Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90.

ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100µM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with unpredicted or allosteric sites, without the need of any binding probes.

Insights into PARP Inhibitors' Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays.

PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD(+)) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD(+)binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.

NMS-E973, a novel synthetic inhibitor of Hsp90 with activity against multiple models of drug resistance to targeted agents, including intracranial metastases.

PURPOSE: Recent developments of second generation Hsp90 inhibitors suggested a potential for development of this class of molecules also in tumors that have become resistant to molecular targeted agents. Disease progression is often due to brain metastases, sometimes related to insufficient drug concentrations within the brain. Our objective was to identify and characterize a novel inhibitor of Hsp90 able to cross the blood-brain barrier (BBB). EXPERIMENTAL DESIGN: Here is described a detailed biochemical and crystallographic characterization of NMS-E973. Mechanism-based anticancer activity was described in cell models, including models of resistance to kinase inhibitors. Pharmacokinetics properties were followed in plasma, tumor, liver, and brain. In vivo activity and pharmacodynamics, as well as the pharmacokinetic/pharmacodynamic relationships, were evaluated in xenografts, including an intracranially implanted melanoma model. RESULTS: NMS-E973, representative of a novel isoxazole-derived class of Hsp90 inhibitors, binds Hsp90α with subnanomolar affinity and high selectivity towards kinases, as well as other ATPases. It possesses potent antiproliferative activity against tumor cell lines and a favorable pharmacokinetic profile, with selective retention in tumor tissue and ability to cross the BBB. NMS-E973 induces tumor shrinkage in different human tumor xenografts, and is highly active in models of resistance to kinase inhibitors. Moreover, consistent with its brain penetration, NMS-E973 is active also in an intracranially implanted melanoma model. CONCLUSIONS: Overall, the efficacy profile of NMS-E973 suggests a potential for development in different clinical settings, including tumors that have become resistant to molecular targeted agents, particularly in cases of tumors which reside beyond the BBB.