LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer.

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs.

Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity.

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1ß (IL-1ß), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and ß-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 µmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)(70) = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.

Efficacy of low-dose oral metronomic dosing of the prodrug of gemcitabine, LY2334737, in human tumor xenografts.

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.

Human carboxylesterase-2 hydrolyzes the prodrug of gemcitabine (LY2334737) and confers prodrug sensitivity to cancer cells.

PURPOSE: The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity. EXPERIMENTAL DESIGN: Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts. RESULTS: Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3-GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P ≤ 0.001). CONCLUSIONS: CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression.

Quantification of gemcitabine incorporation into human DNA by LC/MS/MS as a surrogate measure for target engagement.

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

Kinetic validation of the use of carboxydichlorofluorescein as a drug surrogate for MRP5-mediated transport.

Multidrug resistance protein-5 (MRP5, ABCC5) is a member of the ATP-binding cassette transporter superfamily that effluxes a broad range of natural and xenobiotic compounds such as cyclic GMP, antiviral compounds, and cancer chemotherapeutic agents including nucleoside-based drugs, antifolate agents and platinum compounds. In cellular assays, MRP5 transfectants are less fluorescent after incubation with 5-chloromethylfluorescein diacetate (CMFDA). The present study examines the uptake of a close fluorescent analog, carboxydichlorofluorescein (CDCF), and drug substrates into inside-out membrane vesicles prepared from MRP transfected cells. MRP5-mediated uptake of CDCF was ATP-dependent and GSH-independent and possessed a Km of 12 microM and a Vmax of 56 pmol/min/mg prot. Comparison of kinetic parameters with drug substrates such as methotrexate (MTX), pemetrexed (Alimta), and the metabolite of 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (5-FdUMP) (Km values of 0.3-1.3 mM) indicated that MRP5 has a 25-100-fold higher affinity for CDCF than for these drugs and that they share a common transport binding site. In addition, the potency of MRP5 inhibitors such as probenecid, MK571, and the phosphodiesterase 5 inhibitors correlated well between the uptake of CDCF and MTX. A survey of CDCF uptake by other MRPs revealed that MRP2 (ABCC2) also demonstrated ATP-dependent uptake with a Km of 19 microM and Vmax of 95.5 pmol/min/mg prot, while MRP1 (ABCC1) and MRP4 (ABCC4) had little to no uptake. Taken together, these data indicate that CDCF is a useful fluorescent drug surrogate with which to measure ATP-dependent MRP5-mediated transport.

The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites.

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were approximately 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K(m) of 1.1 mmol/L and V(max) of 439 pmol/min/mg protein. Uptake of 250 micromol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 micromol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters.

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Characterization and application of a vinblastine-selected CACO-2 cell line for evaluation of p-glycoprotein.

The role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to up-regulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.

Identification and characterization of the canine multidrug resistance-associated protein.

Human multidrug resistance protein 1 (MRP1) confers resistance to the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins. It is also capable of binding to and transporting the glutathione S-conjugate leukotriene C4 (LTC4) in isolated membrane vesicles. To explore species differences that exist between MRP orthologs, we cloned and characterized the mRNA encoding a canine ortholog of human MRP1-designated canine MRP1 (canMRP1). The canMRP1 mRNA encodes a protein of identical length as MRP1. Sequence alignment revealed that canMRP1 was 92% identical to MRP1 and 88% identical to murine mrp1. Five polymorphisms were identified in the canMRP1 cDNA coding sequence, including one resulting in an amino acid change from alanine to serine at aa149 (canMRP1-A and B alleles, respectively). canMRP1 was expressed and functionally characterized in HeLa and A2780 cells. Both alleles conferred an increased resistance to vincristine and etoposide and transported LTC4. The compound LY402913, a modulating agent developed against human MRP1, was able to sensitize canMRP1-expressing cells to vincristine. The modulation of canMRP1 by LY402913 was additionally confirmed by the calcein-AM accumulation assay. LY402913 inhibited the efflux of calcein in canMRP1-expressing cells. Thus, canMRP1 is similar to MRP1 in conferring resistance to vincristine and etoposide, transporting calcein-a.m., and being inhibited by LY402913. However, despite the high degree of sequence identity and functional similarity to MRP1, canMRP1 transgene failed to confer resistance to doxorubicin either in HeLa or A2780 cells. Knowledge of species differences between canine and human proteins will aid in the design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical evaluation of MRP1 modulators.