A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

Fatty acid amide hydrolase inhibitors. 3: tetra-substituted azetidine ureas with in vivo activity.

We describe here our attempts to optimise the human fatty acid amide hydrolase (FAAH) inhibition and physicochemical properties of our previously reported tetrasubstituted azetidine urea FAAH inhibitor, VER-156084. We describe the SAR of a series of analogues and conclude with the demonstration of in vivo dose-dependant FAAH inhibition in an anandamide-loading study in rats.

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas.

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.

Application of Off-Rate Screening in the Identification of Novel Pan-Isoform Inhibitors of Pyruvate Dehydrogenase Kinase.

Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.

Mitogen activated protein (MAP) kinases: development of ATP and non-ATP dependent inhibitors.

Extracellular signals regulate most of the body's physiological functions through the MAP kinase signaling pathways. These MAP kinase signaling pathways are normally under tight regulation such that activation and inactivation occurs only when needed. However, aberrant regulation observed with naturally occurring mutations in specific signaling proteins often results in constitutive activation of the MAP kinases and is involved in several pathophysiological conditions, such as cancer, neurodegeneration, and inflammation. As such, much effort has been expended to develop inhibitory molecules of the MAP kinase signaling pathways. Several compounds have been identified that inhibit MAP kinase signaling by targeting receptors or other proteins upstream of the MAP kinases. The development of specific inhibitors of the MAP kinases themselves has been less successful and only a few compounds, which interfere with ATP binding, have been identified. A common problem with kinase inhibitors that compete with ATP binding is their lack of specificity. Thus, alternative approaches to inhibit MAP kinase function are being sought. The MAP kinase proteins contain docking domains that direct the interactions with a variety of substrate proteins. Using the 3-dimensional structure of MAP kinases and computer modeling, molecules that target specific docking domains and selectively disrupt substrate interactions are being developed. This non-ATP interfering approach may allow the selective inhibition of MAP kinase substrates involved in disease processes while preserving MAP kinase functions associated with normal cells.

Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells.

PURPOSE: The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. METHODS: We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. RESULTS: VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI(50)s in the range 5.3-14.4 microM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. CONCLUSION: These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined.

Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design.

The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.

Computational identification of inhibitors of protein-protein interactions.

The ability to control protein-protein interactions (PPIs) for therapeutic purposes is attractive since many processes in cells involve such interactions. Recent successes in the discovery of small molecules that target protein-protein interactions for drug development have shown that targeting these interactions is indeed feasible. In the present review the use of computer-aided drug design (CADD) via database screening or docking algorithms for identifying inhibitors of protein-protein interactions is introduced. The principles of database screening and a practical protocol for targeting PPIs are described. The recent applications of these approaches to different systems involving protein-protein interactions, including BCL-2, S100B, ERK and p56lck, are presented and provide valuable examples of inhibitor discovery and design.

DNA bending induced by carbocyclic sugar analogs constrained to the north conformation.

DNA bending caused by introduction of carbocyclic sugars constrained to the north conformation was studied, using explicit solvent molecular dynamic (MD) simulations. The native Drew-Dickerson (DD) dodecamer and its three modifications containing north carbocyclic sugars in the 7th (T7*), 8th (T8*) or both 7th and 8th (T7T8*) nucleotide positions were examined. Introduction of the carbocyclic sugar results in A-form conformations for the alpha, beta, chi, zeta, and sugar pucker backbone parameters in the modified nucleotides. Increased steric repulsion between the sugar and its parent base in the modified oligonucleotides impacts the roll and cup dinucleotide step parameters, increasing the bending of the oligomer axis. Increased buckling of the substituted nucleotides disrupts the usual stabilizing base stacking interactions. The level of overall bending depends on the number and position of carbocyclic sugars introduced in the DNA sequence. Single sugar substitutions are unable to induce substantial bending due to the neighboring unmodified nucleotides counterbalancing the distortion. Significant bending can, however, be induced by two consecutive north sugars (T7T8*), which is in agreement with experimental results. The modified oligomers populate a wide range of bend angles, indicating that they maintain flexibility in the bent state. The present results suggest that insertion of carbocyclic sugars into DNA or RNA duplexes can be used to engineer bending of the duplexes without impacting the electrostatic or chemical properties of the phosphodiester backbone, thereby serving as excellent tools for experimental elucidation of nucleic acid structure-function relationships.

Characterization of ATP-independent ERK inhibitors identified through in silico analysis of the active ERK2 structure.

The extracellular signal-regulated kinases (ERK1 and ERK2) are important mediators of cell proliferation. Constitutive activation of the ERK proteins plays a critical role in the proliferation of many human cancers. Taking advantage of recently identified substrate docking domains on ERK2, we have used computer-aided drug design (CADD) to identify novel low molecular weight compounds that interact with ERK2 in an ATP-independent manner and disrupt substrate-specific interactions. In the current study, a CADD screen of the 3D structure of active phosphorylated ERK2 protein was used to identify inhibitory compounds. We tested 13 compounds identified by the CADD screen in ERK-specific phosphorylation, cell proliferation, and binding assays. Of the 13 compounds tested, 4 compounds strongly inhibited ERK-mediated phosphorylation of ribosomal S6 kinase-1 (Rsk-1) and/or the transcription factor Elk-1 and inhibited the proliferation of HeLa cervical carcinoma cells with IC(50) values in the 2-10 microM range. These studies demonstrate that CADD can be used to identify lead compounds for development of novel non-ATP-dependent inhibitors selective for active ERK and its interactions with substrates involved in cancer cell proliferation.

CH/pi interactions involving aromatic amino acids: refinement of the CHARMM tryptophan force field.

High-level ab initio calculations have been carried out to study weak CH/pi interactions and as a check of the CHARMM force field for aromatic amino acids. Comparisons with published data indicate that the MP2/cc-pVTZ level of theory is suitable for calculations of CH/pi interaction, including the T-shape benzene dimer. This level of theory was, therefore, applied to investigate CH/pi interactions between ethene or cis-2-butene and benzene in a variety of orientations. In addition, complexes between ethene and a series of model compounds (toluene, methylindole and p-cresol) representing the aromatic amino acids were studied motivated by the presence of CH/pi interactions in biological systems. Ab initio binding energies were compared to the binding energies obtained with the CHARMM22 force field. In the majority of orientations, CHARMM22 reproduces the preferred binding modes, with excellent agreement for the benzene dimer. Small discrepancies found in the calculations involving methylindole along with a survey of published thermodynamic data for the aromatic amino acids prompted additional optimization of the tryptophan force field. Partial atomic charges, Lennard-Jones parameters, and force constants were improved to obtain better intra- and intermolecular properties, with significant improvements obtained in the reproduction of experimental heats of sublimation for indole and free energies of aqueous solvation for methylindole.

Lead validation and SAR development via chemical similarity searching; application to compounds targeting the pY+3 site of the SH2 domain of p56lck.

Compound selection based on chemical similarity has been used to validate active "parent" compounds identified via database searching as viable lead compounds and to obtain initial structure-activity relationships for those leads. Twelve parent compounds that have inhibitory activity against the SH2 domain of the p56 T-cell tyrosine kinase (Lck) are the focus of this study. Lck is involved in the T-cell mediated immune response, and inhibitors of Lck protein-protein interactions could potentially be used to develop novel immunosuppressants. Similarity searches for each parent compound were performed using 2D structural fingerprints on a database containing 1,300,000 commercially available compounds. The inhibitory activity of the selected compounds was assessed using enzyme immunoassay (EIA). In general, the most active parent compounds yield the most high activity similar compounds; however, in two cases low activity parent compounds (i.e. inhibitory activity < 25% at 100 microM) yielded multiple similar compounds with activities > 60%. Such compounds may, therefore, be considered as viable lead compounds for optimization. Structure-activity relationships were explored by examining both ligand structures and their computed bound conformations to the protein. Functional groups common to the active compounds as well as key amino acid residues that form hydrogen bonds with the active compounds were identified. This information will act as the basis for the rational optimization of the lead compounds.

Impact of multiple cation-pi interactions upon calix[4]arene substrate binding and specificity.

The cation-pi interaction influence on the conformation and binding of calix[4]arenes to alkali-metal cations has been studied using a dehydroxylated model. The model allows for the separation of cooperative cation-pi and electrostatic forces commonly found in the binding motifs found in calixarene complexes. Starting from the four well-known calix[4]arene conformations, six conformers for this dehydroxylated model (cone, partial cone, flattened cone, chair, 1,2-alternate, and 1,3-alternate) have been characterized by geometry optimization and frequency analysis using the Becke three-parameter exchange functional with the nonlocal correlation functional of Lee, Yang, and Parr and the 6-31G(d) basis set. Without the stabilization provided by the hydroxyl hydrogen bonds in calix[4]arene, neither the cone nor the 1,2-alternate conformation is computed to be a ground-state structure. The partial cone, flattened cone, chair, and 1,3-alternate conformers have been identified as ground-state structures in a vacuum, with the partial cone and the 1,3-alternate as the lowest energy minima in the aromatic model. The C(4)(v)() cone conformation is found to be a transition structure separating the flattened cone (C(2)(v)()) conformers. The energetic and structural preferences of the calix[4]arene model change dramatically when it is bound to Li(+), Na(+), and K(+). The number of pi-faces, the positioning of these pi-faces with respect to the cations, and the nature of the cation were studied as factors in the binding strength. A detailed study of the distances and angles between the aromatic ring centroids and the cations reveals the energetic advantages of multiple weak cation-pi interactions. The geometries are often far from the optimal cation-pi interaction in which the cation approaches in a perpendicular path the aromatic ring center, where the quadrupole moment is strongest. The results reveal that multiple weaker nonoptimal cation-pi interactions contribute significantly to the overall binding strength. This theoretical analysis underscores the importance of neighboring aromatic faces and provides new insight into the significance of cation-pi binding, not only for calix[4]arenes, but also for other supramolecular and biological systems.

3-chloropropanoic acid (UMB66): a ligand for the gamma-hydroxybutyric acid receptor lacking a 4-hydroxyl group.

Gamma-Hydroxybutyric acid (GHB) has gained in notoriety in recent years due to its association with sexual assaults. GHB is an endogenous ligand for GHB receptors, but its complete pharmacological mechanism of action in vivo remains unclear due to apparent GABAergic components. It has been proposed that the hydroxyl group in the 4-position acts as a hydrogen bond donor to the GHB receptor. Herein we show that 3-chloropropanoic acid possesses significant affinity for the GHB receptor, has no affinity for GABA receptors, and cannot undergo metabolism to GABAergic compounds. UMB66 is thus a selective agent for the study of GHB in vivo. These results, in combination with data from quantum mechanical calculations, suggest that the hydroxyl group of GHB actually acts as a hydrogen bond acceptor in contrast to the currently accepted model. This finding is anticipated to facilitate the rational design of novel agents with selectivity for GHB receptors that may be used to elucidate the mechanism of action of this common drug of abuse.