Structure-guided selection of specificity determining positions in the human kinome

Background: The human kinome contains many important drug targets. It is well-known that inhibitors of protein kinases bind with very different selectivity profiles. This is also the case for inhibitors of many other protein families. The increased availability of protein 3D structures has provided much information on the structural variation within a given protein family. However, the relationship between structural variations and binding specificity is complex and incompletely understood. We have developed a structural bioinformatics approach which provides an analysis of key determinants of binding selectivity as a tool to enhance the rational design of drugs with a specific selectivity profile. Results: We propose a greedy algorithm that computes a subset of residue positions in a multiple sequence alignment such that structural and chemical variation in those positions helps explain known binding affinities. By providing this information, the main purpose of the algorithm is to provide experimentalists with possible insights into how the selectivity profile of certain inhibitors is achieved, which is useful for lead optimization. In addition, the algorithm can also be used to predict binding affinities for structures whose affinity for a given inhibitor is unknown. The algorithm’s performance is demonstrated using an extensive dataset for the human kinome. Conclusion: We show that the binding affinity of 38 different kinase inhibitors can be explained with consistently high precision and accuracy using the variation of at most six residue positions in the kinome binding site. We show for several inhibitors that we are able to identify residues that are known to be functionally important

Evaluation of Modified PEG-Anilinoquinazoline Derivatives as Potential Agents for EGFR Imaging in Cancer by Small Animal PET

Purpose: The in vivo evaluation of three modified polyethylene glycol (PEG)-anilinoquinazoline derivatives labeled with 124 I, 18 F, and 11 C as potential positron emission tomography (PET) bioprobes for visualizing epidermal growth factor receptor (EGFR) in cancer using small animal PET. Procedures: Xenograft mice with the human glioblastoma cell lines U138MG (lacking EGFR expression) and U87MG.wtEGFR (transfected with an overexpressing human wild-type EGFR gene) were used. Static and dynamic PET imaging was conducted for all three PEGylated compounds. Tumor necrosis, microvessel density, and EGFR levels were evaluated by histopathology and enzyme-linked immunosorbent assay. Results: Nineteen animal models were generated (two U138MG, three U87MG, 14 with both U138MG and U87MG bilateral masses). In static images, a slight increase in tracer uptake was observed in tumors, but in general, there was no retention of tracer uptake over time and no difference in uptake between U138MG and U87MG masses. In addition, no significant uptake was demonstrated in dynamic scans of the 18 F-PEG tracer. No necrosis was present except in four animals. MVD was 9.6 and 48 microvessels/×400 field in the U138GM and U87GM masses

LRP-1 Promotes Cancer Cell Invasion by Supporting ERK and Inhibiting JNK Signaling Pathways

Background: The low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor mediating the clearance of various extracellular molecules involved in the dissemination of cancer cells. LRP-1 thus appeared as an attractive receptor for targeting the invasive behavior of malignant cells. However, recent results suggest that LRP-1 may facilitate the development and growth of cancer metastases in vivo, but the precise contribution of the receptor during cancer progression remains to be elucidated. The lack of mechanistic insights into the intracellular signaling networks downstream of LRP-1 has prevented the understanding of its contribution towards cancer. Methodology/Principal Findings: Through a short-hairpin RNA-mediated silencing approach, we identified LRP-1 as a main regulator of ERK and JNK signaling in a tumor cell context. Co-immunoprecipitation experiments revealed that LRP-1 constitutes an intracellular docking site for MAPK containing complexes. By using pharmacological agents, constitutively active and dominant-negative kinases, we demonstrated that LRP-1 maintains malignant cells in an adhesive state that is favorable for invasion by activating ERK and inhibiting JNK. We further demonstrated that the LRP-1-dependent regulation of MAPK signaling organizes the cytoskeletal architecture and mediates adhesive complex turnover in cancer cells. Moreover, we found that LRP-1 is tethered to the actin network and to focal adhesion sites and controls ERK and JNK targeting to talin-rich structures. Conclusions: We identified ERK and JNK as the main molecular relays by which LRP-1 regulates focal adhesion disassembly of malignant cells to support invasion

Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease