Surinder S. Narula

3D solution structure of copper and silver-substituted yeast metallothioneins

3D solution structural calculations for yeast silver(I)‐substituted metallothionein (MT) and native copper(I) MT were completed using experimentally determined NOE and dihedral angle constraints, in conjunction with experimentally derived metal‐to‐Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops separated by a deep cleft containing the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N‐terminus with the overall protein volume conserved to within 6.5%.

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.

Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide

BACKGROUND Recruitment of the intracellular tyrosine kinase Syk to activated immune-response receptors is a critical early step in intracellular signaling. In mast cells, Syk specifically associates with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within the IgE receptor. The mechanism by which Syk recognizes these motifs is not fully understood. Both Syk SH2 (Src homology 2) domains are required for high-affinity binding to these motifs, but the C-terminal SH2 domain (Syk-C) can function independently and can bind, in isolation, to the tyrosine-phosphorylated IgE receptor in vitro. In order to improve understanding of the cellular function of Syk, we have determined the solution structure of Syk-C complexed with a phosphotyrosine peptide derived from the gamma subunit of the IgE receptor. RESULTS The Syk-C:peptide structure is compared with liganded structures of both the SH2 domain of Src and the C-terminal SH2 domain of ZAP-70 (the 70 kDa zeta-associated protein). The topologies of these domains are similar, although significant differences occur in the loop regions. In the Syk-C structure, the phosphotyrosine and leucine residues of the peptide ligand interact with pockets on the protein, and the intervening residues are extended. CONCLUSIONS Syk-C resembles other SH2 domains in its peptide-binding interactions and overall topology, a result that is consistent with its ability to function as an independent SH2 domain in vitro. This result suggests that Syk-C plays a unique role in the intact Syk protein. The determinants of the binding affinity and selectivity of Syk-C may reside in the least-conserved structural elements that comprise the phosphotyrosine- and leucine-binding sites. These structural features can be exploited for the design of Syk-selective SH2 antagonists for the treatment of allergic disorders and asthma.

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.

Structure-based design and synthesis of a novel class of Src SH2 inhibitors

The structure-based design and synthesis of a novel class of 2,4-disubstituted thiazoles as Src SH2 inhibitors is described. Initial results are presented, including the X-ray and NMR analysis of one thiazole inhibitor bound to Lck and Src SH2.

Bone-Targeted Src kinase inhibitors: novel pyrrolo- and pyrazolopyrimidine analogues

Src tyrosine kinase is a therapeutic target for bone diseases that has been validated by gene knockout studies. Furthermore, in vitro cellular studies implicate that Src has a positive regulatory role in osteoclasts and a negative regulatory role in osteoblasts. The potential use of Src inhibitors for osteoporosis therapy has been previously shown by novel bone-targeted ligands of the Src SH2 (e.g., AP22408) and non-bone-targeted, ATP-based inhibitors of Src kinase. Significant to this study, compounds 2-12 exemplify novel analogues of known pyrrolopyrimidine and pyrazolopyrimidine template-based Src kinase inhibitors that incorporate bone-targeting group modifications designed to provide tissue (bone) selectivity and diminished side effects. Accordingly, we report here the structure-based design, synthetic chemistry and biological testing of these compounds and proof-of-concept studies thereof.

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.

An efficient synthesis of a 4'-phosphonodifluoromethyl-3'-formyl-phenylalanine containing SRC SH2 ligand.

A CuBr-mediated, regioselective cross-coupling between methyl 2,5-diiodobenzoate (4) and [(diethoxyphosphinyl)difluoromethyl]zinc bromide is reported. Palladium-catalyzed incorporation of an amino acid side chain, followed by subsequent modifications resulted in the rapid construction of 2. Compound 2 was designed to engage Cys188 of the Src SH2 domain, however, this was not observed spectroscopically.

SMART Drug Design: Novel Phosphopeptide and ATP Mimetic-Based Small Molecule Inhibitors of the Oncogenic Protein Kinase pp60src (Src)

Over the past two decades, the oncogenic protein kinase pp60src (Src) has been the focus of tremendous biological investigations that have identified it to be a promising therapeutic target for both cancer and bone disease drug discovery. The molecular, cellular and in vivo functional properties of Src provide a detailed framework for strategies to advance small molecule inhibitors relative to both its noncatalytic (e.g., SH2) and catalytic (i.e., kinase) domains. This chapter illustrates phoshopeptide mimetic-based small molecule Src SH2 inhibitors and ATP mimetic-based, small molecule Src kinase inhibitors. Key lead compounds exemplifying Src SH2 and Src kinase inhibitors are described with respect to structural biology, drug design and biological activity (in vitro and in vivo). The term SMART refers to small molecule ARIAD therapeutics that has been particularly focused on generating and optimizing novel lead compounds such as AP22408 and AP23464. AP22408 is a prototype bone-targeted Src SH2 inhibitor that blocks binding to phosphorylated ligands and was first to achieve in vivo proof-of-concept in a bone disease model. AP23451 is a second-generation, bone-targeted Src inhibitor and determined to be effective in both osteolytic bone metastasis and osteoporosis in vivo models. AP23464 is a prototype Src kinase inhibitor that is competitive to ATP and is extraordinarily potent in vitro and provides proof-of-concept in Src-dependent, cell assays representing both bone degrading osteoclasts and cancer cells. X-ray crystallographic structures of the aforementioned Src SH2 and Src kinase inhibitors provide insight to SMART drug design strategies. Second-generation Src kinase inhibitors are amidst preclinical and clinical drug development, and such small molecules illustrate varying template classes.