Tamer A. Ahmed

Pyramidamycins A-D and 3-hydroxyquinoline-2-carboxamide; cytotoxic benzamides from Streptomyces sp. DGC1

Four new benzamides, pyramidamycins A-D (2–5) along with the new natural 3-hydroxyquinoline-2-carboxamide (6) were isolated from the crude extract of Streptomyces sp. DGC1. Additionally, five other known compounds, namely 2-aminobenzamide (anthranilamide) (1), 4′,7-dihydroxyisoflavanone (7), 2′-deoxy-thymidine, 2′-deoxy-uridine and adenosine were also isolated and identified. The structures of the new compounds 2–6 were elucidated by 1D and 2D NMR studies along with HR MS analyses. The isolated compounds 1–6 contained the same amide side chain. The isolated compounds 1–7 were biologically evaluated in comparison with landomycin A against a prostate cancer cell line (PC3) and non-small cell lung cancer cell line (H460) for 48 h and against several bacterial strains. Pyramidamycin C (4) was the most active compound against both PC3 and H460 cell lines (GI50=2.473 and 7.339 μM, respectively). Benzamides (1–3) demonstrated inhibitory activity against Kocuria rosea B-1106 (a diameter halo of 13±2 mm for 1; 10±2 mm for 2 and 3). Compound 6 was slightly active against both Escherichia coli DH5α and Micrococcus luteus NRRL B-2618 (diameter halos 8±2 and 9±2 mm, respectively). Taxonomically, the amplified 500-bp 16 S rRNA fragment of the Streptomyces sp. DGC1 had 99% identity (BLAST search) to the 16S rRNA gene of Streptomyces atrovirens strain NRRL B-16357.

Validated LC-MS/MS method for simultaneous determination of SIM and its acid form in human plasma and cell lysate： Pharmacokinetic application

Simvastatin (SIM) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor widely used in hyperlipidemia therapy. SIM has recently been studied for its anticancer activity at doses higher than those used for the hyperlipidemia therapy. This prompted us to study the pharmacokinetics of high-dose SIM in cancer patients. For this purpose, an LC–MS/MS method was developed to measure SIM and its acid form (SIMA) in plasma and peripheral blood mononuclear cells (PBMCs) obtained from patients. Chromatographic analyte separation was carried out on a reverse-phase column using 75:25 (% v/v) acetonitrile:ammonium acetate (0.1 M, pH 5.0) mobile phase. Detection was performed on a triple quadrupole mass spectrometer, equipped with a turbo ion spray source and operated in positive ionization mode. The assay was linear over a range 2.5–500 ng/mL for SIM and 5–500 ng/mL for SIMA in plasma and 2.5–250 ng/mL for SIM and 5–250 ng/mL for SIMA in cell lysate. Recovery was >58% for SIM and >75% for SIMA in both plasma and cell lysate. SIM and SIMA were stable in plasma, cell lysate and the reconstitution solution. This method was successfully applied for the determination of SIM and SIMA in plasma and PBMCs samples collected in the pharmacokinetic study of high-dose SIM in cancer patients.

Saquayamycins G-K, cytotoxic angucyclines from Streptomyces sp. Including two analogues bearing the aminosugar rednose.

Streptomyces sp. KY40-1, a strain isolated from the Kentucky Appalachian foothills, is the producer of moromycins A (18) and B (19). Further investigations of this strain led to the isolation and structure elucidation of the five new saquayamycins G-K (1-5), along with known compounds. Two of the new compounds bear the unusual aminosugar rednose, which was found here for the first time in angucyclines. The different attachment positions of this aminosugar in these two compounds indicate a high acceptor substrate flexibility of the responsible glycosyl transferase or alternatively the involvement of multiple glycosyl transferases. The cytotoxic activity of the isolated compounds was determined using human prostate cancer (PC-3) and non-small-cell lung cancer (H460) cell lines. Cell viability assays showed that saquayamycins J (4), K (5), A (7), and B (8) were most active in PC3 cells, with saquayamycin B (8) showing the highest activity (GI(50) = 0.0075 μM). The aminosugar-containing saquayamycins H (2) and saquayamycin B (8) showed the highest activity against H460 cells, with a GI(50) of 3.3 and 3.9 μM, respectively. The results presented here provide more insights into the structure-activity relationship of saquayamycins with respect to the nature, number, and linkage of sugar residues.

Simvastatin interacts synergistically with tipifarnib to induce apoptosis in leukemia cells through the disruption of RAS membrane localization and ERK pathway inhibition

Tipifarnib, a farnesyltransferase inhibitor (FTI), was initially designed to disrupt RAS farnesylation and membrane localization necessary for RAS function. However, alternative geranylgeranylation has been postulated as an escape mechanism by which RAS bypasses the effect of FTI treatment. In this study, we demonstrate that simvastatin, an HMG-CoA reductase inhibitor, augments the cytotoxic effect of tipifarnib by blocking the alternative geranylgeranylation of RAS. Notably, this effect was accompanied by disruption of RAS membrane localization and ERK downregulation. In addition, the apoptotic effect of this combination was associated with downregulation of the antiapoptotic Mcl-1 protein and activation of the caspase cascade. These findings demonstrate that combining tipifarnib and simvastatin was successful in targeting RAS/ERK signaling and inducing apoptosis in leukemia cells. Both simvastatin and tipifarnib were used at clinically achievable doses, which make the combination promising for future clinical studies.

Abstract 3778: Pharmacokinetic, safety and efficacy of high dose simvastatin in refractory and relapsed chronic lymphocytic leukemia (CLL) patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Statins are established as safe and effective anti-hypercholesterolemia drugs. Preclinical evidence shows that statins exhibit antitumor activity against different tumors by reducing prenylation of small GTPase molecules, which play a critical role in intracellular cancer cell signaling. However, these effects have only been noted at higher concentrations, which cannot be achieved with the typical dosing. In a Phase I trial, simvastatin was well tolerated by patients with relapsed or refractory myeloma or lymphomas at amaximum tolerated dose (MTD) of 7.5mg/kg twice daily. However, the pharmacokinetic (PK) was not defined and it is unknown if simvastatin plasma concentrations can reach the levels necessary for the antitumor activity observed in vitro. Here we evaluated the PK, safety and efficacy of high dose simvastatin in the context of a pilot trial enrolling patients with recurrent and refractory CLL. Methods: Patients received simvastatin orally at 7.5mg/kg twice a day for 7 days during a 21 day cycle for 6 cycles. During study treatment, patients underwent weekly or bi-weekly evaluations that included physical examination, complete blood counts and comprehensive chemistry profiles. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria (version 3.0). The NCI CLL revised guidelines for diagnosis and treatment were utilized to determine the level of clinical response. Blood samples were collected during cycle 1 at predose, 15 min and 1,2,3,6,8, and 12 hr and at predose on day-7. Simvastatin lactone and acid concentrations were measured in plasma and peripheral blood mononuclear cells (PBMCs) using a validated HPLC-MS/MS assay. Biochemical assays assessing the apoptotic effects of simvastatin were also performed in CLL cell samples (the predose and day-7) obtained from the treated patients. Results: Patients were accrued in this pilot trial. High dose simvastatin was well tolerated and there were no signs of serious adverse effects. Simvastatin exposure was dose proportional (AUC and Cmax) as compared to the doses used to treat hyperlipidemia. Exposure was lower than that required for in vitro cytotoxicity against immortalized cells. Simvastatin exposure in both plasma and PBMCs was variable with an AUC12hr (%CV) of 800.6 hr*ng/ml (88.6%) and 11.03 hr*ng/mg (119.4%), respectively. Following 7 days exposure to high dose simvastatin, CLL cells had increased the expression of cleaved PARP, an apoptotic marker. Conclusion: Preliminary results show that high dose simvastatin is well tolerated with no signs of serious side effects. Although plasma concentrations were lower than that required for in-vitro cytotoxicity, the simvastatin concentrations achieved in the clinic induced apoptosis in patient CLL cells. Further studies in CLL patients are warranted to demonstrate its efficacy alone or in combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3778. doi:1538-7445.AM2012-3778

Abstract 1225: A unified model of RAF inhibitor action determines inhibitor activity in BRAF-dependent tumors

The FDA approved RAF inhibitors vemurafenib and dabrafenib have elicited profound responses in melanoma patients with tumors harboring BRAFV600E mutation, but resistance limits their effectiveness. Furthermore, RAF inhibitors exhibit only modest efficacy in colorectal and thyroid BRAFV600E tumors and they are not effective in tumors harboring non-V600 BRAF mutations. While RAF/MEK inhibitor combinations demonstrate increased clinical efficacy, drug resistance develops as well, through similar mechanisms to those identified in RAF inhibitor monotherapy. This suggests that insufficient RAF inhibition limits treatment efficacy and indicates the need for more effective therapeutic approaches to potently and durably target RAF/ERK signaling. Although the unique biochemical properties of RAF inhibitors have been a topic of intensive investigation, a unified model of RAF inhibitor action has been lacking. Our biochemical and structural analysis in a panel of RAF inhibitors with different structural properties revealed that they exert their effects via the combined outcome of two distinct allosteric mechanisms. RAF priming and dimerization are the result of RAF/RAS-GTP interaction upon inhibitor binding to RAF and stabilization of the αC-helix in the IN (active) position. Resistance of the dimeric RAF to vemurafenib and dabrafenib is the result of stabilization of the αC-helix of the drug-bound protomer towards the OUT (inactive) position, a conformation that sterically prevents occupancy of both RAF protomers. Next generation RAF inhibitors that stabilize the αC-helix of RAF towards the IN position (αC-IN RAF inhibitors) allow occupancy of both RAF protomers and thus accomplish potent inhibition of the dimeric form of RAF. We further provide structural, biochemical and cell-based evidence that next generation αC-IN RAF inhibitors (such as MLN480, TAK632, LY3009120, AZ628 and others) will be more effective for the treatment of patients with tumors expressing non-V600 BRAF mutants and in BRAFV600E colorectal and thyroid tumors. However, αC-IN RAF inhibitors are predicted to have a low therapeutic window, since they inhibit dimeric wild-type BRAF also in normal cells. Together our findings provide a blueprint for the development of RAF inhibitors with defined biochemical properties and support therapeutic strategies in which relatively lower, non-toxic concentrations of αC-IN inhibitors will be administered in combination with currently approved RAF and MEK inhibitor regimens for the treatment of BRAFV600E melanoma, colorectal and thyroid cancers, as well as for tumors with BRAF mutations other than V600. Citation Format: Zoi Karoulia, Yang Wu, Tamer A. Ahmed, Qisheng Xin, Julien Bollard, Clemens Krepler, Xuewei Wu, Chao Zhang, Gideon Bollag, Meenhard Herlyn, James A. Fagin, Amaia Lujambio, Evripidis Gavathiotis, Poulikos I. Poulikakos. A unified model of RAF inhibitor action determines inhibitor activity in BRAF-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1225. doi:10.1158/1538-7445.AM2017-1225

Abstract A78: Simvastatin synergistically potentiates tipifarnib cytotoxic and apoptotic effects and disrupts Ras membrane localization in human leukemia cells.

Background: Tipifarnib, a farnesyl transferase inhibitor, was initially designed to prevent Ras farnesylation and its subsequent membrane localization, which is necessary for Ras mediated signaling. Although, the clinical effectiveness of tipifarnib as a single agent in solid tumors was limited, it was modest in hematologic malignancies. Alternative prenylation by geranylgeranyl transferase has been postulated as an escape mechanism by which Ras evades the effect of tipifarnib. Simvastatin, an anti-hyperlipidemic drug that inhibits 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, has been shown in several studies to induce apoptosis in cancer cells through blockade of the geranylgeranylation pathway of small GTPases. Thus, we hypothesized that simvastatin can augment the cytotoxic effect of tipifarnib by blocking the alternative Ras prenylation pathway. Our studies were carried out in a panel of leukemia cell lines. Methods: Studies were carried out using five cell lines of varied leukemic origin including HL60, K562, Molt4, Jurkat and HSB2. Cells were exposed to increasing drug concentrations for 72 hours either as a single or combination treatment with simvastatin and tipifarnib. Cell viability was measured using AlamarBlue (Invitrogen) and the combination index values were calculated based on the Chou-Talalay equations. Apoptosis was evaluated using Annexin V assay. Biochemical assays assessing the expression of caspases, c-PARP and Ras, in both membrane and cytosolic fractions, were also used. Results: The combination of tipifarnib with simvastatin was shown to induce a synergistic cytotoxic effect in all cell lines tested. Notably, our results showed that coadministarion of simvastatin and tipifarnib resulted in the induction of apoptosis through the activation of several apoptotic markers including Caspase 3, 7 and 9 as well as the upregulation of cleaved PARP. These effects were accompanied by disruption of Ras membrane localization and its sequestration into the cytosol. Conclusions: These findings demonstrate that combining simvastatin with tipifarnib effectively disrupts Ras prenylation and induces apoptosis in human leukemia cell lines. These effects were observed with simvastatin and tipifarnib concentrations that can be achieved in the clinic. Thus, the effectiveness of this combination should be explored further in future clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A78.

Abstract C216: Small molecule antitubulin drugs that evade multiple drug resistance mechanisms.

Background: A new library of small molecules with structural features resembling combretastatin analogs was synthesized and two analogues were chosen by the NCI for screening. Both agents exhibited activity in all NCI-60 panel cell lines with GI50 values Methods: Biochemical assays to assess the interaction with tubulin were carried out using purified tubulin. Immunofluorescence staining for beta-tubulin was also evaluated in PC3 cells following drug treatment. Vinblastine and docetaxel were used to demonstrate tubulin destabilizing and stabilizing modes of action, respectively. Cytotoxicity assays (72 hour GI50) were carried out in PC3, DU145, LNCaP and C4/2 prostate cancer cells and in PC3-DR cells (docetaxel resistant PC3 cells). To assess the effect of P-gp, we used NCI/ADR-Res ovarian cancer cells and L-MDR1 porcine epithelial cells and their non-expressing parent lines. Assays were performed in 96-well plates using AlamarBlue cell viability dye. Cell-cycle analysis was performed using propidium iodide staining and flow activated cell sorting. Western blot analyses were used to assess the effect of drug treatment on apoptotic pathway and on cell cycle proteins. Results: The GI50 in all cell lines was in the 1–10 nM range with no apparent resistance in P-gp expressing cells or in docetaxel resistant PC3-DR cells. The lead combretastatin analog showed a concentration dependent antitubulin interaction with a mode of action similar to vinblastine. This suggests that these compounds act by interfering with the microtubule polymerization process. Immunofluorescence staining for beta-tubulin showed diffuse staining patterns with drug increased concentration suggesting the lack of microtubule formation. Cell-cycle analysis demonstrated a G2/M cell cycle accumulation after 24-hr treatment with drug. Interestingly, there was no drug dependent effect on the number of cells in the sub-G1 fraction suggesting that the mode of cell death may be different than apoptosis. This was corroborated with the lack of caspase activation as well as the lack of cleaved-PARP. A concentration dependent p-Bcl2 was noted, however, suggesting that the drug interferes with the anti-apoptotic role of Bcl-2. Nuclear staining with Hoechst 33342 and DAPI demonstrated the presence of multinucleated cells suggesting that mitotic catastrophe could be the primary mode of cell death. This was further corroborated by the concentration dependent centrosome overduplication and presence of multiple spindle poles. Conclusion: These exquisitely potent antitubulin agents appear to overcome multiple modes of drug resistance including P-glycoprotein, p-53 deregulation, and Bcl-2. Further studies are ongoing to verify the exact molecular mechanism mediating cell death and to determine the in-vivo activity of these novel compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C216.