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1.  Modulation of Radiation Injury Response in Retinal Endothelial Cells by Quinic Acid Derivative KZ-41 Involves p38 MAPK 
PLoS ONE  2014;9(6):e100210.
Radiation-induced damage to the retina triggers leukostasis, retinal endothelial cell (REC) death, and subsequent hypoxia. Resultant ischemia leads to visual loss and compensatory retinal neovascularization (RNV). Using human RECs, we demonstrated that radiation induced leukocyte adhesion through mechanisms involving p38MAPK, p53, and ICAM-1 activation. Additional phenotypic changes included p38MAPK-dependent tyrosine phosphorylation of the focal adhesion scaffolding protein, paxillin (Tyr118). The quinic acid derivative KZ-41 lessened leukocyte adhesion and paxillin-dependent proliferation via inhibition of p38MAPK-p53-ICAM-1 signaling. Using the murine oxygen-induced retinopathy (OIR) model, we examined the effect of KZ-41 on pathologic RNV. Daily ocular application of a KZ-41-loaded nanoemulsion significantly reduced both the avascular and neovascular areas in harvested retinal flat mounts when compared to the contralateral eye receiving vehicle alone. Our data highlight the potential benefit of KZ-41 in reducing both the retinal ischemia and neovascularization provoked by genotoxic insults. Further research into how quinic acid derivatives target and mitigate inflammation is needed to fully appreciate their therapeutic potential for the treatment of inflammatory retinal vasculopathies.
PMCID: PMC4067294  PMID: 24956278
2.  Synthesis and Pharmacological Evaluation of the Stereoisomersof 3-Carba Cyclic-Phosphatidic Acid 
Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Here we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA5 GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA5 compared to the R stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.
PMCID: PMC3040411  PMID: 21051230
lysophosphatidic acid; NPP2; autotaxin; GPR92; lysophospholipase D
3.  EDL-360: A Potential Novel Antiglioma Agent 
Glioma is a brain tumor that arises from glial cells or glial progenitor cells, and represents 80% of malignant brain tumor incidence in the United States. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor malignancy with fewer than 8% of patients with GBM surviving for more than 3 years. Over the past 10 years, despite improvement in diagnosis and therapies for cancer, the survival rate for high-grade glioma patients remains dismal. The main focus of our research is to identify potent novel antiglioma small molecules. We previously showed that EDL-360, a tetrahydroisoquinoline (THIQ) analog, as being highly cytotoxic to human glioma cell cultures. Here we show that EDL-360 significantly induced apoptosis in human glioma cell lines (U87 and LN18). However, in normal astrocytic cells, EDL-360 induced a modest G0/G1 cell cycle arrest but did not induce apoptosis. In an attempt to enhance EDL-360 induced cell death, we tested simultaneous treatment with EDL-360 and embelin (an inhibitor of the anti-apoptotic protein, XIAP). We found that, glioma cells had significant lower viability when EDL-360 and embelin were used in combination when compared to EDL-360 alone. We also used combination treatment of EDL-360 with decylubiquinone (dUb), a caspase-9 inhibitor, and found that the combination treatment induced a significant cell death when compared to treatment with EDL-360 alone. This is the first report that suggests that dUb has anticancer activity, and perhaps acts as a XIAP inhibitor. Finally, our in vivo data showed that EDL-360 treatment induced a partial regression in glioma tumorigenesis and induced cell death in the treated tumors as shown by H&E staining. Taken together these data suggests that EDL-360 has a potential therapeutic application for treating glioma, especially when combined with XIAP inhibitors.
PMCID: PMC4285352  PMID: 25574358
Ovarian cancer; PARP inhibitors; Angiogenesis
4.  Synergistic Combination of Novel Tubulin Inhibitor ABI-274 and Vemurafenib Overcome Vemurafenib Acquired Resistance in BRAFV600E Melanoma 
Molecular cancer therapeutics  2013;13(1):16-26.
Acquired clinical resistance to vemurafenib, a selective BRAFV600E inhibitor, arises frequently after short term chemotherapy. Since inhibitions of targets in the RAF-MEK-ERK pathway result in G0/G1 cell cycle arrest, vemurafenib-resistant cancer cells are expected to escape this cell cycle arrest and progress to subsequent G2/M phase. We hypothesized that a combined therapy using vemurafenib with a G2/M phase blocking agent will trap resistant cells and overcome vemurafenib resistance. To test this hypothesis, we first determined the combination index (CI) values of our novel tubulin inhibitor ABI-274 and vemurafenib on parental human A375 and MDA-MB-435 melanoma cell lines to be 0.32 and 0.1, respectively, suggesting strong synergy for the combination. We then developed an A375RF21 subline with significant acquired resistance to vemurafenib and confirmed the strong synergistic effect. Next we studied the potential mechanisms of overcoming vemurafenib resistance. Flow cytometry confirmed that the combination of ABI-274 and vemurafenib synergistically arrested cells in G1/G2/M phase, and significantly increased apoptosis in both parental A375 and the vemurafenib-resistant A375RF21 cells. Western blot analysis revealed that the combination treatment effectively reduced the level of phosphorylated and total AKT, activated the apoptosis cascade, and increased cleaved caspase-3 and cleaved PARP, but had no significant influence on the level of ERK phosphorylation. Finally, in vivo co-administration of vemurafenib with ABI-274 showed strong synergistic efficacy in the vemurafenib-resistant xenograft model in nude mice. Overall, these results offer a rational combination strategy to significantly enhance the therapeutic benefit in melanoma patients who inevitably become resistant to current vemurafenib therapy.
PMCID: PMC3947172  PMID: 24249714
tubulin inhibitor; vemurafenib resistance; synergistic combination
5.  Selective Androgen Receptor Modulators (SARMs) Negatively Regulate Triple-Negative Breast Cancer Growth and Epithelial:Mesenchymal Stem Cell Signaling 
PLoS ONE  2014;9(7):e103202.
The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75–95% of estrogen receptor (ER)-positive and 40–70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer.
Materials and Methods
Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR) were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC) co-culture signaling studies were performed to understand the mechanisms of action.
Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures.
1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.
PMCID: PMC4114483  PMID: 25072326
6.  Discovery of 4-Substituted Methoxybenzoyl-Aryl-Thiazole as Novel Anticancer Agents: Synthesis, Biological Evaluation and Structure-Activity Relationships 
Journal of medicinal chemistry  2009;52(6):1701-1711.
A series of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART) have been discovered and synthesized as a result of structural modifications of the lead compound 2-arylthiazolidine-4-carboxylic acid amides (ATCAA). The antiproliferative activity of the SMART agents against melanoma and prostate cancer cells was improved from μM to low nM range compared with ATCAA series. The structure-activity relationship was discussed from modifications of “A”, “B” “C” rings and the linker. Preliminary mechanism of action studies indicated that these compounds exert their anticancer activity through inhibition of tubulin polymerization.
PMCID: PMC2760094  PMID: 19243174
Thiazolidine; Thiazole; Melanoma; Prostate cancer; Antiproliferative activity; Structure-activity relationship; X-ray Crystal structure; Tubulin polymerization inhibitor
7.  Discovery of 4-Aryl-2-benzoyl-imidazoles As Tubulin Polymerization Inhibitor with Potent Antiproliferative Properties 
Journal of medicinal chemistry  2013;56(8):3318-3329.
A series of 4-aryl-2-benzoyl-imidazoles were designed and synthesized based on our previously reported 2-aryl-4-benzoyl-imidazole (ABI) derivatives. The new structures reversed the aryl group and the benzoyl group of previous ABI structures and were named as reverse ABI (RABI) analogs. RABIs were evaluated for biological activity against 8 cancer cell lines including multidrug-resistant cancer cell lines. In vitro assays indicated that several RABI compounds had excellent antiproliferative properties with IC50 values in the low nanomolar range. The average IC50 of the most active compound 12a is 14 nM. In addition, the mechanism of action of these new analogs was investigated by cell cycle analysis, tubulin polymerization assay, competitive mass spectrometry binding assay and molecular docking studies. These studies confirmed that these new RABI analogs maintain their mechanisms of action by disrupting tubulin polymerization, similar to their parental ABI analogs.
PMCID: PMC3668676  PMID: 23547728
8.  Novel Quinic Acid Derivative KZ-41 Prevents Retinal Endothelial Cell Apoptosis Without Inhibiting Retinoblastoma Cell Death Through p38 Signaling 
To determine whether a novel NF-κB inhibitor, KZ-41, can inhibit melphalan's actions on retinal endothelial cell (REC) inflammation and apoptosis, without eliminating the chemotherapeutic efficacy of melphalan on cell death of retinoblastoma cells (Y79).
RECs were cultured in M131 medium supplemented with growth factors and antibiotics. Once cells reached confluence, they were treated with or without 10 μM KZ-41, following treatment with 4 μg/mL melphalan. Cell proteins were extracted and analyzed for intracellular adhesion molecule 1 (ICAM-1) levels and Cell Death ELISA. RECs were also transfected with or without NF-κB siRNA or treated with SB202190 (p38 [mitogen activated protein kinase] MAPK inhibitor) before melphalan treatment to determine the involvement of NF-κB and p38 MAPK in REC apoptosis and ICAM-1 levels. We also cultured retinoblastoma cells (Y79) in RMPI-1640 medium supplemented with 20% fetal bovine serum and performed a Cell Death ELISA after melphalan + KZ-41 treatment to determine if the treatments altered melphalan's ability to promote cell death of Y79 cells.
KZ-41 inhibited melphalan-stimulation of ICAM-1 levels and REC apoptosis, whereas KZ-41 did not alter melphalan's effects on Y79 cells. KZ-41's protective effects on REC were mediated through p38 MAPK activation. Although KZ-41 blocked both NF-κB- and p38 MAPK–dependent ICAM-1 stimulation; the p38 MAPK/ICAM-1 pathway appears to be the primary pathway involved in melphalan-induced REC apoptosis.
KZ-41 protects REC against melphalan-induced upregulation of ICAM-1 and apoptosis through p38 MAPK–dependent pathways.
Novel quinic acid derivative KZ-41 protects retinal endothelial cells from increased ICAM-1 and apoptosis after treatment with melphalan, a chemotherapy agent used for retinoblastoma. KZ-41 does not alter melphalan's actions on retinoblastoma cells.
PMCID: PMC3762329  PMID: 23942968
KZ-41; NF-κB; Y79; retinal endothelial cell; cell death; inflammation
9.  Discovery and Preclinical Characterization of Novel Small Molecule TRK and ROS1 Tyrosine Kinase Inhibitors for the Treatment of Cancer and Inflammation 
PLoS ONE  2013;8(12):e83380.
Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.
PMCID: PMC3873281  PMID: 24386191
10.  Formulation and Characterization of Polyester/Polycarbonate Nanoparticles for Delivery of a Novel Microtubule Destabilizing Agent 
Pharmaceutical research  2012;29(11):3064-3074.
Since our newly synthesized potent 5-indolyl derivative, (2-(1 H-Indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293), to treat resistant melanoma was hydrophobic, our objective was to synthesize a biodegradable copolymer for formulating this drug into nanoparticles and to determine its anticancer activity and mechanism of action.
Methoxy poly (ethylene glycol)-b-poly (carbonate-colactide) [mPEG-b-P (CB-co-LA)] was synthesized for formulating LY293 into nanoparticles by o/w emulsification and stabilization by solvent evaporation. Particle size, drug release profile, in vitro efficacy in multiple melanoma cells, and mechanism of action of drug-loaded nanoparticles were determined.
LY293-loaded nanoparticles with 170 nm mean size and 2.2 and 4.16% drug loading efficiently inhibited proliferation of A375 and B16F10 cells with IC50 of 12.5 nM and 25 nM, respectively. LY293 circumvented multidrug resistance and inhibited proliferation of Pgp overexpressing MDA-MB435/LCC6 MDR1 melanoma cells. Upon treatment with LY293-loaded nanoparticles, A375 cells underwent cell cycle arrest in G2/M phase and apoptotic cell death. Immunofluorescence images showed inhibition of tubulin polymerization after treatment with LY293.
LY293-loaded mPEG-b-P (CB-co-LA) nanoparticles showed excellent efficacy and induced apoptosis in melanoma cells. These polyester/polycarbonate-based nanoparticles provided an excellent platform to deliver different poorly soluble drugs to melanoma.
PMCID: PMC3646297  PMID: 23054088
LY293; melanoma; polymeric nanoparticles; tubulin polymerization
11.  Orally Bioavailable Tubulin Antagonists for Paclitaxel-Refractory Cancer 
Pharmaceutical research  2012;29(11):3053-3063.
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.
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.
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 IATwas 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.
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.
PMCID: PMC3646298  PMID: 22760659
paclitaxel resistant cancer; P-glycoprotein; pharmacokinetics; tubulin; xenograft
12.  Novel Tubulin Polymerization Inhibitors Overcome Multidrug Resistance and Reduce Melanoma Lung Metastasis 
Pharmaceutical research  2012;29(11):3040-3052.
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.
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.
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.
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.
PMCID: PMC3659804  PMID: 22410804
2-aryl-4-benzoyl-imidazoles (ABI); antimelanoma; melanoma lung metastasis; multidrug resistance; tubulin polymerization inhibitor
13.  An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site 
Pharmaceutical research  2012;29(11):2943-2971.
Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding site is one of the most important pockets for potential tubulin polymerization destabilizers. Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. A large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers. CBSIs are believed to act by a common mechanism via binding to the colchicine site on tubulin. The present review is a synopsis of compounds that have been reported in the past decade that have provided an increase in our understanding of the actions of CBSIs.
PMCID: PMC3667160  PMID: 22814904
antimitotic; cancer; colchicine; multidrug resistance; tubulin polymerization inhibitor
14.  Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI–III) analogues targeting tubulin polymerization as antiproliferative agents 
Journal of medicinal chemistry  2012;55(16):7285-7289.
Novel ABI–III compounds were designed and synthesized based on our previously reported ABI-I and ABI–II analogs. ABI–III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC50 value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp mediated multidrug resistance. ABI–III analogs maintain their mechanisms of action by inhibition of tubulin polymerization.
PMCID: PMC3426659  PMID: 22783954
Melanoma; prostate cancer; multidrug resistance; tubulin polymerization inhibitor; antiproliferative activity
15.  Compound 49b protects against blast-induced retinal injury 
To determine whether Compound 49b, a novel beta-adrenergic receptor agonist, can prevent increased inflammation and apoptosis in mice after exposure to ocular blast.
Eyes of C57/BL6 mice were exposed to a blast of air from a paintball gun at 26 psi (≈0.18 MPa). Eyes were collected 4 hours, 24 hours, and 72 hours after blast exposure. In a subset of mice, Compound 49b eyedrops (1 mM) were applied within 4 hours, 24 hours, or 72 hours of the blast. Three days after blast exposure, all mice were sacrificed. One eye was used to measure levels of retinal proteins (TNFα, IL-1β, Bax, BcL-xL, caspase 3, and cytochrome C). The other eye was used for TUNEL labeling of apoptotic cells, which were co-labeled with NeuN to stain for retinal ganglion cells.
We found that ocular exposure to 26 psi air pressure led to a significant increase in levels of apoptotic and inflammatory mediators within 4 hours, which lasted throughout the period investigated. When Compound 49b was applied within 4 hours or 24 hours of blast injury, levels of apoptotic and inflammatory mediators were significantly reduced. Application of Compound 49b within 72 hours of blast injury reduced levels of inflammatory mediators, but not to untreated levels.
Ocular blast injury produces a significant increase in levels of key inflammatory and apoptotic markers in the retina as early as 4 hours after blast exposure. These levels are significantly reduced if a beta-adrenergic receptor agonist is applied within 24 hours of blast exposure. Data suggest that local application of beta-adrenergic receptor agonists may be beneficial to reduce inflammation and apoptosis.
PMCID: PMC3751549  PMID: 23899290
Apoptosis; Beta-adrenergic receptor agonists; Cytokines
16.  New substituted 4H-chromenes as anticancer agents ☆ 
As a continuation of our efforts to discover and develop small molecules as anticancer agents, we identified GRI-394837 as an initial hit from similarity search on RGD and its analogs. Based on GRI-394837, we designed and synthesized a focused set of novel chromenes (4a–e) in a single step using microwave method. All five compounds showed activity in the nanomolar range (IC50: 7.4–640 nM) in two melanoma, three prostate and four glioma cancer cell lines. The chromene 4e is active against all the cell lines and particularly against the A172 human glioma cell line (IC50: 7.4 nM). Interestingly, in vitro tubulin polymerization assay shows 4e to be a weak tubulin polymerization inhibitor but it shows very strong cytotoxicity in cellular assays, therefore there must be additional unknown mechanism(s) for the anticancer activity. Additionally, the strong antiproliferative activity was verified by one of the selected chromene (4a) by the NCI 60 cell line screen. These results strongly suggest that the novel chromenes could be further developed as a potential therapeutic agent for a variety of aggressive cancers.
PMCID: PMC3659818  PMID: 22608389
Chromenes; RGD; Melanoma; Glioma; Prostate cancer; Tubulin polymerization inhibitors
17.  Design, Synthesis and Biological Action of 20R-Hydroxyvitamin D3 
Journal of Medicinal Chemistry  2012;55(7):3573-3577.
Non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated from semi-preparative HPLC and its structure is characterized. The comparison of 20R isomer to its 20S counterpart in biological evaluation demonstrates they have different behaviours in both antiproliferative and metabolic studies.
PMCID: PMC3332540  PMID: 22404326
20R-hydroxyvitamin D3; 20S-hydroxyvitamin D3; chemical synthesis; NMR; antiproliferative activity; metabolism; CYP11A1; CYP27B1
18.  Controlling cancer through the autotaxin–lysophosphatidic acid receptor axis 
LPA (lysophosphatidic acid, 1-acyl-2-hydroxy-sn-glycero-3-phosphate), is a growth factor-like lipid mediator that regulates many cellular functions, many of which are unique to malignantly transformed cells. The simple chemical structure of LPA and its profound effects in cancer cells has attracted the attention of the cancer therapeutics field and drives the development of therapeutics based on the LPA scaffold. In biological fluids, LPA is generated by ATX (autotaxin), a lysophospholipase D that cleaves the choline/serine headgroup from lysophosphatidylcholine and lysophosphatidylserine to generate LPA. In the present article, we review some of the key findings that make the ATX–LPA signalling axis an emerging target for cancer therapy.
PMCID: PMC3590848  PMID: 22260662
autotaxin; cancer; drug discovery; lysophosphatidic acid (LPA); 4-pentadecylbenzylphosphonic acid (4-PBA); therapy
19.  Novel vitamin D photoproducts and their precursors in the skin 
Dermato-endocrinology  2013;5(1):7-19.
Novel metabolic pathways initiated by the enzymatic action of CYP11A1 on 7DHC (7-dehydrocholesterol), ergosterol, vitamins D3 and D2 were characterized with help of chemical synthesis, UV and mass spectrometry and NMR analyses. The first pathway follows the sequence 7DHC→22(OH)7DHC → 20,22(OH)27DHC → 7DHP (7-dehydropregnenolone), which can further be metabolized by steroidogenic enzymes. The resulting 5,7-dienes can be transformed by UVB to corresponding, biologically active, secosteroids. Action of CYP11A1 on vitamin D3 and D2 produces novel hydroxyderivatives with OH added at positions C17, C20, C22, C23 and C24, some of which can be hydroxylated by CYP27B1 and/or by CYP27A1 and/ or by CYP24A1.The main products of these pathways are biologically active with a potency related to their chemical structure and the target cell type. Main products of CYP11A1-mediated metabolism on vitamin D are non-calcemic and non-toxic at relatively high doses and serve as partial agonists on the vitamin D receptor. New secosteroids are excellent candidates for therapy of fibrosing, inflammatory or hyperproliferative disorders including cancers and psoriasis.
PMCID: PMC3897599  PMID: 24494038
skin; keratinocytes; melanocytes; melanoma cells; dermal fibroblasts; vitamin D; 5,7-dienes
20.  Effects of sidechain length and composition on the kinetic conversion and product distribution of vitamin D analogs determined by real-time NMR 
Dermato-endocrinology  2013;5(1):142-149.
Novel pregna-5, 7-dienes were synthesized and subjected to UVB irradiation to generate the corresponding pre-D intermediates, tachysterol and lumisterol analogs. The kinetics of the conversion from each of the pre-D intermediates to the corresponding novel D analogs was investigated by using real time 1H NMR measurements inside the NMR magnet. Both the length and composition of the side chains were found to affect the rate of the kinetic conversion from pre-D intermediates to the thermodynamically more stable D analogs. Compound 7cc which has both a long side chain and a tertiary alcohol moiety showed the highest conversion rate, while compound 4a-S which has a very short side chain without the tertiary alcohol had the lowest conversion rate among the 13 tested compounds. We also determined product distributions for these 5,7-dienes upon UVB irradiation followed by thermodynamic equilibration. No clear correlations between product distribution and side chain length or composition were identifiable under the current experimental conditions, suggesting there are other factors affecting the kinetics during the photochemical reactions for these 5,7-dienes. To the best of our knowledge, this is the first time the influences of side chain length and composition on the real time conversion kinetics from pre-D to D are studied. This study could serve as step-stones in future kinetic studies of novel biologically active 5,7-dienes and their corresponding D analogs under more physiologically relevant ex vivo or in vivo conditions, as well as providing important insights into optimizing yields of the desired active products during their organic syntheses.
PMCID: PMC3897582  PMID: 24494047
kinetics; pre-D; vitamin D; UVB; real-time NMR
21.  Benzyl and Naphthalene-Methyl Phosphonic Acid Inhibitors of Autotaxin with Anti-invasive and Anti-metastatic Actions 
ChemMedChem  2011;6(5):922-935.
Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) via a lysophospholipase D activity that leads to the generation of the growth factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Here we report the synthesis and pharmacological characterization of inhibitors of ATX based on the 4-tetradecanoylaminobenzyl phosphonic acid scaffold that was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzyl phosphonic acid and 6-substituted naphthalen-2-yl-methyl phosphonic acid analogs blocked ATX with Ki values in the low-micromolar-nanomolar range against FS-3, LPC, and nucleotide substrates through a mixed-mode mechanism of inhibition. None of the compounds tested inhibited the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor subtypes. Analogs 22 and 30b, the two most potent ATX inhibitors, dose-dependently inhibited the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro. The average terminal half-life for compound 22 was 10h ± 5.4h and it caused a long-lasting reduction plasma LPA levels. Compounds 22 and 30b significantly reduced lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The described 4-substituted benzyl phosphonic acids and 6-substituted naphthalen-2-yl-methyl phosphonic acids represent new lead compounds that effectively inhibit the ATX-LPA-LPA receptor axis both in vitro and in vivo.
PMCID: PMC3517046  PMID: 21465666
ATX inhibitors; LPA receptors; 4-substituted benzyl phosphonic acids; 6-substituted naphthalen-2-yl-methyl phosphonic acids; structure-activity relationships
22.  Compound 49b Prevents Diabetes-Induced Apoptosis through Increased IGFBP-3 Levels 
To determine whether Compound 49b, a novel PKA-activating drug, can prevent diabetic-like changes in the rat retina through increased insulin-like growth factor binding protein-3 (IGFBP-3) levels.
For the cell culture studies, we used both human retinal endothelial cells (REC) and retinal Müller cells in either 5 mM (normal) or 25 mM (high) glucose. Cells were treated with 50 nM Compound 49b alone of following treatment with protein kinase A (PKA) siRNA or IGFBP-3 siRNA. Western blotting and ELISA analyses were done to verify PKA and IGFBP-3 knockdown, as well as to measure apoptotic markers. For animal studies, we used streptozotocin-treated rats after 2 and 8 months of diabetes. Some rats were treated topically with 1 mM Compound 49b. Analyses were done for retinal thickness, cell numbers in the ganglion cell layer, pericyte ghosts, and numbers of degenerate capillaries, as well as electroretinogram and heart morphology.
Compound 49b requires active PKA and IGFBP-3 to prevent apoptosis of REC. Compound 49b significantly reduced the numbers of degenerate capillaries and pericyte ghosts, while preventing the decreased retinal thickness and loss of cells in the ganglion cell layer. Compound 49b maintained a normal electroretinogram, with no changes in blood pressure, intraocular pressure, or heart morphological changes.
Topical Compound 49b is able to prevent diabetic-like changes in the rat retina, without producing systemic changes. Compound 49b is able to prevent REC apoptosis through increasing IGFBP-3 levels, which are reduced in response to hyperglycemia.
We have developed a novel beta-adrenergic receptor agonist that prevents diabetic-like changes in the rat retina without systemic side effects. We have also determined the mechanism by which Compound 49b can prevent retinal endothelial cell apoptosis.
PMCID: PMC3378083  PMID: 22467575
23.  The Lysophosphatidic Acid Type 2 Receptor Is Required for Protection Against Radiation-Induced Intestinal Injury 
Gastroenterology  2007;132(5):1834-1851.
Background & Aims
We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo
The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA1 and LPA2 knockout mice exposed to different apoptotic stimuli
OTP was more efficacious than LPA in reducing gamma irradiation–, camptothecin-, or tumor necrosis factor α/cycloheximide–induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3–positive cells but was ineffective in LPA2 knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA2 knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)100/30 radiation by 50%.
Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype.
PMCID: PMC3446791  PMID: 17484878
24.  Lysophosphatidic acid induces prostate cancer PC3 cell migration via activation of LPA1, p42 and p38α 
Biochimica et biophysica acta  2007;1771(7):883-892.
Prostate cancer cell migration is an essential event both in the progression of prostate cancer and in the steps leading to metastasis. We report here that lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces prostate cancer PC3 cell migration via the activation of the LPA1 receptor, which is linked to a PTX-sensitive activation mechanism of the mitogen-activated protein kinases (MAPK). Our results demonstrate that parallel activation of ERK1/2 and p38, but not JNK, is responsible for LPA-stimulated PC3 cell migration. Furthermore, using small interfering RNA (siRNA) technology, and overexpressing dominant-negative mutants of p38 MAPK isotypes of α, β, γ and δ, we have identified that the activation of ERK2 (p42) and p38α, but not of ERK1 and the other isoforms of p38 MAPK, is required for LPA-induced migration. Our study provides the first evidence for a functional role of p42 and p38α in LPA-induced mammalian cell migration, and also demonstrates, for the first time, that the receptor LPA1 mediates prostate cancer cell migration. The results of the present study suggest that LPA, the receptor LPA1, ERK2 and p38α are important regulators for prostate cancer cell invasion and thus could play a significant role in the development of metastasis.
PMCID: PMC3446792  PMID: 17531530
Lysophosphatidic acid; Receptors; Cell migration; Protein kinases and prostate cancer cells
25.  Synthesis and antiproliferative activity of novel 2-aryl-4-benzoyl-imidazole derivatives targeting tubulin polymerization 
Bioorganic & medicinal chemistry  2011;19(16):4782-4795.
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.
PMCID: PMC3152597  PMID: 21775150

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