Microtubins: a novel class of small synthetic microtubule targeting drugs that inhibit cancer cell proliferation.

Microtubule targeting drugs like taxanes, vinca alkaloids, and epothilones are widely-used and effective chemotherapeutic agents that target the dynamic instability of microtubules and inhibit spindle functioning. However, these drugs have limitations associated with their production, solubility, efficacy and unwanted toxicities, thus driving the need to identify novel antimitotic drugs that can be used as anticancer agents. We have discovered and characterized the Microtubins (Microtubule inhibitors), a novel class of small synthetic compounds, which target tubulin to inhibit microtubule polymerization, arrest cancer cells predominantly in mitosis, activate the spindle assembly checkpoint and trigger an apoptotic cell death. Importantly, the Microtubins do not compete for the known vinca or colchicine binding sites. Additionally, through chemical synthesis and structure-activity relationship studies, we have determined that specific modifications to the Microtubin phenyl ring can activate or inhibit its bioactivity. Combined, these data define the Microtubins as a novel class of compounds that inhibit cancer cell proliferation by perturbing microtubule polymerization and they could be used to develop novel cancer therapeutics.

Increased hemoglobin-oxygen affinity ameliorates bleomycin-induced hypoxemia and pulmonary fibrosis.

Although exertional dyspnea and worsening hypoxia are hallmark clinical features of idiopathic pulmonary fibrosis (IPF), no drug currently available could treat them. GBT1118 is a novel orally bioavailable small molecule that binds to hemoglobin and produces a concentration-dependent left shift of the oxygen-hemoglobin dissociation curve with subsequent increase in hemoglobin-oxygen affinity and arterial oxygen loading. To assess whether pharmacological modification of hemoglobin-oxygen affinity could ameliorate hypoxemia associated with lung fibrosis, we evaluated GBT1118 in a bleomycin-induced mouse model of hypoxemia and fibrosis. After pulmonary fibrosis and hypoxemia were induced, GBT1118 was administered for eight consecutive days. Hypoxemia was determined by monitoring arterial oxygen saturation, while the severity of pulmonary fibrosis was assessed by histopathological evaluation and determination of collagen and leukocyte levels in bronchoalveolar lavage fluid. We found that hemoglobin modification by GBT1118 had strong antihypoxemic therapeutic effects with improved arterial oxygen saturation to near normal level. Moreover, GBT1118 treatment significantly attenuated bleomycin-induced lung fibrosis, collagen accumulation, body weight loss, and leukocyte infiltration. This study is the first to suggest the beneficial effects of hemoglobin modification in fibrotic lungs and offers a promising and novel therapeutic strategy for the treatment of hypoxemia associated with chronic fibrotic lung disorders in human, including IPF.

Virtual-Reality Simulator System for Double Interventional Cardiac Catheterization Using Fractional-Order Vascular Access Tracker and Haptic Force Producer.

This study proposes virtual-reality (VR) simulator system for double interventional cardiac catheterization (ICC) using fractional-order vascular access tracker and haptic force producer. An endoscope or a catheter for diagnosis and surgery of cardiovascular disease has been commonly used in minimally invasive surgery. It needs specific skills and experiences for young surgeons or postgraduate year (PGY) students to operate a Berman catheter and a pigtail catheter in the inside of the human body and requires avoiding damaging vessels. To improve the training in inserting catheters, a double-catheter mechanism is designed for the ICC procedures. A fractional-order vascular access tracker is used to trace the senior surgeons' consoled trajectories and transmit the frictional feedback and visual feedback during the insertion of catheters. Based on the clinical feeling through the aortic arch, vein into the ventricle, or tortuous blood vessels, haptic force producer is used to mock the elasticity of the vessel wall using voice coil motors (VCMs). The VR establishment with surgeons' consoled vessel trajectories and hand feeling is achieved, and the experimental results show the effectiveness for the double ICC procedures.

Large-scale chemical similarity networks for target profiling of compounds identified in cell-based chemical screens.

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60-70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/).

Potential Association of Urinary N7-(2-Carbamoyl-2-hydroxyethyl) Guanine with Dietary Acrylamide Intake of Smokers and Nonsmokers.

Acrylamide (AA), a rodent carcinogen, is widely used in industry and present in cigarette smoke as well as in foods processed at high temperatures. The metabolic activation of AA to glycidamide (GA) could be critical for AA carcinogenicity since GA causes DNA adduct formation in vivo. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct of AA, is subjected to spontaneous and enzymatic depurination and excreted through urine. Urinary N7-GAG analysis can confirm AA genotoxicity and identify active species of AA metabolites in humans, thereby serving as a risk-associated biomarker for molecular epidemiology studies. This study aimed to develop an isotope-dilution solid-phase extraction liquid chromatography tandem mass spectrometry method to comparatively analyze urinary N7-GAG levels in nonsmokers and smokers. Urinary N-acetyl-S-(propionamide)-cysteine (AAMA), a metabolite of AA, was also analyzed as a biomarker for current AA exposure. Urinary N7-GAG was quantified by monitoring m/z 239 → 152 for N7-GAG and m/z 242 → 152 for (13)C3-labeled N7-GAG under positive electron spray ionization and multiple reaction mode. The median urinary N7-GAG level was 0.93 µg/g creatinine in nonsmokers (n = 33) and 1.41 µg/g creatinine in smokers (n = 30). Multiple linear regression analysis of data revealed that N7-GAG levels were only significantly associated with AAMA levels. These results demonstrate that urinary N7-GAG of nonsmokers and smokers is significantly associated with a very low level of dietary AA intake, assessed by analyzing urinary AAMA.

Design, synthesis, and biological evaluation of stable colchicine binding site tubulin inhibitors as potential anticancer agents.

To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure-activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring.

Spontaneous bilateral perirenal hemorrhage following prolonged fever: an uncommon presentation of polyarteritis nodosa.

A 68-year-old man presented with a spontaneous bilateral perirenal hemorrhage following a 2-month fever of unknown origin. A renal biopsy for a pathologic diagnosis seemed very risky because of the patient's bilateral perirenal hemorrhage. Therefore, we diagnosed polyarteritis nodosa using an abdominal computed tomography scan, a renal angiogram, and American College of Rheumatology criteria. The patient's multiple symptoms then responded well to the prescribed immunosuppressive regimen. This case is an uncommon presentation of polyarteritis nodosa with fever of unknown origin before a spontaneous bilateral perirenal hemorrhage.

Role and pharmacologic significance of cytochrome P-450 2D6 in oxidative metabolism of toremifene and tamoxifen.

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P-450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N-desmethyl (NDM) and 4-hydroxy (4-OH) metabolites during steady-state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4-OH TAM and 4-OH-NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4-OH-NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4-hydroxylation of TOR by CYP2D6. The 4-OH and 4-OH-NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30-fold greater affinity were 35- to 187-fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360-fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4-OH TAM and 4-OH-NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6-inhibiting medications.

Orally bioavailable tubulin antagonists for paclitaxel-refractory cancer.

PURPOSE: To evaluate the efficacy and oral activity of two promising indoles, (2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound II] and (2-(1H-indol-5-ylamino)-thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound IAT], in paclitaxel- and docetaxel-resistant tumor models in vitro and in vivo. METHODS: The in vitro drug-like properties, including potency, solubility, metabolic stability, and drug-drug interactions were examined for our two active compounds. An in vivo pharmacokinetic study and antitumor efficacy study were also completed to compare their efficacy with docetaxel. RESULTS: Both compounds bound to the colchicine-binding site on tubulin, and inhibited tubulin polymerization, resulting in highly potent cytotoxic activity in vitro. While the potency of paclitaxel and docetaxel was compromised in a multidrug-resistant cell line that overexpresses P-glycoprotein, the potency of compounds II and IAT was maintained. Both compounds had favorable drug-like properties, and acceptable oral bioavailability (21-50 %) in mice, rats, and dogs. Tumor growth inhibition of greater than 100 % was achieved when immunodeficient mice with rapidly growing paclitaxel-resistant prostate cancer cells were treated orally at doses of 3-30 mg/kg of II or IAT. CONCLUSIONS: These studies highlight the potent and broad anticancer activity of two orally bioavailable compounds, offering significant pharmacologic advantage over existing drugs of this class for multidrug resistant or taxane-refractory cancers.

Novel tubulin polymerization inhibitors overcome multidrug resistance and reduce melanoma lung metastasis.

PURPOSE: To evaluate abilities of 2-aryl-4-benzoyl-imidazoles (ABI) to overcome multidrug resistance (MDR), define their cellular target, and assess in vivo antimelanoma efficacy. METHODS: MDR cell lines that overexpressed P-glycoprotein, MDR-associated proteins, and breast cancer resistance protein were used to evaluate ABI ability to overcome MDR. Cell cycle analysis, molecular modeling, and microtubule imaging were used to define ABI cellular target. SHO mice bearing A375 human melanoma xenograft were used to evaluate ABI in vivo antitumor activity. B16-F10/C57BL mouse melanoma lung metastasis model was used to test ABI efficacy to inhibit tumor lung metastasis. RESULTS: ABIs showed similar potency to MDR cells compared to matching parent cells. ABIs were identified to target tubulin on the colchicine binding site. After 31 days of treatment, ABI-288 dosed at 25 mg/kg inhibited melanoma tumor growth by 69%; dacarbazine at 60 mg/kg inhibited growth by 52%. ABI-274 dosed at 25 mg/kg showed better lung metastasis inhibition than dacarbazine at 60 mg/kg. CONCLUSIONS: This new class of antimitotic compounds can overcome several clinically important drug resistant mechanisms in vitro and are effective in inhibiting melanoma lung metastasis in vivo, supporting their further development.

Pharmacokinetic optimization of 4-substituted methoxybenzoyl-aryl-thiazole and 2-aryl-4-benzoyl-imidazole for improving oral bioavailability.

Microtubules are critical components of the cytoskeleton. Perturbing their function arrests the growth of a broad spectrum of cancer cell lines, making microtubules an excellent and established target for chemotherapy. All of the U.S. Food and Drug Administration-approved antitubulin agents bind to paclitaxel or vinblastine binding sites in tubulin. Because of the complexity of their structures, it is difficult to structurally modify the vinca alkaloids and taxanes and develop orally bioavailable agents. Antitubulin agents that target the colchicine-binding site in tubulin may provide a better opportunity to be developed for oral use because of their relatively simple structures and physicochemical properties. A potent antitubulin agent, 4-(3,4,5-trimethoxybenzoyl)-2-phenyl-thiazole (SMART-H), binding to the colchicine-binding site, was discovered in our laboratory. However, the bioavailability of SMART-H was low because of its poor solubility. Structural modification of SMART-H led to the development of 2-aryl-4-benzoyl-imidazole analog (ABI-274), with improved bioavailability and potency but still considerable first-pass metabolism. A chlorine derivative (ABI-286), replacing the methyl site of ABI-274, resulted in 1.5-fold higher metabolic stability in vitro and 1.8-fold lower clearance in rats in vivo, indicating that metabolic stability of ABI-274 can be extended by blocking benzylic hydroxylation. Overall, ABI-274 and ABI-286 provided 2.4- and 5.5-fold increases in exposure (area under the curve) after oral dosing in rats compared with SMART-H. Most importantly, the structural modifications did not compromise potency. ABI-286 exhibited moderate clearance, moderate volume of distribution, and acceptable oral bioavailability. This study provided the first evidence that ABI-286 may be the first member of a new class of orally bioavailable antitubulin agents.

Synthesis and antiproliferative activity of novel 2-aryl-4-benzoyl-imidazole derivatives targeting tubulin polymerization.

We previously reported the discovery of 2-aryl-4-benzoyl-imidazoles (ABI-I) as potent antiproliferative agents for melanoma. To further understand the structural requirements for the potency of ABI analogs, gain insight in the structure-activity relationships (SAR), and investigate metabolic stability for these compounds, we report extensive SAR studies on the ABI-I scaffold. Compared with the previous set of ABI-I analogs, the newly synthesized ABI-II analogs have lower potency in general, but some of the new analogs have comparable potency to the most active compounds in the previous set when tested in two melanoma and four prostate cancer cell lines. These SAR studies indicated that the antiproliferative activity was very sensitive to subtle changes in the ligand. Tested compounds 3ab and 8a are equally active against highly paclitaxel resistant cancer cell lines and their parental cell lines, indicating that drugs developed based on ABI-I analogs may have therapeutic advantages over paclitaxel in treating resistant tumors. Metabolic stability studies of compound 3ab revealed that N-methyl imidazole failed to extend stability as literature reported because de-methylation was found as the major metabolic pathway in rat and mouse liver microsomes. However, this sheds light on the possibility for many modifications on imidazole ring for further lead optimization since the modification on imidazole, such as compound 3ab, did not impact the potency.

Design, synthesis, and SAR studies of 4-substituted methoxylbenzoyl-aryl-thiazoles analogues as potent and orally bioavailable anticancer agents.

In a continued effort to improve upon the previously published 4-substituted methoxybenzoyl-aryl-thiazole (SMART) template, we explored chemodiverse "B" rings and "B" to "C" ring linkage. Further, to overcome the poor aqueous solubility of this series of agents, we introduced polar and ionizable hydrophilic groups to obtain water-soluble compounds. For instance, based on in vivo pharmacokinetic (PK) studies, an orally bioavailable phenyl-amino-thiazole (PAT) template was designed and synthesized in which an amino linkage was inserted between "A" and "B" rings of compound 1. The PAT template maintained nanomolar (nM) range potency against cancer cell lines via inhibiting tubulin polymerization and was not susceptible to P-glycoprotein mediated multidrug resistance in vitro, and markedly improved solubility and bioavailability compared with the SMART template (45a-c (PAT) vs 1 (SMART)).

Biotransformation of a novel antimitotic agent, I-387, by mouse, rat, dog, monkey, and human liver microsomes and in vivo pharmacokinetics in mice.

3-(1H-Indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel indole compound with antitubulin action and potent antitumor activity in various preclinical models. I-387 avoids drug resistance mediated by P-glycoprotein and showed less neurotoxicity than vinca alkaloids during in vivo studies. We examined the pharmacokinetics and metabolism of I-387 in mice as a component of our preclinical development of this compound and continued interest in structure-activity relationships for antitubulin agents. After a 1 mg/kg intravenous dose, noncompartmental pharmacokinetic analysis in plasma showed that clearance (CL), volume of distribution at steady state (Vd(ss)), and terminal half-life (t(1/2)) of I-387 were 27 ml per min/kg, 5.3 l/kg, and 7 h, respectively. In the in vitro metabolic stability study, half-lives of I-387 were between 10 and 54 min by mouse, rat, dog, monkey, and human liver microsomes in the presence of NADPH, demonstrating interspecies variability. I-387 was most stable in rat liver microsomes and degraded quickly in monkey liver microsomes. Liquid chromatography-tandem mass spectrometry was used to identify phase I metabolites. Hydroxylation, reduction of a ketone group, and O-demethylation were the major metabolites formed by the liver microsomes of the five species. The carbonyl group of I-387 was reduced and identified as the most labile site in human liver microsomes. The results of these drug metabolism and pharmacokinetic studies provide the foundation for future structural modification of this pharmacophore to improve stability of drugs with potent anticancer effects in cancer patients.

Biological activity of 4-substituted methoxybenzoyl-aryl-thiazole: an active microtubule inhibitor.

Formation of microtubules is a dynamic process that involves polymerization and depolymerization of αß-tubulin heterodimers. Drugs that enhance or inhibit tubulin polymerization can destroy this dynamic process, arresting cells in the G(2)/M phase of the cell cycle. Although drugs that target tubulin generally demonstrate cytotoxic potency in the subnanomolar range, resistance due to drug efflux is a common phenomenon among the antitubulin agents. We recently reported a class of 4-substituted methoxybenzoyl-aryl-thiazoles (SMART) that exhibited great in vitro potency and broad spectrum cellular cytotoxicity. Evaluation of the in vitro and in vivo anticancer activities of 3 SMART compounds, SMART-H (H), SMART-F (F), and SMART-OH (OH), with varying substituents at the 4-position of aryl ring, demonstrated that they bind potently to the colchicine-binding site in tubulin, inhibit tubulin polymerization, arrest cancer cells in G(2)/M phase of the cell cycle, and induce their apoptosis. The SMART compounds also equipotently inhibit the growth of parental and MDR-overexpressing cells in vitro, indicating that they can overcome multidrug resistance. In vivo antitumor efficacy studies in human prostate (PC-3) and melanoma (A375) cancer xenograft models demonstrated that SMART-H and SMART-F treatments resulted in %T/C values ranging from 4% to 30%. In addition, in vivo SMART-H treatment for 21 days at the higher dose (15 mg/kg) failed to produce any apparent neurotoxicity. These studies provide the first in vivo evidence and proof-of-concept that SMART compounds are similarly efficacious to currently FDA approved antitubulin drugs for cancer treatment, but they can circumvent P-glycoprotein-mediated drug resistance.

Discovery of novel 2-aryl-4-benzoyl-imidazoles targeting the colchicines binding site in tubulin as potential anticancer agents.

A series of 2-aryl-4-benzoyl-imidazoles (ABI) was synthesized as a result of structural modifications based on the previous set of 2-aryl-imidazole-4-carboxylic amide (AICA) derivatives and 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART). The average IC(50) of the most active compound (5da) was 15.7 nM. ABI analogues have substantially improved aqueous solubility (48.9 µg/mL for 5ga vs 0.909 µg/mL for SMART-1, 0.137 µg/mL for paclitaxel, and 1.04 µg/mL for combretastatin A4). Mechanism of action studies indicate that the anticancer activity of ABI analogues is through inhibition of tubulin polymerization by interacting with the colchicine binding site. Unlike paclitaxel and colchicine, the ABI compounds were equally potent against multidrug resistant cancer cells and the sensitive parental melanoma cancer cells. In vivo results indicated that 5cb was more effective than DTIC in inhibiting melanoma xenograph tumor growth. Our results suggest that the novel ABI compounds may be developed to effectively treat drug-resistant tumors.

I-387, a novel antimitotic indole, displays a potent in vitro and in vivo antitumor activity with less neurotoxicity.

(3-(1H-indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel synthetic compound that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models. I-387 inhibited the in vitro growth of several human cancer cell lines with IC50 values in the range of 15 to 39 nmol/L. Nanomolar concentrations of the compound induced apoptosis and caused phosphorylation of the antiapoptotic protein Bcl-2. I-387 induced a strong and concentration-dependent G2-M arrest in PC-3 cells by constitutive activation of Cdc2/cyclin B1 complex and destabilized polymerization of purified tubulin in vitro by binding to the colchicine-binding site. In vivo, I-387 treatment effectively inhibited tumor growth in mice bearing PC-3 tumor xenografts. In vitro studies of nerve growth factor-dependent neurite outgrowth in PC12 pheochromocytoma cells and in vivo studies of mouse behavior showed that I-387 was less neurotoxic than vinblastine and vincristine, tubulin destabilizers with known neurotoxicity. Interestingly, multidrug-resistant cell lines that overexpressed P-glycoprotein (P-gp), multidrug resistance-associated proteins, and breast cancer resistance protein were rendered resistant to docetaxel, vinblastine, SN-38, and doxorubicin, but not to I-387. I-387 dosed at 10 mg/kg was equally effective with 76% tumor growth inhibition in xenograft models using MES-SA uterine sarcoma cells and MES-SA/DX5 cells overexpressing P-gp. In contrast, docetaxel and vinblastine were not effective in MES-SA/DX5 xenograft models. The potent in vitro and in vivo antitumor activity of I-387 suggests that it may represent a new antimitotic agent for management of various malignancies, particularly for patients with drug-resistant cancer.

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin.

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non-radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine-, vinblastine- and paclitaxel-binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin-binding site of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART-H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART-H specifically and reversibly bound only to the colchicine-binding site, but not to vinblastine- or paclitaxel sites. This new non-radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors.

2-Arylthiazolidine-4-carboxylic acid amides (ATCAA) target dual pathways in cancer cells: 5'-AMP-activated protein kinase (AMPK)/mTOR and PI3K/Akt/mTOR pathways.

Phosphatidylinositol-3-kinase (PI3K)/Akt and 5'-AMP-activated protein kinase (AMPK) are attractive targets for anti-cancer drug development. Inhibition of Akt or activation of AMPK is cytotoxic to human cancer cells in vitro and in vivo. We previously demonstrated that 2-arylthiazolidine-4-carboxylic acid amides (ATCAA) are effective cytotoxic agents in prostate and melanoma cancer cell lines, with IC(50) values in the low/sub micromolar range. Using in vitro and in vivo studies, we further characterized the anti-cancer efficacy and mechanism of action of ATCAA-10, a potent lead. ATCAA-10 exhibited equal potency on both MES/SA and P-glycoprotein over-expressing multidrug resistant MES/SA/Dx5 cells, suggesting that ATCAA-10 may overcome multiple drug resistance. Cell-free kinase binding assays excluded the direct binding of ATCAA-10 to several kinases, including IGF-1R, EGFR, FGFR and PDGFR. However, in A549 and HeLa cells, ATCAA-10 effectively dephosphorylated Akt, with concomitant phosphorylation of AMPK. Determination of intracellular ATP and AMP concentrations revealed that ATCAA-10 activated AMPK by altering the intracellular AMP/ATP ratio. ATCAA-10 exhibited favorable pharmacokinetic properties in both mice and rats, including low clearance, low hepatic extraction rate, moderate volume of distribution and long half-life. In addition, ATCAA-10 inhibited A549 tumor xenograft growth with 46% tumor growth inhibition (TGI) at 20 mg/kg dose. Taken together; these results suggest that ATCAA-10 modulates the activity of two signaling pathways, PI3K/AKT/mTOR and AMPK/mTOR, resulting in the inhibition of cancer cell growth.

Drug metabolism and pharmacokinetics of 4-substituted methoxybenzoyl-aryl-thiazoles.

Tubulins are some of the oldest and most extensively studied therapeutic targets for cancer. Although many tubulin polymerizing and depolymerizing agents are known, the search for improved agents continues. We screened a class of tubulins targeting small molecules and identified 4-(3,4,5-trimethoxybenzoyl)-2-phenyl-thiazole (SMART-H) as our lead compound. SMART-H inhibited the proliferation of a variety of cancer cells in vitro, at subnanomolar IC(50), and in vivo, in nude mice xenografts, with near 100% tumor growth inhibition. Metabolic stability studies with SMART-H in liver microsomes of four species (mouse, rat, dog, and human) revealed half-lives between <5 and 30 min, demonstrating an interspecies variability. The clearance predicted based on in vitro data correlated well with in vivo clearance obtained from mouse, rat, and dog in vivo pharmacokinetic studies. SMART-H underwent four major metabolic processes, including ketone reduction, demethylation, combination of ketone reduction and demethylation, and hydroxylation in human liver microsomes. Metabolite identification studies revealed that the ketone and the methoxy groups of SMART-H were most labile and that ketone reduction was the dominant metabolism reaction in human liver microsomes. We designed and tested four derivatives of SMART-H to improve the metabolic stability. The oxime and hydrazide derivatives, replacing the ketone site, demonstrated a 2- to 3-fold improved half-life in human liver microsomes, indicating that our prediction regarding metabolic stability of SMART-H can be extended by blocking ketone reduction. These studies led us to the next generation of SMART compounds with greater metabolic stability and higher pharmacologic potency.