Catalytic Site Inhibitors Research Articles

Abstract 1789: Auristatin-based antibody-drug conjugates are synergistic in combination with PI3K-AKT-mTOR pathway inhibitors in hematologic malignancies and carcinoma

Abstract Antibody-drug conjugates (ADCs) that couple the potent anti-mitotic agents monomethyl auristatin E or F (MMAE or MMAF) to tumor antigen specific monoclonal antibodies have demonstrated antitumor activity in preclinical models. We report that the combination of auristatin-based ADCs with inhibitors of the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signaling pathway improves cytotoxic activity in Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and renal cell carcinoma (RCC) cell line models. Implicated in tumor cell growth and survival, the PI3K-AKT-mTOR pathway is frequently hyperactivated in carcinomas and hematologic malignancies with elevated AKT activity reported in ∼64% of Hodgkin Reed-Sternberg (HRS) cells and 25-52% of primary patient diffuse large B-cell lymphomas. We produced dose response curves for auristatin ADCs and PI3K-AKT-mTOR pathway inhibitors, alone and in combination, and then performed combination index (CI) analysis using the Chou-Talalay method. The CD30-targeting ADC brentuximab vedotin (SGN-35) and the CD19-targeting ADC (SGN-19A) were synergistic (CI < 0.7) with the allosteric mTORC1 inhibitors sirolimus and temsirolimus in panel of lymphoma cell lines. Similarly, a CD70 targeting ADC (SGN-75) was synergistic with sirolimus and temsirolimus in RCC cell lines. Unconjugated MMAE drug was synergistic in combination with sirolimus and temsirolimus, proving the auristatin component of the ADC is responsible for enhanced combinatorial activity. Next, we found that brentuximab vedotin, SGN-19A, and SGN-75 were each synergistic with NVP-BEZ235, a dual specificity PI3K and mTORC1/2 catalytic site inhibitor. Therefore, the synergy observed with auristatin ADCs is generalizable to inhibitors of the PI3K-AKT-mTOR pathway and not unique to rapamycin analogs. Finally, the brentuximab vedotin combination with temsirolimus was evaluated in a L428 mouse xenograft model of B-like HL. Temsirolimus and brentuximab vedotin alone showed antitumor activity in the L428 model; however, the brentuximab vedotin plus temsirolimus combination had significantly greater (P-value = 0.001) antitumor activity than either drug alone. The mechanism for the synergy observed in these combinations may involve reduced expression of antiapoptotic proteins by mTOR pathway inhibitors, which sensitizes cancer cells to auristatin ADCs. Collectively, these data suggest the potential for auristatin ADC plus mTOR inhibitor cotherapy in PI3K-AKT-mTOR pathway hyperactivated lymphomas and carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1789. doi:10.1158/1538-7445.AM2011-1789

Abstract 4463: OSI-027, a dual mTORC1/mTORC2 inhibitor, induces autophagy in cancer cells

Abstract Inhibitors of mTORC1 have proven clinical activity in renal cell carcinoma (RCC), indicating that mTOR is an important signaling hub in this tumor type. mTOR exists in two functionally distinct protein complexes: mTORC1 and mTORC2. mTORC1 regulates multiple cellular functions including protein translation and tumor metabolism While this complex binds rapamycin some aspects of mTORC1 function are rapamycin insensitive. mTORC2 controls distinct cellular functions including survival and is not directly inhibited by rapalogs. In order to evaluate the relative contribution of mTORC1 and mTORC2 to the survival of RCC cells, we compared the effects of rapamycin, an allosteric inhibitor of mTORC1, to OSI-027, a selective catalytic-site inhibitor of mTORC1/mTORC2. In RCC cell lines, OSI-027 provided greater maximal inhibition of proliferation than rapamycin, with reduction of cell viability to less than baseline in 5 of 12 cell lines, however caspase 3/7-dependent apoptosis was observed only in one cell line. We identified a subset of RCC cell lines which were sensitive to OSI-027 but not rapamycin, and another set which was relatively sensitive to both. Comparison of gene expression profiles of these groups identified MAP1LC3, encoding LC3, a critical effector of autophagy, as significantly upregulated in cell lines which were preferentially sensitive to OSI-027. We therefore sought to further define the role of autophagy in response to OSI-027 treatment. Autophagy is a catabolic process by which cells consume proteins and organelles to promote survival under adverse conditions, and which can result in cell death. Both mTORC1 and mTORC2 have been shown to negatively regulate autophagy, and inhibition of mTOR has been shown to induce formation of autophagic vesicles. Treatment in vitro with OSI-027 resulted in the conversion of LC3 to the lipidated form LC3-II as well as accumulation of LC3 in autophagosomes, robust indicators of autophagy. Increased autophagosome content occurred in a dose-dependent manner and is inversely proportional to viable cell number although induction of apoptosis was not observed, implying that in these cells OSI-027-induced autophagy leads to complete growth arrest or apoptosis-independent cell death. OSI-027-mediated induction of autophagy occurred in the majority of RCC cell lines tested, whereas little or no autophagy resulted from rapamycin treatment. In xenograft tumors sensitive to OSI-027 but not rapamycin, we analyzed induction of LC3-II and apoptosis in tumor lysates. Consistent with our observations in vitro, we observed that treatment with OSI-027 but not rapamycin resulted in induction of autophagy in vivo, while neither compound induced apoptosis. Together these data indicate that autophagy may play a key role in mediating OSI-027 but not rapamycin efficacy and supports further exploration of the utility of dual mTORC1/mTORC2 inhibitors for the treatment of renal cell carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4463. doi:10.1158/1538-7445.AM2011-4463

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Abstract 1632: Co-targeting mTOR and IGF-1R/IR results in synergistic activity against a broad array of tumor cell lines, independent of KRAS mutation status

Abstract The IGF-1 receptor (IGF-1R) and insulin receptor (IR) are known to couple via IRS-1 to the PI3K signaling axis. Upon activation, these receptor tyrosine kinases stimulate PI3K, initiating a cascade of ser/thr kinases, including mTOR. mTOR is associated with two complexes: mTORC1, rapamycin sensitive, and mTORC2, which does not bind rapamycin. Since mTOR functions downstream of IGF-1R and IR, it is thought that tumor cells sensitive to IGF-1R inhibition would also respond to mTORC1/mTORC2 inhibition. To test this hypothesis we evaluated the effects of OSI-906, a selective small molecule dual kinase inhibitor of IGF-1R and IR and OSI-027, a selective catalytic-site inhibitor of mTORC1/mTORC2, alone and in combination in cell proliferation assays using a broad array of tumor cell lines. Across multiple tumor types, cells with activating KRAS mutations were typically less sensitive to OSI-027 than those expressing wild-type KRAS. In contrast, mutant KRAS did not reduce sensitivity to OSI-906. Cell lines with activating mutations in PIK3CA or loss of PTEN were sensitive to OSI-027 but the presence of a concurrent mutation in KRAS decreased their sensitivity to OSI-027. In KRAS mutant cells, OSI-906 was more effective than OSI-027 at reducing phospho-AKT levels. Conversely, in tumor cell lines with wild-type KRAS cells, OSI-027 more effectively reduced phospho-AKT levels compared to OSI-906. Similar effects were observed in a pair of isogenic cell lines engineered to express either wild-type or mutant KRAS. The observation that tumor cell lines exhibit differential sensitivity to inhibitors of IGF-1R/IR or mTORC1/mTORC2 supports the concept that IGF-1R and IR can activate multiple pathways and are not limited to signaling through the mTOR axis. Also, mTOR is a central signaling hub which integrates signaling from multiple inputs, not only the IGF-1R/IR cascade. The combination of OSI-906 and OSI-027 synergistically inhibited the proliferation of cancer cell lines, including those expressing mutant KRAS. In some cases, the combination also resulted in synergistic induction of apoptosis. These data provide support for further exploration of the potential utility of combining OSI-906 (dual IGF-1R/IR inhibitor) with OSI-027 (dual mTORC1/mTORC2 inhibitor) for the treatment of human cancers, including those with activating mutations in KRAS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1632.

1-(3-Deoxy-3-fluoro-β- d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic, crystallographic and modelling studies

Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3′ position of the glucose moiety and a variety of pyrimidine derivatives at the 1′ position. The best of this carbohydrate-based family of five inhibitors displays a K i value of 46 μM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284–286 of the 280s loop, Ala383 of the 380s loop, and His341 of the β-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on β- d-glucose pyrimidine derivatives, tautomeric forms of ligands 1– 5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using α- d-glucose as a benchmark model, a series of substitutions for glucose –OH at the 3′ (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native –OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors.

Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I—Inhibitor Design and Validation

A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

Abstract B139: Molecular pharmacology and antitumor activity of PHT-427 a novel AKT/PDPK1 pleckstrin homology domain inhibitor

Abstract The pleckstrin homology (PH) domain is a highly conserved three dimensional superfold found in many high valued pharmaceutical target proteins. While for the majority of PH domain proteins PtdIns binding is weak and non-specific, a subclass of approximately 40 PH domain containing proteins show high affinity for phosphatidylinositol-3-phosphates (PtdIns-3-P) causing their translocation to the plasma membrane and/or activation. Akt and PDKP1 are two such PH domain containing serine threonine kinase members of the PtdIns-3-kinase signaling pathway that plays a critical role in activating survival and anti-apoptotic signaling in cancer cells. This pathway is constitutively activated through mutation, overexpression and loss of the tumor suppressor PTEN in many different cancer types. There is emerging evidence that Akt and PDKP1 play complimentary yet independent roles in signaling by the pathway. Attempts to develop ATP catalytic site inhibitors of Akt and PDKP1 have generally resulted in toxic agents because of inhibition of off-target kinase family members. Through a process of reiterative molecular docking and structure refinement using a proprietary computational platform, and measurement of binding affinities by surface plasmon resonance (SPR) spectroscopy, we have developed inhibitors of these proteins that bind to the PH domain. We report here the development of PHT-427 a compound that binds to the PH domain of AKT and PDKP1. To develop PHT-427 an initial pharmacophore that bound to the PH domain PtdIns binding pocket was first identified. During the docking/modeling we identified a channel whithin the PH domains of both AKT and PDPK1 that could accommodate the binding of an alkyl chain. A series of analogs with C-4 to C-16 alkyl chain length were therefore synthesized and PHT-427 (C-12 chain) was found to bind with the highest affinity to the PH domains of both PDPK1 and AKT. PHT-427 gives a transient inhibition of AKT signaling in cells and tumor xenografts and a longer lasting inhibition of PDPK1 signaling. This inhibition correlated with decreased activation of downstream signaling targets for both proteins. PHT-427 given ias an oral formulation on a twice daily schedule inhibited the growth rate of human tumor xenografts in immunodeficient mice with up to an 80% inhibition in the most sensitive tumors and showed greater activity than analogs with C4, C6 or C8 alkyl chains. Tumors with a K-Ras mutation were less sensitive to PHT-427 as it has been seen for other inhibitors of the PtdIns-3-K signaling pathway. Combination antitumor studies showed that PHT-427 has greater than additive activity with paclitaxel in breast cancer and with erlotinib in non small cell lung cancer. There was minimal toxicity of PH-427 at doses that gave antitumor activity. PHT-427 given twice daily for 5 days caused no weight loss or change in blood chemistry. In summary we have identified a novel PH domain binding inhibitor of PDPK1/AKT signaling that has minimal toxicity and antitumor activity against human tumor xenografts.. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B139.

Cyclic Nucleotide Binding GAF Domains from Phosphodiesterases: Structural and Mechanistic Insights

GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, -10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

Structure-Based Virtual Ligand Screening: Recent Success Stories

Today, computational methods are commonly used in all areas of health science research. Among these methods, virtual ligand screening has become an established technique for hit discovery and optimization. In this review, we first introduce structure-based virtual ligand screening and briefly comment on compound collections and target preparations. We also provide the readers with a list of resources, from chemoinformatics packages to compound collections, which could be helpful to implement a structure-based virtual screening platform. Then we discuss seventeen recent success stories obtained with various receptor-based in silico methods, performed on experimental structures (X-ray crystallography, 12 cases) or homology models (5 cases) and concerning different target classes, from the design of catalytic site inhibitors to drug-like compounds impeding macromolecular interactions. In light of these results, some suggestions are made about areas that present opportunities for improvements.

Nonproteolytic Induction of Catalytic Activity into the Single-Chain Form of Urokinase-Type Plasminogen Activator by Dipeptides

Serine proteases are initially synthesized as single-chain proenzymes with activities that are many orders of magnitude lower than those of the mature enzyme. Proteolytic cleavage of an exposed loop liberates a new amino terminus that inserts into a hydrophobic pocket and forms a stabilizing salt bridge with a ubiquitously conserved aspartate residue, resulting in a conformational change organizing the mature oxyanion hole. In a decisive 1976 work, Huber and Bode [Bode, W., and Huber, R. (1976) FEBS Lett. 68, 231-236] demonstrated that peptides sequentially similar to the new amino terminus in combination with a catalytic site inhibitor could specifically induce a trypsin-like conformation in trypsinogen. We now demonstrate that an Ile-Ile or Ile-Val dipeptide can induce limited enzyme activity in the single-chain zymogen form of urokinase-type plasminogen activator (uPA) or its K158A variant, which cannot be activated proteolytically. Furthermore, the slow formation of a covalent serpin-protease complex between single-chain uPA and PAI-1 is significantly accelerated in the presence of specific dipeptide sequences. The technique of using a dipeptide mimic as a surrogate for the liberated amino terminus further provides a novel means by which to covalently label the immature active site of single-chain uPA with a fluorescent probe, permitting fluorescence approaches for direct observations of conformational changes within the protease domain during zymogen activation. These data demonstrate the structural plasticity of the protease domain, reinforce the notion of "molecular sexuality", and provide a novel way of studying conformational changes of zymogens during proteolytic activation.

Assay platform for screening and identifying allosteric and catalytic site inhibitors of viral polymerases.

An assay platform consisting of two luminescent‐based assays has been developed for screening inhibitors of viral polymerases and for studying mechanisms of action of the prospective pharmaceutical compounds. The assays from this platform are designed to measure the activity of DNA‐directed DNA polymerases, RNA‐directed DNA polymerases, and RNA‐directed RNA polymerases, and can be used for identifying the mechanism of action of a prospective viral polymerase inhibitor in a single volume experiment and operating in real‐time detection mode. The assays have been demonstrated for detection of allosteric vs. catalytic site inhibition (e.g., NNRTI vs. NRTI) of HIV‐1 reverse transcriptase and for the study of the inhibition of RNA‐directed RNA polymerases. The assay platform addresses the drawbacks of the existing methods in that it reduces the cost of reagents and reduces the assay time. The technology is adaptable to high‐throughput format for application in large and small scale drug discovery projects.

Activation of p38 Mitogen-Activated Protein Kinase Contributes to the Early Cardiodepressant Action of Tumor Necrosis Factor

The purpose of this study was to determine whether p38 mitogen-activated protein kinase (p38-MAPK) contributes to tumor necrosis factor-alpha (TNFalpha)-induced contractile depression. Tumor necrosis factor has both beneficial and detrimental consequences that may result from the activation of different downstream pathways. Tumor necrosis factor activates p38-MAPK, a stress-responsive kinase implicated in contractile depression and cardiac injury. In isolated hearts from mice lacking the p38-MAPK activator, MAPK kinase 3 (MKK3), perfused at constant coronary pressure or flow, we measured the left ventricular developed pressure (LVDP) and the relationship between end-diastolic volume and LVDP in the presence and absence of 10 ng/ml TNFalpha. Within 15 min at constant pressure, TNFalpha significantly reduced LVDP and coronary flow in outbred and mkk3(+/+) mice. This early negative inotropic effect was associated with a marked phosphorylation of both p38-MAPK and its indirect substrate, HSP27. In hearts lacking MKK3, TNFalpha failed to activate p38-MAPK or to cause significant contractile dysfunction. The actions of TNFalpha were similarly attenuated in MAPK-activated protein kinase 2 (MK2)-deficient hearts, which have a marked reduction in myocardial p38-MAPK protein content, and by the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/l). Under conditions of constant coronary flow, the p38-MAPK activation and contractile depression induced by TNFalpha, though attenuated, remained sensitive to the absence of MKK3 or the presence of SB203580. The role of p38-MAPK in TNFalpha-induced contractile depression was confirmed in isolated murine cardiac myocytes exposed to SB203580 or lacking MKK3. Tumor necrosis factor activates p38-MAPK in the intact heart and in isolated cardiac myocytes through MKK3. This activation likely contributes to the early cardiodepressant action of TNFalpha.

Structural determinants of phosphodiesterase 6 response on binding catalytic site inhibitors

To predict the response of retinal phosphodiesterase on binding catalytic site inhibitors, a homology model of the catalytic domain of subunit α of type 6 phosphodiesterase has been built by selecting an experimental structure of type 5 phosphodiesterase as template. Guanosine monophosphate and inhibitors (sildenafil, zaprinast) docked to the type 6 phosphodiesterase binding crevice similarly to the experimental conformations of guanosine monophosphate and sildenafil in the catalytic domain of type 5 phosphodiesterase. Inhibitors, but not guanosine monophosphate, interacted with Phe778 and Met759 (sildenafil) or Met759 (zaprinast), the key residues involved in the interaction between the catalytic binding domain and the inhibitory γ subunit of type 6 phosphodiesterase. Agreeing with predictions obtained by modelling binding, both inhibitors (1 and 10 μM) enhanced the amplitude of electric light responses of the isolated rat retina, however, the enhancement was smaller for the more efficacious inhibitor sildenafil. These paradoxical responses can be explained as a result of the enhancement of light activation of PDE6 through the competition between the catalytic site inhibitors and the γ subunit residues for catalytic domain residues Phe778 and Met759.

Glycogen phosphorylase inhibitors: A free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.

P38 MAPK Autophosphorylation Drives Macrophage IL-12 Production during Intracellular Infection

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mphi). We used synthetic inhibitors and dominant-negative Mphi mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3(-/-) Mphi, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38alpha MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38alpha MAPK association with adaptor protein, TGF-beta-activated protein kinase 1-binding protein-1. We observed TGF-beta-activated protein kinase 1-binding protein-1-p38alpha MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mphi. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38alpha MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.

Diverse mechanisms of myocardial p38 mitogen-activated protein kinase activation: evidence for MKK-independent activation by a TAB1-associated mechanism contributing to injury during myocardial ischemia.

The ischemic activation of p38alpha mitogen-activated protein kinase (p38alpha-MAPK) is thought to contribute to myocardial injury. Under other circumstances, activation is through dual phosphorylation by MAPK kinase 3 (MKK3). Therefore, the mkk3-/- murine heart should be protected during ischemia. In retrogradely perfused mkk3-/- and mkk3+/+ mouse hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion, infarction/risk volume was similar (50+/-5 versus 51+/-4, P=0.93, respectively), as was intraischemic p38-MAPK phosphorylation (10 minutes ischemia as percent basal, 608+/-224 versus 384+/-104, P=0.43, respectively). This occurred despite undetectable activation of MKK3/6 in mkk3-/- hearts. However, tumor necrosis factor (TNF)-induced p38-MAPK phosphorylation was markedly diminished in mkk3-/- vs mkk3+/+ hearts (percent basal, 127+/-23 versus 540+/-267, respectively, P=0.04), suggesting an MKK-independent activation mechanism by ischemia. Hence, we examined p38-MAPK activation by TAB1-associated autophosphorylation. In wild-type mice and mkk3-/- mice, the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/L) diminished phosphorylation during ischemia versus control (10 minutes ischemia as percent basal, 143+/-2 versus 436+/-96, P=0.003, and 122+/-25 versus 623+/-176, P=0.05, respectively) and reduced infarction volume (infarction/risk volume, 57+/-5 versus 36+/-3, P<0.001, and 50+/-5 versus 29+/-3, P=0.003, respectively) but did not alter TNF-induced activation, although in homogenates of ischemic hearts but not TNF-exposed hearts, p38-MAPK was associated with TAB1. Furthermore, adenovirally expressed wild-type and drug-resistant p38alpha-MAPK, lacking the SB203580 binding site, was phosphorylated when H9c2 myoblasts were subjected to simulated ischemia. However, SB203580 (1 micromol/L) did not prevent the phosphorylation of resistant p38alpha-MAPK. These findings suggest the ischemic activation of p38-MAPK contributing to myocardial injury is by TAB1-associated autophosphorylation.

N, N-Dibenzyl- N′-benzylidenehydrazines: Potent Competitive Glucose-6-phosphatase Catalytic Enzyme Inhibitors

A novel class of N, N-dibenzyl- N′-benzylidenehydrazines as potent and competitive glucose-6-phosphatase catalytic site inhibitors are described. Optimisation of this series identified compounds with IC 50 values as low as 170 nM.

Protein Phosphatase 2A Is Associated with Class C L-type Calcium Channels (Cav1.2) and Antagonizes Channel Phosphorylation by cAMP-dependent Protein Kinase

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.

Glucose-6-phosphatase Catalytic Enzyme Inhibitors: Synthesis and In Vitro Evaluation of Novel 4,5,6,7-Tetrahydrothieno[3,2- c]- and -[2,3- c]pyridines

The discovery of the first class of potent glucose-6-phosphatase catalytic site inhibitors, substituted 4,5,6,7-tetrahydrothieno[3,2- c]- and -[2,3- c]pyridines, is described. Optimisation of this series involved solution phase combinatorial synthesis and very potent compounds were prepared with IC 50 values down to 140 nM. The structure–activity relationship (SAR) of these compounds indicates that: a tetrahydrothieno[3,2- c]pyridine core ring system and the isomeric [2,3- c] system are equipotent and much better than the corresponding benzo analogue, 1,2,3,4-tetrahydro-isoquinoline. The 4-substituent of the tetrahydrothieno[3,2- c]pyridine ring has to be a phenyl group, optionally substituted with a lipophilic 4-substituent, such as trifluoromethoxy or chloro. The 5-substituent of the tetrahydrothieno[3,2- c]pyridine ring has to be a substituted benzoyl; anisoyl and ( E)-3-furan-3-ylacryloyl are the best of the investigated groups. Substitution in the benzoyl ortho position seems to be forbidden, whereas substitution in the meta position is tolerated only if a methoxy para substituent is present. These SAR findings were parallel to those obtained in the 4,5,6,7-tetrahydrothieno[2,3- c]pyridine system. Enantioselectivity in enzyme recognition was observed and the activity resided in all cases only in one of the enantiomers.