The overarching goal of this proposal is to develop a new class of Ribonucleotide Reductase (RR) inhibitors that are more effective than any of the existing small molecule therapies, and can overcome resistance to Hydroxyurea (HU), the only FDA approved drug for targeting RR- based cancers. In addition, this new class of inhibitors will ameliorate the severe iron chelation side effects associated with 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine.), a compound that is currently being tested in clinical trials. A lead compound, COH20, with low-<M range inhibition was developed through modification of a hit identified from screening a diverse compound library from NCI Developmental Therapeutics Program (DTP). COH20 demonstrated 90.2% inhibitory activity of recombinant human RRM2/RRM1 enzyme complex at 9<M in vitro. Moreover, at less than 10<M range, growth inhibitions of human oropharyngeal cancer KB cell line, human leukemia cell line REH, MOLT-4, and human prostate cancer LNCaP cell line in cell culture were observed. Importantly, COH20 shows significantly less toxicity to human normal fibroblast cells when compared to 3-AP. Results from computational modeling and biophysical measurements suggest that COH20 interferes with the radical transfer pathway at the interface between subunits RRM1 and RRM2, hence disrupting RRM1 and RRM2 protein-protein interaction. This represents a novel inhibition mechanism that is completely different from existing known inhibitors. Biacore binding analysis, computational docking, as well as NMR Saturation Transfer Difference (STD) studies has been performed to verify COH20 binding is specific to RRM2. These studies suggest a V-shaped binding pocket of RRM2 with COH20. Additional Structure Activity Relation (SAR) studies are proposed which will guide future refinement and optimization. Preliminary data suggest that COH20 represents a promising new class of RR inhibitors with the advantages of (a) a novel inhibition mechanism and target specificity that interferes with the radical transfer pathway at the RRM1-RRM2 interface, (b) much greater potency than HU with an IC50 at the low-<M range, and (c) ameliorating iron chelation-related side effects as seen with 3-AP. Therefore, our objectives are to optimize the properties of COH20 into a viable candidate as an anticancer drug targeting overexpressed RR-based human cancers. The specific aims for this proposal are outlined as follows: Specific Aim I. Determine the Cytotoxicity of COH20 in Human Cancer Cells In Vitro. Specific Aim II. Confirm the Efficacy of COH20 using In Vivo Mouse Models. Specific Aim III. Refinement and Validation of the Binding Mode of COH20 Using Computer Docking, Biacore Analysis, NMR Analysis and Medicinal Chemistry. In summary, we propose a multidisciplinary translational program to develop a new and promising class of RRM2 inhibitors from bench to bedside against human cancers. The current lead compound, COH20, which was synthesized by our group, may circumvent potential drug resistance problems caused by the overexpression of RRM2 as well as avoid undesirable side effects seen from the currently undergoing trials of 3-AP. COH20 is believed to interfere at the RRM1 and RRM2 binding interface and interrupt with the radical transfer process. The bis- catechol pharmacophore is also novel and represents a new possible quenching mechanism underlying free radical inhibition. Taken together, these findings establish a new direction for the future development of RR inhibitors. PUBLIC HEALTH RELEVANCE: Ribonucleotide Reductase (RR) is an important cancer drug target, however the development of inhibitors directed at it lags behind. Hydroxyurea (HU) is the only drug specifically targeting RR that is commercially available. It has shortcomings in potency and is easily susceptible to resistance development. While more potent, 3-AP chelates iron from red blood cell resulting in hypoxia, respiratory distress and Methemoglobulin as shown in human clinical trials. As the clinical principal investigator of these trials, our experience has led us to realize that a good RR inhibitor should have a) greater potency than HU, b) significantly less iron chelating ability than 3-AP, and c) specific targeting of RRM2. Very recently, our group has successfully identified "hits" from a selected NCI chemical DTP library and synthetically optimized the initial hits to obtain a significantly more potent lead compound COH20. This process was accomplished via an in vitro semi-high throughput assay using two subunits of recombinant human RR (RRM2/RRM1) and their [3H] CDP reduction activity. COH20 targets RRM2 is significant. The specificity of COH20 with RRM2 was further confirmed by computational modeling, site-directed mutagenesis studies of RRM2 protein, Biacore analysis, and NMR STD experiments. COH20 is believed to reside at the V-shaped pocket at the interface between RRM1 and RRM2 and is likely to block the free radical transfer pathway though a novel catechol radical stabilization mechanism. Considering the size and chemical composition of COH20 and the distance to the dinuclear iron center, the bound ligand (in this pocket) does not appear to be susceptible to iron chelation as 3-AP or involved in the direct quenching of the initially formed tyrosyl free radical as is the case with HU. COH20 represents a unique inhibitor that accomplishes the task of higher antitumoral activity as well as establishing a new drug design in developing RR inhibitors for the future.
NIH ExPorter
NIH ExPorter