Pharmacophore Hybridization To Discover Novel Topoisomerase II Poisons with Promising Antiproliferative Activity

We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide and generated new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative activity on human cancer cells, endorsing their further exploration for anticancer drug discovery

Effective treatment of experimental ES-2 human ovarian cancers with a cytotoxic analog of luteinizing hormone-releasing hormone AN-207

The receptors for luteinizing hormone-releasing hormone (LHRH) are found in 80% of human ovarian carcinomas. These receptors can be used for targeted chemotherapy with cytotoxic analogs of LHRH, such as AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to [D-Lys ]LHRH. We investigated the effects of AN-207 and AN-201 on the growth of LHRH receptor-positive ES-2 human ovarian cancers. The effects of the treatment on mRNA and protein levels of human epidermal growth factor (EGF) receptors (EGFR and HER-2) in ovarian tumors were determined by RT-PCR and immunoblotting. In Experiment 1, nude mice bearing ES-2 ovarian tumors were injected i.v. with 250 nmol/kg doses of AN-207, AN-201, the carrier [D-Lys ]LHRH, an unconjugated mixture of AN-201 and [D-Lys ]LHRH or vehicle. AN-207 caused a significant ( <0.01) 59.5% inhibition in tumor growth while its components were ineffective. In Experiment 2, mice with large ES-2 tumors were treated with AN-207 or AN-201 at 250 nmol/kg. Again, AN-207, but not AN-201, inhibited tumor growth. In Experiment 3, the site of action of AN-207 was investigated. The blockade of LHRH receptors with Cetrorelix partially suppressed the antitumor effect of AN-207. Treatment with AN-207 significantly ( <0.01) decreased the expression of mRNA for EGFR, and HER-2 by 27 and 34%, respectively, as compared to controls and reduced the receptor protein levels of EGFR and HER-2 by 35 and 36%, respectively ( <0.05). The results indicate that cytotoxic LHRH analog AN-207 could be considered for chemotherapy of ovarian cancers expressing LHRH receptors

Targeted cytotoxic analogue of somatostatin AN-238 inhibits growth of androgen-independent dunning R-3327-AT-1 prostate cancer in rats at nontoxic doses

Receptors for somatostatin (SST) that are found on prostate cancers might be used for targeting of chemotherapeutic agents. Thus, doxorubicin derivative 2-pyrrolinodoxorubicin (AN-201) can be linked to SST analogue RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) to form targeted cytotoxic SST analogue AN-238. In this study, we evaluated the effects of AN-238 on the growth of SST receptor (SSTR)-positive androgen-independent Dunning R-3327-AT-1 prostate cancers in Copenhagen rats. The dose range and tumor growth-inhibitory effects of AN-238 and AN-201 were investigated in preliminary experiments. Administration of cytotoxic radical AN-201 at single i.v. doses of 110, 125, and 150 nmol/kg resulted in 0, 77.7, and 100% mortality, respectively, within 6-10 days. Four weeks after the injection of 110 nmol/kg AN-201, mean tumor volume was reduced by 35.1% (P < 0.05), as compared with controls. In contrast, a single i.v. injection of analogue AN-238 at a dose of 300 nmol/kg was nontoxic and remarkably potent in inhibiting the growth of Dunning AT-1 tumors, resulting in a 85.9% (/' < 0.01) reduction in tumor volume after 4 weeks. Treatment with AN-238 extended the survival time of tumor-bearing rats from 52.0 ±3.75 to 91.8 ±3.70 days, corresponding to a 76.5% (P < 0.01) increase. In a comprehensive experiment, we compared the effects of radical AN-201 at 115 nmol/kg, analogue AN-238 at 115 and 300 nmol/kg, carrier SST analogue RC-121 at 300 nmol/kg, and a mixture of AN-201 and RC-121 at doses of 300 nmol/kg administered i.v. Administration of AN-201 at 115 nmol/kg led to 90.0% mortality in 12 days, but animals treated with 115 nmol/kg of AN-238 showed no signs of toxicity, their tumor volume was reduced by 40.0% (/' < 0.05), and their tumor weight was reduced by 42.8% (P < 0.01) after 4 weeks, as com pared with controls. The dose of 300 nmol/kg of AN-238 was also nontoxic and diminished tumor volume by 80.9% (/' < 0.01) and tumor weight by 82.0% (P < 0.01). No reduction in tumor growth or toxic effects was observed with carrier RC-121, but after the injection of unconjugated mixture of AN-201 and RC-121 at doses of 300 nmol/kg, all rats died within 4 days. Specific high-affinity receptors for SST were found on Dunning R-3327-AT-1 tumor membranes by radioligand binding assay and were identified by reverse transcription-PCR as SSTR2. Our study indicates that cytotoxic SST analogue AN-238 can be targeted to SSTRs on tumors and produces a powerful inhibition of the growth of DunningAT-1 rat prostate cancer at doses that are nontoxic, whereas its cytotoxic component, 2-pyrrolinodoxorubicin, is toxic and ineffective

Lid domain plasticity and lipid flexibility modulate enzyme specificity in human monoacylglycerol lipase

Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason, MAGL inhibition is a promising strategy to treat pain, cancer, and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation, cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly, the enzymatic binding site of MAGL is confined by a 75-amino-acid-long, flexible cap domain, named ‘lid domain’, which is structurally similar to that found in several other lipases. However, it is unclear how lid domain plasticity affects catalysis in MAGL. By integrating extensive molecular dynamics simulations and free-energy calculations with mutagenesis and kinetic experiments, we here define a lid-domain-mediated mechanism for substrate selection and binding in MAGL catalysis. In particular, we clarify the key role of Phe159 and Ile179, two conserved residues within the lid domain, in regulating substrate specificity in MAGL. We conclude by proposing that other structurally related lipases may share this lid-domain-mediated mechanism for substrate specificity

Inhibition of growth of ES-2 human ovarian cancers by bombesin antagonist RC-3095, and luteinizing hormone-releasing hormone antagonist Cetrorelix

We evaluated the effects of the bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095, and the luteinizing hormone–releasing hormone (LH–RH) antagonist Cetrorelix, administered singly or in combination, on the growth of human ovarian carcinoma cell line ES-2, xenografted into nude mice. RC-3095 at a dose of 20 μg/day and Cetrorelix (100 μg/day), significantly reduced the volume of ES-2 tumors by 63.0% ( P<0.01) and 38.0% ( P<0.05) respectively, after 44 days of treatment, as compared with controls. The combination of RC-3095 with Cetrorelix inhibited the growth of ES-2 tumors by 66.2% ( P<0.01). Serum levels of LH were significantly decreased in the groups treated with Cetrorelix alone and/or in combination with RC-3095. RT-PCR analyses revealed that the expression of mRNA for receptors of GRP (GRPR/BRS-1) and Neuromedin B (NMBR/BRS-2) on tumors was significantly decreased in all the treated groups. The expression of mRNA for epidermal growth factor receptors (EGFR) on tumors was reduced by 36.5 % ( P<0.05) in the animals treated with Cetrorelix and by 72.5% ( P<0.05) in the group that received the combination of RC-3095 with Cetrorelix. Our results indicate that the bombesin antagonist RC-3095 and the LH-RH antagonist Cetrorelix inhibit effectively the growth of ES-2 ovarian cancers in nude mice. These antagonists and their combination could be considered for the therapy of patients with ovarian cancer

Targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 inhibits the growth of PC-82 human prostate cancer in nude mice

BACKGROUND Receptors for luteinizing hormone-releasing hormone (LH-RH) found in prostate cancers might be used for targeting of chemotherapeutic agents. Doxorubicin derivative 2-pyrrolinodoxorubicin (AN-201) can be linked to carrier analog [D-Lys6]LH-RH to form the targeted cytotoxic analog of LH-RH, AN-207. METHODS We evaluated the effects of AN-207 and its components on the growth of LH-RH receptor-positive human prostate cancer PC-82 xenografted into nude mice. Analog AN-207, radical AN-201, carrier [D-Lys6]LH-RH, or a mixture of [D-Lys6]LH-RH and AN-201 were injected intravenously once at doses of 200 nmol/kg. Tumor growth, body weight, total WBC counts, and serum prostate-specific antigen (PSA) were determined. Receptors for LH-RH on PC-82 tumors were evaluated, and the expression of mRNA for LH-RH receptors was assessed by RT-PCR. RESULTS Eight weeks after administration of cytotoxic analog AN-207, there was a 67.8% reduction in tumor volume (P < 0.01), 70.7% decrease in tumor burden (P < 0.01), and 36.5% decrease in serum PSA levels (P < 0.01) as compared with controls. Only one of 8 animals treated with AN-207 died. Cytotoxic radical AN-201 caused a 34.2% (not significant, NS) reduction in tumor volume with no change in serum PSA, and killed 3 of 8 mice due to toxicity. Carrier [D-Lys6]LH-RH and the unconjugated mixture of [D-Lys6]LH-RH and AN-201 had no effect on tumor growth. LH-RH receptors as well as the expression of their mRNA were found in PC-82 tumors. CONCLUSIONS The cytotoxic analog of LH-RH AN-207 powerfully inhibited growth of PC-82 human prostate cancer with only minor toxicity. Targeted cytotoxic LH-RH analogs may be useful for treatment of prostate cancer. Prostate 38:151–158, 1999. © 1999 Wiley-Liss, Inc

Inhibition of the UCI‐107 human ovarian carcinoma cell line by a targeted cytotoxic analog of somatostatin, AN‐238

BACKGROUND Cytotoxic analogs of somatostatin (SST), such as AN‐238, which consists of 2‐pyrrolinodoxorubicin (AN‐201) linked to the SST carrier RC‐121, can be targeted to tumors that express SST receptors. Because SST receptors are present in ovarian carcinoma cells, the authors evaluated the effect of AN‐238 on the UCI‐107 ovarian carcinoma cell line. METHODS An analysis of microsatellite alleles in cocultured SST receptor positive and receptor negative cells was used for the demonstration of in vitro targeting. The toxicity and antitumor effects of AN‐238 in nude mice bearing UCI‐107 human ovarian tumors were investigated with or without pharmacologic inhibition of serum carboxylesterases (CE). The expression of SST receptor subtypes was determined by reverse transcriptase‐polymerase chain reaction analysis, and the binding affinity of AN‐238 to SST receptors was determined by radioligand assays. RESULTS The proliferation of SST receptor positive UCI‐107 cells in vitro was inhibited preferentially by AN‐238. AN‐238 showed high‐affinity binding to UCI‐107 tumor membranes at a 50% inhibition concentration of 3.39 nM ± 0.74 nM. In vivo, the volume and weights of UCI‐107 tumors treated with AN‐238 were decreased by more than 60% (P < 0.05) compared with controls. Cytotoxic radical AN‐201 or the unconjugated mixture of AN‐201 with carrier RC‐121 had no significant effects on tumors and were toxic. In mice with inhibited serum CE activity, AN‐201 at 400 nmol/kg was lethal, whereas AN‐238 at a total dose of 800 nmol/kg caused only 22% mortality and reduced tumor weight by 69% and volume by 70% (P < 0.05 vs. control). CONCLUSIONS Targeted chemotherapy with the SST conjugate AN‐238 inhibits SST receptor positive experimental ovarian tumors. AN‐238 may provide a new treatment modality for patients with advanced ovarian carcinoma. Cancer 2001;92:1168–76. © 2001 American Cancer Society. The effectiveness and toxicity of the targeted, cytotoxic analog of somatostatin, AN‐238, was evaluated in a UCI‐107 model of human ovarian carcinoma cells. The study demonstrates that AN‐238 inhibits the growth of ovarian carcinoma cells that express somatostatin receptors, whereas its cytotoxic radical, 2‐pyrrolinodoxorubicin (AN‐201), is ineffective and toxic