Gary Griffiths

Discovery and characterization of 2-anilino-4- (thiazol-5-yl)pyrimidine transcriptional CDK inhibitors as anticancer agents.

The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e., CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mitotic inhibitor groups. We describe the characterization of the transcriptional inhibitor class in terms of kinase inhibition profile, cellular mode of action, and selectivity for transformed cells. A structural selectivity rationale was used to optimize potency and biopharmaceutical properties and led to thexa0development of a transcriptional inhibitor, 3,4-dimethyl-5-[2-(4-piperazin-1-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one, with anticancer activity in animal models.

Discovery of N-Phenyl-4-(thiazol-5-yl)pyrimidin-2-amine Aurora Kinase Inhibitors

Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds. The anticancer effects of lead compound 4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine (18; K(i) values of 8.0 and 9.2 nM for aurora A and B, respectively) were shown to emanate from cell death following mitotic failure and increased polyploidy as a consequence of cellular inhibition of aurora A and B kinases. Preliminary in vivo assessment showed that compound 18 was orally bioavailable and possessed anticancer activity. Compound 18 (CYC116) is currently undergoing phase I clinical evaluation in cancer patients.

Collateral sensitivity of multidrug-resistant cells to the orphan drug tiopronin.

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues.

Design, Synthesis, and Evaluation of 2-Methyl- and 2-Amino-N-aryl-4,5-dihydrothiazolo-[4,5-h]quinazolin-8-amines as Ring-Constrained 2-Anilino-4-(thiazol-5-yl)pyrimidine Cyclin-Dependent Kinase Inhibitors

Following the recent discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin dependent kinase (CDK) inhibitors, a program was initiated to evaluate related ring-constrained analogues, specifically, 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines for inhibition of CDKs. Here we report the rational design, synthesis, structure-activity relationships (SARs), and cellular mode-of-action profile of these second generation CDK inhibitors. Many of the analogues from this chemical series inhibit CDKs with very low nanomolar K(i) values. The most potent compound reported in this study inhibits CDK2 with an IC(50) of 0.7 nM ([ATP] = 100 microM). Furthermore, an X-ray crystal structure of 2-methyl-N-(3-(nitro)phenyl)-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine (11g), a representative from the chemical series in complex with cyclin A-CDK2, is reported, confirming the design rationale and expected binding mode within the CDK2 ATP binding pocket.

(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

PURPOSEnTo develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy.nnnMATERIALS AND METHODSnThis study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses.nnnRESULTSn(89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7).nnnCONCLUSIONnWe have developed a (89)Zr-oxine complex cell tracking technique for use with PET that is applicable to a broad range of cell types and could be a valuable tool with which to evaluate various cell-based therapies.

Preparation and long-term biodistribution studies of a PAMAM dendrimer G5–Gd-BnDOTA conjugate for lymphatic imaging

AIMSnTo demonstrate the use of gadolinium (Gd)-labeled dendrimers as lymphatic imaging agents and establish the long-term biodistribution (90-day) of this type of agent in mice.nnnMATERIALS & METHODSnA G5-Gd-BnDOTA dendrimer was prepared and injected into mice and monkeys for MR lymphangiography, and long-term biodistribution of the conjugate was studied.nnnRESULTSnAdministration of G5-Gd-BnDOTA in mice demonstrated a rapid uptake in the deep lymphatic system while injection in monkeys showed enhanced internal iliac nodes, indicating its general utility for lymphatic tracking. Biodistribution studies to 90 days showed that gadolinium conjugate is slowly being eliminated from the liver and other organs.nnnCONCLUSIONnThe use of G5-Gd-BnDOTA holds great promise for lymphatic imaging, but its slow clearance from the body might hamper its eventual clinical translation.

Immuno-PET Imaging of Tumor Endothelial Marker 8 (TEM8)

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with 89Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. 89Zr-df–L2mAb was synthesized using a desferioxamine–L2mAb conjugate (df–L2mAb); 125I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. 89Zr-df–L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. 89Zr-df–L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. 125I-L2mAb and 89Zr-df–L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of 89Zr-df–L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. 89Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies 89Zr-df–L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. 89Zr-df–L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, 89Zr-df–L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as 89Zr-df–L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.