Biological activity of a stable 6-aryl-2-benzoyl-pyridine colchicine-binding site inhibitor, 60c, in metastatic, triple-negative breast cancer.

BACKGROUND: Improving survival for patients diagnosed with metastatic disease and overcoming chemoresistance remain significant clinical challenges in treating breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of therapeutically targetable receptors (ER/PR/HER2). TNBC therapy includes a combination of cytotoxic chemotherapies, including microtubule-targeting agents (MTAs) like paclitaxel (taxane class) or eribulin (vinca class); however, there are currently no FDA-approved MTAs that bind to the colchicine-binding site. Approximately 70 % of patients who initially respond to paclitaxel will develop taxane resistance (TxR). We previously reported that an orally bioavailable colchicine-binding site inhibitor (CBSI), VERU-111, inhibits TNBC tumor growth and treats pre-established metastatic disease. To further improve the potency and metabolic stability of VERU-111, we created next-generation derivatives of its scaffold, including 60c. RESULTS: 60c shows improved in vitro potency compared to VERU-111 for taxane-sensitive and TxR TNBC models, and suppress TxR primary tumor growth without gross toxicity. 60c also suppressed the expansion of axillary lymph node metastases existing prior to treatment. Comparative analysis of excised organs for metastasis between 60c and VERU-111 suggested that 60c has unique anti-metastatic tropism. 60c completely suppressed metastases to the spleen and was more potent to reduce metastatic burden in the leg bones and kidney. In contrast, VERU-111 preferentially inhibited liver metastases and lung metastasis repression was similar. Together, these results position 60c as an additional promising CBSI for TNBC therapy, particularly for patients with TxR disease.

ABCB1-dependent collateral sensitivity of multidrug-resistant colorectal cancer cells to the survivin inhibitor MX106-4C.

AIMS: To investigate the collateral sensitivity (CS) of ABCB1-positive multidrug resistant (MDR) colorectal cancer cells to the survivin inhibitor MX106-4C and the mechanism. METHODS: Biochemical assays (MTT, ATPase, drug accumulation/efflux, Western blot, RT-qPCR, immunofluorescence, flow cytometry) and bioinformatic analyses (mRNA-sequencing, reversed-phase protein array) were performed to investigate the hypersensitivity of ABCB1 overexpressing colorectal cancer cells to MX106-4C and the mechanisms. Synergism assay, long-term selection, and 3D tumor spheroid test were used to evaluate the anti-cancer efficacy of MX106-4C. RESULTS: MX106-4C selectively killed ABCB1-positive colorectal cancer cells, which could be reversed by an ABCB1 inhibitor, knockout of ABCB1, or loss-of-function ABCB1 mutation, indicating an ABCB1 expression and function-dependent mechanism. MX106-4C's selective toxicity was associated with cell cycle arrest and apoptosis through ABCB1-dependent survivin inhibition and activation on caspases-3/7 as well as modulation on p21-CDK4/6-pRb pathway. MX106-4C had good selectivity against ABCB1-positive colorectal cancer cells and retained this in multicellular tumor spheroids. In addition, MX106-4C could exert a synergistic anti-cancer effect with doxorubicin or re-sensitize ABCB1-positive cancer cells to doxorubicin by reducing ABCB1 expression in the cell population via long-term exposure. CONCLUSIONS: MX106-4C selectively kills ABCB1-positive MDR colorectal cancer cells via a novel ABCB1-dependent survivin inhibition mechanism, providing a clue for designing CS compound as an alternative strategy to overcome ABCB1-mediated colorectal cancer MDR.

Colchicine-Binding Site Agent CH-2-77 as a Potent Tubulin Inhibitor Suppressing Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer. Unlike other subtypes of breast cancer, TNBC lacks hormone and growth factor receptor targets. Colchicine-binding site inhibitors (CBSI) targeting tubulin have been recognized as attractive agents for cancer therapy, but there are no CBSI drugs currently FDA approved. CH-2-77 has been reported to have potent antiproliferative activity against a panel of cancer cells in vitro and efficacious antitumor effects on melanoma xenografts, yet, its anticancer activity specifically against TNBC is unknown. Herein, we demonstrate that CH-2-77 inhibits the proliferation of both paclitaxel-sensitive and paclitaxel-resistant TNBC cells with an average IC50 of 3 nmol/L. CH-2-77 also efficiently disrupts the microtubule assembly, inhibits the migration and invasion of TNBC cells, and induces G2-M cell-cycle arrest. The increased number of apoptotic cells and the pattern of expression of apoptosis-related proteins in treated MDA-MB-231 cells suggest that CH-2-77 induces cell apoptosis through the intrinsic apoptotic pathway. In vivo, CH-2-77 shows acceptable overall pharmacokinetics and strongly suppresses the growth of orthotopic MDA-MB-231 xenografts without gross cumulative toxicities when administered 5 times a week. The in vivo efficacy of CH-2-77 (20 mg/kg) is comparable with that of CA4P (28 mg/kg), a CBSI that went through clinical trials. Importantly, CH-2-77 prevents lung metastasis originating from the mammary fat pad in a dose-dependent manner. Our data demonstrate that CH-2-77 is a promising new generation of tubulin inhibitors that inhibit the growth and metastasis of TNBC, and it is worthy of further development as an anticancer agent.

Design, Synthesis, and Biological Evaluation of Stable Colchicine-Binding Site Tubulin Inhibitors 6-Aryl-2-benzoyl-pyridines as Potential Anticancer Agents.

We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.

Angiogenesis in primary colorectal cancer and matched metastatic tissues: Biological and clinical implications for anti-angiogenic therapies.

Metastasis remains a notable issue in patients with newly diagnosed colorectal carcinomas (CRC). Although anti-angiogenic therapies target metastatic diseases, hypoxia-inducible factor-1 α (HIF-1α) and vascular endothelial growth factor (VEGF) status are routinely evaluated in primary tumors as metastatic sites are infrequently biopsied. The present study aimed to investigate the expression and significance of HIF-1α, VEGF and microvascular density (MVD) in primary tumors and corresponding metastatic CRC tissues. HIF-1α, VEGF and CD34 status were analyzed via immunohistochemistry analysis in 46 patients who underwent surgical resection of primary CRC (35 colon and 11 rectum) and matched metastases (lymph node and liver metastases) in Shandong Cancer Hospital. The association between selected biomarker status and clinicopathological characteristics was analyzed, and expression levels in primary tumors and corresponding metastases were compared. A total of 46 paired colorectal primary tumor and synchronous metastases samples were acquired for analysis using a standardized HIF-1α, VEGF and CD34 immunohistochemical procedure. The results demonstrated that the positive rates of HIF-1α and VEGF in primary CRC were 70 and 73.9%, respectively. HIF-1α (60.9%) and VEGF (58.7%) expression decreased in the lymph metastatic samples compared with primary CRC. Conversely, the level of MVD in primary tumors was significantly higher compared with metastatic tumors. No significant differences were demonstrated between HIF-1α and VEGF expression and the different clinicopathological features in primary CRC and corresponding metastases. Primary carcinomas and matched metastatic tissues demonstrated a moderate level of consistent immunoreactivity for HIF-1α and VEGF. HIF-1α, VEGF and CD34 were expressed in both primary tumors and corresponding metastases of CRC, suggesting that they may be involved in the development of metastasis. HIF-1α and VEGF expression in primary sites was consistent with that observed in metastases; however, it varied from that exhibited in MVD. The current analysis will improve the current understanding of the metastasis models and provide further evidence for evaluating the response to HIF-1α and VEGF inhibitors.

Hsa_circ_0020095 Promotes Oncogenesis and Cisplatin Resistance in Colon Cancer by Sponging miR-487a-3p and Modulating SOX9.

OBJECTIVES: Colon cancer (CC) currently ranks as the third most common human cancer worldwide with an increasing incidence and a poor prognosis. Recently, circular RNAs have been reported to regulate the progression of diverse human cancers. However, the role of circRNA hsa_circ_0020095 in CC remains largely unclear. METHODS: Expression levels of the related circRNAs, microRNAs and mRNA in CC tissues and cells were determined. The impacts of circ_0020095 or miR-487a-3p on CC cells were examined at the indicated times after transfection. Meanwhile, a luciferase-reporter experiment was employed to validate the interplay between miR-487a-3p and circ_002009695 or SOX9. Moreover, the in vivo tumor growth assay was applied to further evaluate the effects of circ_0020095 knockdown on CC progression. RESULTS: We demonstrated that circ_0020095 was highly expressed in CC tissues and cells. The proliferation, migration, invasion, and cisplatin resistance of CC were suppressed by silencing circ_0020095 in vitro and in vivo or by ectopic expression of miR-487a-3p in vitro. Mechanistically, circ_0020095 could directly bind to miR-487a-3p and subsequently act as a miR-487a-3p sponge to modulate the activity by targeting the 3'-UTR of SOX9. Interestingly, overexpression of circ_0020095 dramatically reversed the suppressive effects of miR-487a-3p mimics on CC cells. CONCLUSION: Circ_0020095 functions as an oncogene to accelerate CC cell proliferation, invasion, migration and cisplatin resistance through the miR-487a-3p/SOX9 axis, which could be a promising target for CC treatment.

Structure-Activity Relationship Study of Novel 6-Aryl-2-benzoyl-pyridines as Tubulin Polymerization Inhibitors with Potent Antiproliferative Properties.

We recently reported the crystal structure of tubulin in complex with a colchicine binding site inhibitor (CBSI), ABI-231, having 2-aryl-4-benzoyl-imidazole (ABI). Based on this and additional crystal structures, here we report the structure-activity relationship study of a novel series of pyridine analogues of ABI-231, with compound 4v being the most potent one (average IC50 ∼ 1.8 nM) against a panel of cancer cell lines. We determined the crystal structures of another potent CBSI ABI-274 and 4v in complex with tubulin and confirmed their direct binding at the colchicine site. 4v inhibited tubulin polymerization, strongly suppressed A375 melanoma tumor growth, induced tumor necrosis, disrupted tumor angiogenesis, and led to tumor cell apoptosis in vivo. Collectively, these studies suggest that 4v represents a promising new generation of tubulin inhibitors.

Structure-Guided Design, Synthesis, and Biological Evaluation of (2-(1H-Indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) Methanone (ABI-231) Analogues Targeting the Colchicine Binding Site in Tubulin.

ABI-231 is a potent, orally bioavailable tubulin inhibitor that interacts with the colchicine binding site and is currently undergoing clinical trials for prostate cancer. Guided by the crystal structure of ABI-231 in complex with tubulin, we performed structure-activity relationship studies around the 3-indole moiety that led to the discovery of several potent ABI-231 analogues, most notably 10ab and 10bb. The crystal structures of 10ab and 10bb in complex with tubulin confirmed their improved molecular interactions to the colchicine site. In vitro, biological studies showed that new ABI-231 analogues disrupt tubulin polymerization, promote microtubule fragmentation, and inhibit cancer cell migration. In vivo, analogue 10bb not only significantly inhibits primary tumor growth and decreases tumor metastasis in melanoma xenograft models but also shows a significant ability to overcome paclitaxel resistance in a taxane-resistant PC-3/TxR model. In addition, pharmacological screening suggested that 10bb has a low risk of potential off-target function.

Colchicine Binding Site Agent DJ95 Overcomes Drug Resistance and Exhibits Antitumor Efficacy.

Interfering with microtubule dynamics is a well-established strategy in cancer treatment; however, many microtubule-targeting agents are associated with drug resistance and adverse effects. Substantial evidence points to ATP-binding cassette (ABC) transporters as critical players in the development of resistance. Herein, we demonstrate the efficacy of DJ95 (2-(1H-indol-6-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine), a novel tubulin inhibitor, in a variety of cancer cell lines, including malignant melanomas, drug-selected resistant cell lines, specific ABC transporter-overexpressing cell lines, and the National Cancer Institute 60 cell line panel. DJ95 treatment inhibited cancer cell migration, caused morphologic changes to the microtubule network foundation, and severely disrupted mitotic spindle formation of mitotic cells. The high-resolution crystal structure of DJ95 in complex with tubulin protein and the detailed molecular interactions confirmed its direct binding to the colchicine site. In vitro pharmacological screening of DJ95 using SafetyScreen44 (Eurofins Cerep-Panlabs) revealed no significant off-target interactions, and pharmacokinetic analysis showed that DJ95 was maintained at therapeutically relevant plasma concentrations for up to 24 hours in mice. In an A375 xenograft model in nude mice, DJ95 inhibited tumor growth and disrupted tumor vasculature in xenograft tumors. These results demonstrate that DJ95 is potent against a variety of cell lines, demonstrated greater potency to ABC transporter-overexpressing cell lines than existing tubulin inhibitors, directly targets the colchicine binding domain, exhibits significant antitumor efficacy, and demonstrates vascular-disrupting properties. Collectively, these data suggest that DJ95 has great potential as a cancer therapeutic, particularly for multidrug resistance phenotypes, and warrants further development. SIGNIFICANCE STATEMENT: Paclitaxel is a widely used tubulin inhibitor for cancer therapy, but its clinical efficacy is often limited by the development of multidrug resistance. In this study, we reported the preclinical characterization of a new tubulin inhibitor DJ95, and demonstrated its abilities to overcome paclitaxel resistance, disrupt tumor vasculature, and exhibit significant antitumor efficacy.

Octreotide treatment of cancer chemoradiotherapy-induced diarrhoea: a meta-analysis of randomized controlled trials.

BACKGROUND: Patients receiving radiotherapy and chemotherapy have a high risk developing to an acute chemoradiotherapy-induced diarrhea (RID). The clinical efficacy of octreotide in controlling chemoradiotherapy-induced diarrhea remains controversial. We performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the efficacy of octreotide for treatment the chemoradiotherapy-induced diarrhoea. METHODS: Relevant RCTs studies assessing the effect of octreotide on clinical outcomes compared with placebo were searched in Cochrane Library, PubMed, EMBASE and Web of Science (up to December 2018). Heterogeneity was assessed with I2, and publication bias was evaluated using sensitive analysis. RESULTS: Eight trials, a total of 594 participants. We found octreotide was significantly effective compared with the control group (OR =3.17; 95% CI, 1.28-7.85; P<0.0001). The overall effect of octreotide was 62.5% (220/352), while that of the control group was 49.3% (168/341). We found octreotide group was effective compared with the control group in 24, 48, and 96 h (OR =16.02; 95% CI, 3.51-73.15; P=0.0003), (OR =4.70; 95% CI, 1.65-13.42; P=0.004) and (OR =14.49; 95% CI, 6.24-33.65; P<0.00001). CONCLUSIONS: Octreotide is superior to conventional therapy in the duration and effectiveness for chemoradiotherapy-induced diarrhea.

c.1439delA frameshift deletion mutation in familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a rare autosomal dominant genetic disease related to germline mutations of the APC gene. The clinical features of this disease most commonly include hundreds of adenomas or polyps. If not treated in a timely fashion, FAP can eventually result in colorectal carcinoma. In this report, clinical manifestations, family history, relevant auxiliary examinations and gene detection from patient blood led us to discover a novel frameshift mutation in exon 12 of the APC gene. The deletion of adenine in c.1439 resulted in the formation of codon 480. The occurrence of this frameshift deletion may lead to inexpressibility of the main functional regions in APC and may affect gene function. In addition, colonoscopy and histopathology showed malignant changes in the colon and rectum. There have been no reports of this frameshift mutation, but it can be considered in case of APC mutations and FAP in patients with clinical manifestations; auxiliary examination may be related, and it may be used as a reference for preventive clinical treatment in the future.

Structural Modification of the 3,4,5-Trimethoxyphenyl Moiety in the Tubulin Inhibitor VERU-111 Leads to Improved Antiproliferative Activities.

Colchicine binding site inhibitors (CBSIs) hold great potential in developing new generations of antimitotic drugs. Unlike existing tubulin inhibitors such as paclitaxel, they are generally much less susceptible to resistance caused by the overexpression of drug efflux pumps. The 3,4,5-trimethoxyphenyl (TMP) moiety is a critical component present in many CBSIs, playing an important role in maintaining suitable molecular conformations of CBSIs and contributing to their high binding affinities to tubulin. Previously reported modifications to the TMP moiety in a variety of scaffolds of CBSIs have usually resulted in reduced antiproliferative potency. We previously reported a potent CBSI, VERU-111, that also contains the TMP moiety. Herein, we report the discovery of a VERU-111 analogue 13f that is significantly more potent than VERU-111. The X-ray crystal structure of 13f in complex with tubulin confirms its direct binding to the colchicine site. In addition, 13f exhibited a strong inhibitory effect on tumor growth in vivo.

Primary ovarian small cell carcinoma of pulmonary type with coexisting endometrial carcinoma in a breast cancer patient receiving tamoxifen: A case report and literature review.

RATIONALE: Small cell carcinoma of the ovary (SCCO) is a rare and aggressive extra-pulmonary variant of small cell tumors of uncertain histogenesis. The pathogenesis and optimal treatment of SCCO is unclear. We present a very rare case of a synchronous primary ovarian small cell carcinoma and endometrioid adenocarcinoma of the uterus in a patient after 2 years of tamoxifen treatment for breast cancer. This is the first such report in the English literature. PATIENT CONCERNS: A 46-year-old woman had a history of left breast cancer that was treated with a simple mastectomy and sentinel lymph node biopsy in 2013. The post-operative pathology was invasive ductal carcinoma of the left breast. she had been taking tamoxifen for 2 years. The patient underwent an exploratory laparotomy to reduce the tumor burden, improve bowel compression symptoms, and promote defecation in 2015. The post-operative pathology revealed a rare, simultaneous occurrence of two tumors (endometrial adenocarcinoma and SCCO [pulmonary type]). DIAGNOSES: Primary ovarian small cell carcinoma of pulmonary type with coexisting endometrial carcinoma in a breast cancer patient. INTERVENTIONS: The patient received 3 courses of chemotherapy after operation. The effect was not apparent and the general health status was poor. OUTCOMES: The patient died of progressive disease 7 months post-operatively. LESSONS: The present case suggests that tamoxifen use might be among many etiologic factors in SCCO development. Despite its rarity, SCCO requires a high degree of attention in clinical work because it is an aggressive tumor that has a poor prognosis.

Synthesis and biological evaluation of indole-based UC-112 analogs as potent and selective survivin inhibitors.

The anti-apoptotic protein survivin is highly expressed in cancer cells but has a very low expression in fully differentiated adult cells. Overexpression of survivin is positively correlated with cancer cell resistance to chemotherapy and radiotherapy, cancer cell metastasis, and poor patient prognosis. Therefore, selective targeting survivin represents an attractive strategy for the development of anticancer therapeutics. Herein, we reported the extensive structural modification of our recently discovered selective survivin inhibitor UC-112 and the synthesis of thirty-three new analogs. The structure-activity relationship (SAR) study indicated that replacement of the benzyloxy moeity in UC-112 with an indole moiety was preferred to other moieties. Among these UC-112 analogs, 10f, 10h, 10k, 10n showed the most potent antiproliferative activities. Interestingly, they were more potent against the P-glycoprotein overexpressing cancer cell lines compared with the parental cancer cell lines. Mechanistic studies confirmed that new analogs maintained their unique selectivity against survivin among the IAP family members. In vivo study using 10f in a human A375 melanoma xenograft model revealed that it effectively inhibited melanoma tumor growth without observable acute toxicity. Collectively, this study strongly supports the further preclinical development of selective survivin inhibitors based on the UC-112 scaffold.

CYP27A1 acts on the pre-vitamin D3 photoproduct, lumisterol, producing biologically active hydroxy-metabolites.

Prolonged exposure of the skin to UV radiation causes previtamin D3, the initial photoproduct formed by opening of the B ring of 7-dehydrocholesterol, to undergo a second photochemical reaction where the B-ring is reformed giving lumisterol3 (L3), a stereoisomer of 7-dehydrocholesterol. L3 was believed to be an inactive photoproduct of excessive UV radiation whose formation prevents excessive vitamin D production. Recently, we reported that L3 is present in serum and that CYP11A1 can act on L3 producing monohydroxy- and dihydroxy-metabolites which inhibit skin cell proliferation similarly to 1α,25-dihydroxyvitamin D3. In this study we tested the ability of human CYP27A1 to hydroxylate L3. L3 was metabolized by purified CYP27A1 to 3 major products identified as 25-hydroxyL3, (25R)-27-hydroxyL3 and (25S)-27-hydroxyL3, by NMR. These three products were also seen when mouse liver mitochondria containing CYP27A1 were incubated with L3. The requirement for CYP27A1 for their formation by mitochondria was confirmed by the inhibition of their synthesis by 5ß-cholestane-3α,7α,12α-triol, an intermediate in bile acid synthesis which serves as an efficient competitive substrate for CYP27A1. CYP27A1 displayed a high kcat for the metabolism of L3 (76 mol product/min/mol CYP27A1) and a catalytic efficiency (kcat/Km) that was 260-fold higher than that for vitamin D3. The CYP27A1-derived hydroxy-derivatives inhibited the proliferation of cultured human melanoma cells and colony formation with IC50 values in the nM range. Thus, L3 is metabolized efficiently by CYP27A1 with hydroxylation at C25 or C27 producing metabolites potent in their ability to inhibit melanoma cell proliferation, supporting that L3 is a prohormone which can be activated by CYP-dependent hydroxylations.

Assessment of the Drug-Drug Interaction Potential Between Theacrine and Caffeine in Humans.

Objective: Theacrine, a methylurate class purine alkaloid, triggers diverse pharmacologic responses, including psychostimulatory activity by modulation of adenosinergic and dopaminergic pathways. In a double-blind, placebo-controlled study, theacrine increased energy, concentration, and mood, while reducing fatigue. Because caffeine, a methylxanthine purine alkaloid, is frequently coadministered with theacrine, we sought to determine if a pharmacokinetic and/or pharmacodynamic interaction existed between theacrine and caffeine. Methods: Eight healthy adults received theacrine, as TeaCrine® (25 or 125 mg), caffeine (150 mg), or a combination of theacrine (125 mg) and caffeine (150 mg) in a randomized, double-blind crossover study. Blood samples were collected over a 24-hour period and analyzed by Liquid chromatrography-mass spectrometry/mass spectrometry (LC-MS/MS) for theacrine, caffeine, and paraxanthine. Pharmacodynamic response markers, heart rate and blood pressure, were recorded. Results: Theacrine pharmacokinetics was similar following administration of theacrine alone. Caffeine coadministration increased maximum plasma concentration and area under the curve of theacrine without altering theacrine half-life. Theacrine had no impact on caffeine or paraxanthine pharmacokinetics. There was no difference between treatment groups with regard to heart rate or systolic/diastolic blood pressure. Conclusions: Coadministration of theacrine and caffeine results in a clinically significant pharmacokinetic interaction, viz., increased theacrine exposure. Enhanced oral bioavailability is the most likely mechanism by which caffeine alters theacrine exposure. However, further studies examining the contribution of presystemic elimination mechanisms, for example, efflux transport and/or gut metabolism, to theacrine bioavailability are needed to confirm the exact mechanism(s). Hemodynamic parameters were unaltered despite the pharmacokinetic interaction, suggesting that coadministration of caffeine and theacrine is safe at the doses administered.

Synthesis and Biological Evaluation of Vitamin D3 Metabolite 20S,23S-Dihydroxyvitamin D3 and Its 23R Epimer.

The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory, and antiproliferative activities.

Design, Synthesis and Biological Evaluation of Novel 5H-Chromenopyridines as Potential Anti-Cancer Agents.

A novel series of 5H-chromenopyridines was identified as anticancer agents in our continuing effort to discover and develop new small molecule anti-proliferative agents. Based on our initial lead SP-6-27 compound, we designed and synthesized novel tricyclic 5H-thiochromenopyridine and 5H-chromenopyridine analogs to evaluate the impact of an additional ring, as well as conformational flexibility on cytotoxic activity against human melanoma and glioma cell lines. All of the 5H-thiochromenopyridines have been achieved in good yields (89%-93%) using a single-step, three-component cyclization without the need for purification. The 5H-chromenopyridine analog of the potent 5H-thiochromenopyride was obtained in a good yield upon purification. All newly-prepared 5H-thiochromenopyridines showed good to moderate cytotoxicity against three melanoma and two glioma cell lines (3-15 µM). However, the 5H-chromenopyridine analogue that we prepared in our laboratory lost cytotoxic activity. The moderate cytotoxic activity of 5H-thiochromenopyridines shows the promise of developing chromenopyridines as potential anticancer agents.

Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human CYP24A1.

CYP11A1 hydroxylates vitamin D3 producing 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-dihydroxyvitamin D3 [20,23(OH)2D3] as the major and most characterized metabolites. Both display immuno-regulatory and anti-cancer properties while being non-calcemic. A previous study indicated 20(OH)D3 can be metabolized by rat CYP24A1 to products including 20S,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20S,25-dihydroxyvitamin D3, with both producing greater inhibition of melanoma colony formation than 20(OH)D3. The aim of this study was to characterize the ability of rat and human CYP24A1 to metabolize 20(OH)D3 and 20,23(OH)2D3. Both isoforms metabolized 20(OH)D3 to the same dihydroxyvitamin D species with no secondary metabolites being observed. Hydroxylation at C24 produced both enantiomers of 20,24(OH)2D3. For rat CYP24A1 the preferred initial site of hydroxylation was at C24 whereas the human enzyme preferred C25. 20,23(OH)2D3 was initially metabolized to 20S,23,24-trihydroxyvitamin D3 and 20S,23,25-trihydroxyvitamin D3 by rat and human CYP24A1 as determined by NMR, with both isoforms showing a preference for initial hydroxylation at C25. CYP24A1 was able to further oxidize these metabolites in a series of reactions which included the cleavage of C23-C24 bond, as indicated by high resolution mass spectrometry of the products, analogous to the catabolism of 1,25(OH)2D3 via the C24-oxidation pathway. Similar catalytic efficiencies were observed for the metabolism of 20(OH)D3 and 20,23(OH)2D3 by human CYP24A1 and were lower than for the metabolism of 1,25(OH)2D3. We conclude that rat and human CYP24A1 metabolizes 20(OH)D3 producing only dihydroxyvitamin D3 species as products which retain biological activity, whereas 20,23(OH)2D3 undergoes multiple oxidations which include cleavage of the side chain.

The transiently ordered regions in intrinsically disordered ExsE are correlated with structural elements involved in chaperone binding.

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to deliver protein effectors to target host cells. Transcriptional control of T3SS gene expression is generally coupled to secretion through the release of a regulatory protein. T3SS gene expression in Pseudomonas aeruginosa is regulated by extracellular secretion of ExsE. ExsE is a small 81 residue protein that appears to lack a stable structural core as indicated by previous studies. In this study, we employed various NMR methods to characterize the structure of ExsE alone and when bound to its secretion chaperone ExsC. We found that ExsE is largely unfolded throughout the polypeptide chain, belonging to a class of proteins that are intrinsically disordered. The unfolded, extended conformation of ExsE may expedite efficient secretion through the narrow path of the T3SS secretion channel to activate gene expression in a timely manner. We also found that the structurally flexible ExsE samples through conformations with localized structurally ordered regions. Importantly, these transiently ordered elements are related to the secondary structures involved in binding ExsC based on a prior crystal structure of the ExsC-ExsE complex. These findings support the notion that preexisting structured elements facilitate binding of intrinsically disordered proteins to their targets.