Alfredo Velena

Novel HDAC Inhibitors with Radiosensitizing Properties

Abstract Jung, M., Velena, A., Chen, B., Petukhov, P. A., Kozikowski, A. P. and Dritschilo, A. Novel HDAC Inhibitors with Radiosensitizing Properties. Radiat. Res. 163, 488–493 (2005). The members of the histone deacetylase (HDAC) family play important roles in various cellular processes, including transcriptional regulation, cell proliferation, differentiation and apoptosis. Inhibitors of histone deacetylases are emerging as an important new class of chemotherapeutic agents. As such, identifying stable and potent chemical HDAC inhibitory compounds is an important focus for translational research. Here we report the results of a rational drug design of novel HDAC inhibitors with potential for sensitizing cancer cells to radiation therapy. Over 60 HDAC inhibitor analogues incorporating a urea backbone and the hydroxamic acid end moiety were designed and screened. Six were found to confer 50% inhibition of HDAC enzyme activity at nanomolar concentrations. These candidate HDAC inhibitors inhibited cell proliferation at the ranges of IC50 10–50 μM in various cancer cells, including prostate (PC-3), breast (MCF-7) and head and neck squamous carcinoma (SQ-20B). Furthermore, radiation clonogenic survival assays revealed that these compounds possess radiosensitizing properties that are cell type-specific. The data support the further investigation of these HDAC inhibitors for use as sensitizing agents with potential for clinical application.

Histone Deacetylase Cytoplasmic Trapping by a Novel Fluorescent HDAC Inhibitor

Inhibitors of histone deacetylases (HDAC) are an important emerging class of drugs for the treatment of cancers. HDAC inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combinations with chemotherapies and radiation therapy. Although these drugs have important effects on cancer cell growth and functions, the mechanisms underlying HDAC inhibitor activities remain to be fully defined. By using rational drug design, compound 2, a fluorescent class II HDAC targeting inhibitor, was synthesized and observed to accumulate in the cytoplasmic compartments of treated cells, but not in the nuclei. Furthermore, immunostaining of inhibitor exposed cells for HDAC4 showed accumulation of this enzyme in the cytoplasmic compartment with concomitant increased acetylation of tubulin and nuclear histones. These observations support a mechanism by which nuclear histone acetylation is increased as a result of HDAC4 trapping and sequestration in the cytoplasm after binding to compound 2. The HDAC inhibitor offers potential as a novel theranostic agent, combining diagnostic and therapeutic properties in the same molecule. Mol Cancer Ther; 10(9); 1591–9. ©2011 AACR.

ADAMANTANYL-HISTONE DEACETYLASE INHIBITOR H6CAHA EXHIBITS FAVORABLE PHARMACOKINETICS AND AUGMENTS PROSTATE CANCER RADIATION SENSITIVITY

PURPOSE To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to γ-radiation in vitro and in vivo. METHODS AND MATERIALS H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice. RESULTS H6CAHA demonstrated good solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 ± 0.91 μM × h, and its half-life was 11.17 ± 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of γH2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with γ-radiation (10 × 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days. CONCLUSION These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation.

Pharmacokinetics-pharmacodynamics and antitumor activity of mercaptoacetamide-based histone deacetylase inhibitors.

Structurally diverse histone deacetylase inhibitors (HDACI) have emerged as chemotherapeutic agents. Here, we report the first mercaptoacetamide HDACIs (coded 6MAQH and 5MABMA) for use in treatment against prostate cancer cells in vitro and in vivo and correlate their plasma pharmacokinetics and tissue-pharmacodynamics with tumor sensitivity. HDACIs were assessed for in vitro microsomal stability and growth inhibition against prostate cancer and nonmalignant cells. Antitumor activity was determined following i.p. administration of 6MAQH and 5MABMA (0.5 and 5 mg/Kg) using mice bearing PC3 tumor xenografts (n = 10). The plasma pharmacokinetics of 6MAQH and 5MABMA and their effects on the acetylation of histone H4 in tissues were determined in athymic mice. Both HDACIs significantly inhibited the growth of cancer cells while exerting limited effect on nonmalignant cells. They exhibited stability in human, dog, and rat microsomes [t1/2 (min) = 83, 72, and 66 for 6MAQH and 68, 43, and 70 for 5MABMA, respectively]. Both HDACIs (0.5 mg/Kg) led to tumor regression (P < 0.01), which was sustained for at least 60 days. In vivo data show favorable plasma pharmacokinetics with the area under the curve of 4.97 ± 0.6 μmol/L × h for 6MAQH and 4.23 ± 0.43 μmol/L × h for 5MABMA. The clearance rates for 6MAQH and 5MABMA were 4.05 ± 0.15 and 4.87 ± 0.2 L/h, whereas the half-lives were 2.2 ± 0.33 and 1.98 ± 0.21 h, respectively. Both HDACIs markedly enhanced the acetylation of histone H4 within 30 minutes in tissues, including the brain, liver, and spleen. Taken together, the results provide a rationale for further investigation of these mercaptoacetamide HDACIs as potent anticancer agents. [Mol Cancer Ther 2009;8(10):2844–51]

Abstract 4054: Development of dual function small molecules as therapeutic agents for cancer treatment

Inhibitors of histone deactylases (HDACs) are emerging as potent anti-cancer agents for treatment of patients with hematopoietic and solid tumors. However, most of these compounds exhibit limitations, including off-targets and toxicity. To improve the efficacy and target specificity of drugs, we developed the compound, SP-1-161 conferring HDAC inhibition activity and ataxia-telangiectasia mutated (ATM) activation. The purpose of this study is to develop a therapy that optimizes the protective effects of ATM activation in normal tissue with the sensitizing effects of HDAC inhibition in cancerous tissue into a single molecule. The compound SP-1-161 was rationally designed by optimizing ATM activation by the cap domain of a hydroximic acid, screened against a panel of Class I and Class II HDAC enzymes, and identified as a pan-HDAC inhibitor with nanomolar potency (IC 50 = 8 nM). Western analysis confirms that SP-1-161 increases acetylated histone H3/H4 and α-tubulin and ATM activation in MCF7 cells. Phosphorylated ATM was gradually increased within 1 - 4 hrs in a time-dependent manner. SP-1-161 was then tested in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines to determine its cytotoxicity and effect on radiation clonogenic survival in combination with graded radiation exposure. The data showed that the cytotoxicity values (IC 50 ) were low micro-molar ranges. Furthermore, SP-1-161 protected 184A1 cells (from D 0 = 1 Gy to D 0 = 1.4 Gy) while increasing sensitivity of MCF7 cells to IR (from D 0 =1.6 Gy to D 0 =1.12 Gy). Together, our results demonstrate that SP-1-161 is an unprecedented radio-chemo therapeutic agent for treatment of cancers while protecting normal cells. Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Development of dual function small molecules as therapeutic agents for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2017-4054

Abstract 2613: Assessment of pharmacokinetics-pharmacodynamics and antitumor activities of hydroxamate- and mercaptoacetamide-based histone deacetylase inhibitors

Structurally diverse histone deacetylase inhibitors (HDACIs) have emerged as chemotherapeutic agents and have shown to cause differentiation, cell cycle arrest and apoptosis. As an initial step in identifying a clinically applicable HDACI, we evaluated the in vitro and the in vivo properties of two mercaptoacetamide (6MAQH and 5MABMA) and a hydroxamate (H6CAHA)-based HDACIs: 1) The growth inhibitory activities of these inhibitors were evaluated and compared against a subset of prostate cancer and non-malignant epithelial cell lines. 2) The in vitro permeability, solubility and lipophilicity properties of the HDACIs were assessed. 3) In vivo, the antitumor activities were determined following i.p. administration of the inhibitors (0.4 mg/Kg) using mice bearing PC3 tumor xenografts (n = 10). 4) The plasma pharmacokinetics of the HDACIs and their effect on the acetylation of histone H4 in tissues were determined in nude mice. The data demonstrated that mercaptoacetamides preferentially inhibited the growth of cancer than nonmalignant cells, whereas the hydroxamate showed toxicity at higher concentrations in both cancer and normal cells. The mercaptoacetamides also conferred higher solubility under the acidic environment of the stomach (pH 1.2) and lower under the neutral of the small intestine (pH 7.4) than the hydroxamate. All the HDACIs exhibited high permeability as well as lipophilicity values. In vivo, HDACIs treatment led to tumor growth inhibition (p Citation 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 2613.