COH29 (RNR Inhibitor COH29) is an orally available, aromatically substituted thiazole and inhibitor of the human ribonucleotide reductase (RNR), with potential antineoplastic activity. Upon oral administration, the RNR inhibitor COH29 binds to the ligand-binding pocket of the RNR M2 subunit (hRRM2) near the C-terminal tail. Inhibition of RNR activity decreases the pool of deoxyribonucleotide triphosphates available for DNA synthesis. The resulting decrease in DNA synthesis causes cell cycle arrest and growth inhibition. In addition, this agent may inhibit the nuclear enzyme poly (ADP-ribose) polymerase (PARP) 1, which prevents the repair of damaged DNA and causes both the accumulation of single and double strand DNA breaks and the induction of apoptosis.

RNR:16 μM.

COH29成功克服了Y细胞对吉西他滨和羟基脲的抗药性。在NCI 60人类Y系列的大多数细胞系中，它强效抑制增殖，但对正常成纤维细胞或内皮细胞几乎无影响。NMR、定点突变技术和表面等离子体共振生物传感器研究确认COH29与提议的配体结合口袋结合，并为RRM1-RRM2四聚体结构的组装阻断提供了证据[1]。

COH29 (50/100 mg/kg, b.i.d., p.o.)在MOLT-4肿瘤移植模型中显示出剂量依赖性的抑制增长效果，尤其在治疗第12天时抑制效果显著。在携带TOV11D肿瘤移植物的小鼠中，连续7天使用COH29 (200/300/400 mg/kg/天)同样展示出剂量依赖性的肿瘤生长抑制效果。与对照组相比，肿瘤增长被显著抑制[1]。

For kinase assays following immunoprecipitation of FLAG-CDK7 protein from HCT116 or FLAG-CDK12 from 293A cellular lysates, cells are first treated with THZ1, THZ1-R, or DMSO for 4 hrs at 37°C. Cells are then harvested by lysis in 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, and protease/phosphatase cocktails. Exogenous CDK7 or CDK12 proteins are immunoprecipitated from cellular lysates using FLAG antibody- conjugated agarose beads. Precipitated proteins are washed with lysis buffer 6 times, followed by 2 washes with kinase buffer (40 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5% glycerol) and subjected to in vitro kinase assays at 30°C for 45 minutes using 1 μg of the large subunit of RNAPII (RPB1) as substrate and 25 μM ATP and 10 μCi of 32P ATP. Kinase assays using recombinant CDK7/TFIIH/MAT1 are conducted in the manner as described above using 25 ng of CAK complex per reaction. For kinase assays designed to test time-dependent inactivation of CDK7 kinase activity, CAK complex is pre-incubated with indicated concentrations of THZ1, THZ1-R, or DMSO in kinase buffer without ATP for 4 hrs at 30°C prior to being subjected to kinase assay conditions[1].

Cells is seeded into 96-well plates in 100 μL of complete medium at 2000 to 5000 cells per well, depending on the cell line's growth rate. After overnight incubation, test compound is added to each well at various concentrations in 50 μL of culture medium. After a further incubation for 96 hours at 37°C, fluorescein diacetate (final concentration: 10 mg/mL) and eosin Y [final concentration: 0.1% (w/v)] is added to each well, and the cells is incubated for an additional 20 minutes at 37°C. Cytotoxicity is assessed by Digital Imaging Microscopy System detection Viability is assessed using MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] as previously described[1].