast cancer. c-Src is an oncogenic non-receptor tyrosine kinase that is up-regulated in approximately half of all breast cancer. However the efficacy of existing multi-kinase src inhibitors in breast cancer has been limited. KX-01 (Kinex Pharmaceuticals) is a novel class of clinical non-ATP src inhibitor that targets the peptide binding site. In a panel of breast cancer cell lines, KX-01 resulted in dose dependent inhibition of growth and induction of apoptosis that was independent of p53 status, and was preceded by rapid inhibition of src activity. KX-01 induced apoptosis in MDA-MB-468 cells and BT-549 cell lines reported to be resistant to multi-kinase src inhibitors. KX-01 (50 nM, 6 hours) resulted in significant accumulation of MDA-MB-231 cells (ERα−/PR−/HER2/neu−) and MCF-7 cells (ERα+) in G2/M phase. Immunofluorescent staining for mitotic phase marker phospho-histone 3 indicated that cells had arrested in mitotic phase and many of the mitotic arrested cells were undergoing apoptosis (TUNEL), a novel cell death for a small molecule tyrosine kinase inhibitor. KX-01 induced nuclear accumulation of cyclin B1 and activation of CDK1, MPM2 and Cdc25C that is required for progression past the G2/M checkpoint. KX-01 resulted in cytochrome C release and activation of caspases 6, 7, 8 and 9. Combinations of KX-01 (5-75 nM) with tamoxifen (TAM), paclitaxel (PAC) or doxorubicin (DOX) resulted in synergistic growth inhibition of MCF-7 cells (KX-01 + TAM) and MDA-MB-231 cells (KX-01 + DOX or PAC). In addition, synergistic induction of apoptosis was achieved with combination of KX-01 with DOX, PAC or TAM. c-Src induces phosphorylation of ERα at serines 118 and 167, sites required for full ERα activity. KX-01 combined with TAM resulted in decreased serine 167 phosphorylation and reduced ERα transcriptional activity. In tumor xenografts, KX-01 resulted in a dose dependent inhibition of MDA-MB-231 and MCF-7 tumor growth (1, 2.5 or 5 mg/kg body weight, twice daily by oral gavage). KX-01 exhibited synergistic growth inhibition of MCF-7 tumor xenografts when combined with tamoxifen and caused regression of MDA-MB-231 tumor xenografts when combined with paclitaxel or doxorubicin. Immunohistochemical (IHC) analysis in MCF-7 xenografts revealed that KX-01 + TAM induced robust apoptosis and inhibited neovascularization. KX-01 reduced metastasis to bone (femur) and lung as measured by PCR for detection of human chromosome 17. Interestingly, KX-01 resulted in reexpression of ERα and E-cadherin in MDA-MB-231 xenografts. These data define KX-01 as a potent and unique src kinase inhibitor that synergizes with endocrine therapy and chemotherapy to induce robust cell death, tumor growth inhibition/regression and inhibit metastasis.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2535.

TOTAL SYNTHESIS OF PSEUDOGUAIANOLIDES—II: (±)-AROMATICIN††Dedicated to the memory of Prof. R. B. Woodward, whose artistic creativity and painstaking utilization of strategic planning has inspired and guided our endeavors in the organosynthetic domain.

(±)-Aromaticin (5), a helenanolide with five chiral centers, has been assembled from 2-methyl-1,3-cyclopentanedione in a linear synthesis characterized by high regio- and stereochemistry at all stages.