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The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

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Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme's ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.

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Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with ∼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of ∼2-8% and crystal structures from ∼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).

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OBJECTIVE: Pathologically increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the associated Ca2+-leak from the sarcoplasmic reticulum are recognized to be important novel pharmacotherapeutic targets in heart failure and cardiac arrhythmias. However, CaMKII-inhibitory compounds for therapeutic use are still lacking. We now report on the cellular and molecular effects of a novel pyrimidine-based CaMKII inhibitor developed towards clinical use. METHODS AND RESULTS: Our findings demonstrate that AS105 is a high-affinity ATP-competitive CaMKII-inhibitor that by its mode of action is also effective against autophosphorylated CaMKII (in contrast to the commonly used allosteric CaMKII-inhibitor KN-93). In isolated atrial cardiomyocytes from human donors and ventricular myocytes from CaMKIIδC-overexpressing mice with heart failure, AS105 effectively reduced diastolic SR Ca2+ leak by 38% to 65% as measured by Ca2+-sparks or tetracaine-sensitive shift in [Ca2+]i. Consistent with this, we found that AS105 suppressed arrhythmogenic spontaneous cardiomyocyte Ca2+-release (by 53%). Also, the ability of the SR to accumulate Ca2+ was enhanced by AS105, as indicated by improved post-rest potentiation of Ca2+-transient amplitudes and increased SR Ca2+-content in the murine cells. Accordingly, these cells had improved systolic Ca2+-transient amplitudes and contractility during basal stimulation. Importantly, CaMKII inhibition did not compromise systolic fractional Ca2+-release, diastolic SR Ca2+-reuptake via SERCA2a or Ca2+-extrusion via NCX. CONCLUSION: AS105 is a novel, highly potent ATP-competitive CaMKII inhibitor. In vitro, it effectively reduced SR Ca2+-leak, thus improving SR Ca2+-accumulation and reducing cellular arrhythmogenic correlates, without negatively influencing excitation-contraction coupling. These findings further validate CaMKII as a key target in cardiovascular disease, implicated by genetic, allosteric inhibitors, and pseudo-substrate inhibitors.

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The cardiac field has benefited from the availability of several CaMKII inhibitors serving as research tools to test putative CaMKII pathways associated with cardiovascular physiology and pathophysiology. Successful demonstrations of its critical pathophysiological roles have elevated CaMKII as a key target in heart failure, arrhythmia, and other forms of heart disease. This has caught the attention of the pharmaceutical industry, which is now racing to develop CaMKII inhibitors as safe and effective therapeutic agents. While the first generation of CaMKII inhibitor development is focused on blocking its activity based on ATP binding to its catalytic site, future inhibitors can also target sites affecting its regulation by Ca(2+)/CaM or translocation to some of its protein substrates. The recent availability of crystal structures of the kinase in the autoinhibited and activated state, and of the dodecameric holoenzyme, provides insights into the mechanism of action of existing inhibitors. It is also accelerating the design and development of better pharmacological inhibitors. This review examines the structure of the kinase and suggests possible sites for its inhibition. It also analyzes the uses and limitations of current research tools. Development of new inhibitors will enable preclinical proof of concept tests and clinical development of successful lead compounds, as well as improved research tools to more accurately examine and extend knowledge of the role of CaMKII in cardiac health and disease.

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The dodecameric holoenzyme of calcium-calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca(2+) pulses to become Ca(2+) independent. A simple coincidence-detector model for Ca(2+)-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca(2+)-calmodulin is cooperative, with a Hill coefficient of approximately 3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments.

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Hemoproteins carry out diverse functions utilizing a wide range of chemical reactivity while employing the same heme prosthetic group. It is clear from high-resolution crystal structures and biochemical studies that protein-bound hemes are not planar and adopt diverse conformations. The crystal structure of an H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) contains the most distorted heme reported to date. In this study, Tt H-NOX was engineered to adopt a flatter heme by mutating proline 115, a conserved residue in the H-NOX family, to alanine. Decreasing heme distortion in Tt H-NOX increases affinity for oxygen and decreases the reduction potential of the heme iron. Additionally, flattening the heme is associated with significant shifts in the N-terminus of the protein. These results show a clear link between the heme conformation and Tt H-NOX structure and demonstrate that heme distortion is an important determinant for maintaining biochemical properties in H-NOX proteins.

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Protein-protein interactions involving the catalytic domain of protein kinases are likely to be generally important in the regulation of signal transduction pathways, but are rather sparsely represented in crystal structures. Recently determined structures of the kinase domains of the mitogen-activated protein kinase Fus3, the RNA-dependent kinase PKR, the epidermal growth factor receptor and Ca(2+)/calmodulin-dependent protein kinase II have revealed unexpected and distinct mechanisms by which interactions with the catalytic domain can modulate kinase activity.

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The p21-activated kinases (PAKs) are serine/threonine kinases that are involved in a wide variety of cellular functions including cytoskeletal motility, apoptosis, and cell cycle regulation. PAKs are inactivated by blockage of the active site of the kinase domain by an N-terminal regulatory domain. GTP-bound forms of Cdc42 and Rac bind to the regulatory domain and displace it, thereby allowing phosphorylation of the kinase domain and maximal activation. A key step in the activation process is the phosphorylation of the activation loop of one PAK kinase domain by another, but little is known about the underlying recognition events that make this phosphorylation specific. We show that the phosphorylated kinase domain of PAK2 dimerizes in solution and that this association is prevented by addition of a substrate peptide. We have identified a crystallographic dimer in a previously determined crystal structure of activated PAK1 in which two kinase domains are arranged face to face and interact through a surface on the large lobe of the kinase domain that is exposed upon release of the auto-inhibitory domain. The crystallographic dimer is suggestive of an engagement that mediates trans-autophosphorylation. Mutations at the predicted dimerization interface block dimerization and reduce the rate of autophosphorylation, supporting the role of this interface in PAK activation.

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The Abl and Src tyrosine kinases are key signaling proteins that are of considerable interest as drug targets in cancer and many other diseases. The regulatory mechanisms that control the activity of these proteins are complex, and involve large-scale conformational changes in response to phosphorylation and other modulatory signals. The success of the Abl inhibitor imatinib in the treatment of chronic myelogenous leukemia has shown the potential of kinase inhibitors, but the rise of drug resistance in patients has also shown that drugs with alternative modes of binding to the kinase are needed. The detailed understanding of mechanisms of protein-drug interaction and drug resistance through biophysical methods demands a method for the production of active protein on the milligram scale. We have developed a bacterial expression system for the kinase domains of c-Abl and c-Src, which allows for the quick expression and purification of active wild-type and mutant kinase domains by coexpression with the YopH tyrosine phosphatase. This method makes practical the use of isotopic labeling of c-Abl and c-Src for NMR studies, and is also applicable for constructs containing the SH2 and SH3 domains of the kinases.

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Soluble guanylate cyclases are nitric oxide-responsive signaling proteins in which the nitric oxide sensor is a heme-binding domain of unknown structure that we have termed the heme-NO and oxygen binding (H-NOX) domain. H-NOX domains are also found in bacteria, either as isolated domains, or are fused through a membrane-spanning region to methyl-accepting chemotaxis proteins. We have determined the crystal structure of an oxygen-binding H-NOX domain of one such signaling protein from the obligate anaerobe Thermoanaerobacter tengcongensis at 1.77-angstroms resolution, revealing a protein fold unrelated to known structures. Particularly striking is the structure of the protoporphyrin IX group, which is distorted from planarity to an extent not seen before in protein-bound heme groups. Comparison of the structure of the H-NOX domain in two different crystal forms suggests a mechanism whereby alteration in the degree of distortion of the heme group is coupled to changes on the molecular surface of the H-NOX domain and potentially to changes in intermolecular interactions.

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Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

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Cas is a member of the focal adhesion complex. Phosphorylation of Cas by Src is an important event leading to cell transformation. Using mass spectrometry, we have mapped 11 sites in Cas that are phosphorylated by Src. These sites are all located between residues 132 and 414 of Cas, in a region that is required for binding to a number of other proteins including Crk. We tested synthetic peptides modeled on Cas phosphorylation sites, and found that the sequence containing tyrosine 253 was phosphorylated by Src most efficiently. Using cells derived from Cas-deficient mice, we confirmed that Cas greatly enhanced the ability of Src to transform cells. Phosphorylation of Cas on tyrosine 253 was not required for Src to increase growth rate, suppress contact inhibition, or suppress anchorage dependence. Yet, in contrast to these growth characteristics, phosphorylation of Cas on tyrosine 253 was required for Src to promote cell migration. Thus, a single phosphorylation site on this focal adhesion adaptor protein can effectively separate cell migration from other transformed growth characteristics.

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The inadvertent fusion of the bcr gene with the abl gene results in a constitutively active tyrosine kinase (Bcr-Abl) that transforms cells and causes chronic myelogenous leukemia. Small molecule inhibitors of Bcr-Abl that bind to the kinase domain can be used to treat chronic myelogenous leukemia. We report crystal structures of the kinase domain of Abl in complex with two such inhibitors, imatinib (also known as STI-571 and Gleevec) and PD173955 (Parke-Davis). Both compounds bind to the canonical ATP-binding site of the kinase domain, but they do so in different ways. As shown previously in a crystal structure of Abl bound to a smaller variant of STI-571, STI-571 captures a specific inactive conformation of the activation loop of Abl in which the loop mimics bound peptide substrate. In contrast, PD173955 binds to a conformation of Abl in which the activation loop resembles that of an active kinase. The structure suggests that PD173955 would be insensitive to whether the conformation of the activation loop corresponds to active kinases or to that seen in the STI-571 complex. In vitro kinase assays confirm that this is the case and indicate that PD173955 is at least 10-fold more inhibitory than STI-571. The structures suggest that PD173955 achieves its greater potency over STI-571 by being able to target multiple forms of Abl (active or inactive), whereas STI-571 requires a specific inactive conformation of Abl.

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Signal transduction molecules translate extracellular inputs into their corresponding intracellular responses. Given the complexity and number of signaling pathways present in the eukaryotic cell, it is not surprising that the functions of signaling molecules are often tightly regulated. Autoinhibition is a prevalent mechanism for governing the function of signaling molecules. The relationship between the viral, oncogenic form of Src (v-Src) and the corresponding cellular proto-oncogene (c-Src) highlights the importance of inhibitory intramolecular interactions. Src provides an example of the dramatic cellular consequences arising from the loss of autoregulation.

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