Marie Rose van Schravendijk

Structural basis of Src tyrosine kinase inhibition with a new class of potent and selective trisubstituted purine-based compounds.

The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including osteoporosis and cancer metastases. Src has thus emerged as a molecular target for the discovery of small‐molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. Here, we present crystal structures of the kinase domain of Src in complex with two purine‐based inhibitors: AP23451, a small‐molecule inhibitor designed to inhibit Src‐dependent bone resorption, and AP23464, a small‐molecule inhibitor designed to inhibit the Src‐dependent metastatic spread of cancer. In each case, a trisubstituted purine template core was elaborated using structure‐based drug design to yield a potent Src kinase inhibitor. These structures represent early examples of high affinity purine‐based Src family kinase–inhibitor complexes, and they provide a detailed view of the specific protein–ligand interactions that lead to potent inhibition of Src. In particular, the 3‐hydroxyphenethyl N9 substituent of AP23464 forms unique interactions with the protein that are critical to the picomolar affinity of this compound for Src. The comparison of these new structures with two relevant kinase–inhibitor complexes provides a structural basis for the observed kinase inhibitory selectivity. Further comparisons reveal a concerted induced‐fit movement between the N‐ and C‐terminal lobes of the kinase that correlates with the affinity of the ligand. Binding of the most potent inhibitor, AP23464, results in the largest induced‐fit movement, which can be directly linked to interactions of the hydrophenethyl N9 substituent with a region at the interface between the two lobes. A less pronounced induced‐fit movement is also observed in the Src–AP23451 complex. These new structures illustrate how the combination of structural, computational, and medicinal chemistry can be used to rationalize the process of developing high affinity, selective tyrosine kinase inhibitors as potential therapeutic agents.

Targeting protein kinases for bone disease: Discovery and development of Src inhibitors

The dynamic and highly regulated processes of bone remodeling involve two major cells, osteoclasts and osteoblasts, both of which command a multitude of cellular signaling pathways involving protein kinases. Of the possible kinases in these cells, Src tyrosine kinase stands out as a promising therapeutic target for bone disease as validated by Src knockout mouse studies and in vitro cellular experiments, suggesting a regulatory role for Src in both osteoclasts (positive) and osteoblasts (negative). Advances in structural studies involving both Src and non-Src family kinases, in activated and unactivated protein states, have uncovered key binding site interactions that have led to the design of potent Src inhibitors. The lead compounds originate from a variety of synthetic templates and have demonstrated nM potency in enzymatic/binding assays and efficacy in animal models of bone disease. This review will provide a current understanding of critical Src signalling pathways in osteoclasts and osteoblasts, while detailing the structure-based design and screening-based lead discovery of Src inhibitors to be developed as therapeutic agents for bone disease.

Bone-Targeted 2,6,9-Trisubstituted Purines: Novel Inhibitors of Src Tyrosine Kinase for the Treatment of Bone Diseases

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.

Bone-targeted pyrido[2,3-d]pyrimidin-7-ones: potent inhibitors of Src tyrosine kinase as novel antiresorptive agents.

The design of bone-targeted pyrido[2,3-d]pyrimidin-7-ones as Src tyrosine kinase inhibitors is described. Leveraging SAR from known compounds and using structure-based methods, we were able to rapidly incorporate bone binding components, which maintained, and even increased potency against the target enzyme. Compound 4 displayed a high affinity for hydroxyapatite, a major constituent of bone, and demonstrated antiresoprtive activity in our cell-based assay.

SAR of carbon-linked, 2-substituted purines: synthesis and characterization of AP23451 as a novel bone-targeted inhibitor of Src tyrosine kinase with in vivo anti-resorptive activity.

Targeted disruption of the pp60src (Src) gene has implicated this tyrosine kinase in osteoclast‐mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone‐related diseases. Here, we describe structure activity relationships of a novel series of carbon‐linked, 2‐substituted purines that led to the identification of AP23451 as a potent inhibitor of Src tyrosine kinase with antiresorptive activity in vivo. AP23451 features the use of an arylphosphinylmethylphosphinic acid moiety which confers bone‐targeting properties to the molecule, thereby increasing local concentrations of the inhibitor to actively resorbing osteoclasts at the bone interface. AP23451 exhibited an IC50 = 68 nm against Src kinase; an X‐ray crystal structure of the molecule complexed with Src detailed the molecular interactions responsible for its Src inhibition. In vivo, AP23451 demonstrated a dose‐dependent decrease in PTH‐induced hypercalcemia. Moreover, AP23517, a structurally and biochemically similar molecule with comparable activity (IC50 = 73 nm) except devoid of the bone‐targeting element, demonstrated significantly reduced in vivo efficacy, suggesting that Src activity was necessary but not sufficient for in vivo activity in this series of compounds.

Production of secreted, soluble human two-domain CD4 protein in Escherichia coli

The two-domain form of recombinant soluble human CD4 (rsCD4(183)) has been used for structural studies and to probe the interaction of CD4 with its ligands. rsCD4(183) has generally been produced in Escherichia coli in the form of inclusion bodies. The generation of conformationally native protein from these inclusion bodies is a time-consuming and inefficient process, requiring a refolding step. Here, we describe a procedure for producing 2-4 mg of secreted, conformationally native rsCD4(183) per liter of E. coli, completely bypassing the requirement for protein refolding in vitro. Furthermore, the yield of active protein is comparable to that reported for expression systems that generate inclusion bodies.