Small-molecule Allosteric Modulators Research Articles

The Muscarinic Acetylcholine Receptor M5: Therapeutic Implications and Allosteric Modulation.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) was the most recent mAChR to be cloned and has since emerged as a potential therapeutic target for a number of indications. Early studies with knockout animals have provided clues to the receptor's role in physiological processes related to Alzheimer's disease, schizophrenia, and addiction, and until recently, useful subtype-selective tools to further probe the pharmacology of M5 have remained elusive. Small-molecule allosteric modulators have since gained traction as a means by which to selectively examine muscarinic pharmacology. This review highlights the discovery and optimization of M5 positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs).

In vitro and in vivo pharmacokinetic characterization of mavacamten, a first-in-class small molecule allosteric modulator of beta cardiac myosin

Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug–drug interaction potential.Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug–drug interaction risk.In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating.Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species.Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.

Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators

Transcriptional coactivators are a molecular recognition marvel because a single domain within these proteins, the activator binding domain or ABD, interacts with multiple compositionally diverse transcriptional activators. Also remarkable is the structural diversity among ABDs, which range from conformationally dynamic helical motifs to those with a stable core such as a β-barrel. A significant objective is to define conserved properties of ABDs that allow them to interact with disparate activator sequences. The ABD of the coactivator Med25 (activator interaction domain or AcID) is unique in that it contains secondary structural elements that are on both ends of the spectrum: helices and loops that display significant conformational mobility and a seven-stranded β-barrel core that is structurally rigid. Using biophysical approaches, we build a mechanistic model of how AcID forms binary and ternary complexes with three distinct activators; despite its static core, Med25 forms short-lived, conformationally mobile, and structurally distinct complexes with each of the cognate partners. Further, ternary complex formation is facilitated by allosteric communication between binding surfaces on opposing faces of the β-barrel. The model emerging suggests that the conformational shifts and cooperative binding is mediated by a flexible substructure comprised of two dynamic helices and flanking loops, indicating a conserved mechanistic model of activator engagement across ABDs. Targeting a region of this substructure with a small-molecule covalent cochaperone modulates ternary complex formation. Our data support a general strategy for the identification of allosteric small-molecule modulators of ABDs, which are key targets for mechanistic studies as well as therapeutic applications.

Small Molecule Allosteric Modulators of G-Protein-Coupled Receptors: Drug-Target Interactions.

G-protein-coupled receptors (GPCRs) are the largest class of signaling receptors that are most frequently targeted by therapeutic drugs. Allosteric modulators bound to GPCRs at allosteric sites provide the potential for differential selectivity and improved safety compared with traditional orthosteric ligands. The recent breakthroughs in GPCR structural biology have made structures of GPCRs from classes A, B, C, and F complexed with small-molecule allosteric modulators available. Knowledge of the detailed receptor-modulator interactions at the allosteric sites is useful for structure-based GPCR drug design of novel therapeutics. This Perspective comprehensively summarizes the current status of structural complexes between GPCRs and their small-molecule allosteric modulators, particularly the key receptor-modulator interactions at the allosteric sites. Then, the structural diversity of allosteric sites across four GPCR subfamilies is compared. This study is expected to contribute to the design of GPCR allosteric drugs with an improved therapeutic action.

Translation from SCD Mouse to Man: Chronic Administration of Tucaresol Reduces RBC Sickling and Improves Hematological Parameters, but Does Not Impact Inflammatory Markers in SCD Model Mice

Sickle cell disease is a hematological disorder with significant unmet need that affects approximately ten million people worldwide. Currently, treatment for SCD is limited to a single approved drug, Hydroxyurea, and palliative treatments such as hydration and pain management. The limited treatment options have led to considerable interest in developing new therapies for SCD. One strategy for a disease-modifying treatment for SCD is stabilization of the oxygenated form of hemoglobin (OxyHb) by a small molecule allosteric modulator (Zaugg et. Al, JBC, 1977). One such compound, Tucaresol was advanced into clinical trials on the strength of the pre-clinical data, however a SCD mouse model was unavailable when the trial was initiated. The renewed interest in stabilizing OxyHb with a small molecule presents an opportunity to use Tucaresol as a mechanism for understanding the translation between effects seen in an animal model and clinical endpoints in patients. In this study, we treated Townes SCD mice with Tucaresol for 15 days, to evaluate its effect on hematology, oxygen affinity, sickling and markers of inflammation in vivo. These results are compared to the clinical data reported in the original trials.Tucaresol is a substituted benzaldehyde derived from a class of molecules known to preferentially bind to hemoglobin and stabilize the oxygenated state by formation of a Schiff base linkage at a site on the a-chains (Rolan et. Al, Br. J. Clin. Pharm., 1995). Earlier molecules acting by this mechanism had been shown to reduce Hb S polymerization and RBC sickling in vitro, and it was theorized that long-term treatment with such molecules would impact hemolysis and occurrence of vaso-occlusive crisis (VOC) (Beddell et. Al, Br. J. Pharmac. 1984). Preclinical studies demonstrated Tucaresol modification of Hb S delays the polymerization process and inhibits RBC sickling. In clinical trials, Tucaresol positively impacted hematological markers including hemoglobin and reticulocyte counts, and reduced the percentage of irreversibly sickled cells. However, trials of Tucaresol were halted due to a significant immune response and specific inflammatory markers were not measured during the trial.To study these effects in Townes SCD model, mice were dosed with Tucaresol at 30 mg/kg twice a day, for fifteen days. The dose was selected to be consistent with clinical dosing regimens. Blood was collected prior to study start from individual animals to obtain baseline levels for hematological parameters and soluble cell adhesion molecules in plasma. Blood was sampled at day 3 and day 10 of dosing to understand drug exposure and accumulation. At this dose, Tucaresol demonstrated a steady state achieved by day 10 and the blood concentration ranged from 0.5-1.6 mM on day 15. Following two week dosing with Tucaresol, we observed a 50% increase in hemoglobin, and 24% increase in mean corpuscular hemoglobin concentration (MCHC), relative to vehicle treated animals. Reticulocytes, a clinical indicator of hemolytic anemia, are highly elevated in SCD patients and mice, also showed a 47% decrease in Tucaresol treated relative to vehicle treated animals. Oxygen affinity measurements showed a decrease in P50 and P20 values for treated animals, indicating increased oxygen affinity, consistent with the proposed mode of action of Tucaresol- stabilization of the oxygenated state. The percentage of sickled erythrocytes was also decreased by 38% in Tucaresol treated SCD mice when compared to vehicle controls. The decrease in sickling is consistent with the clinical observation of a 52% (mean) decrease in irreversibly sickled cells. In contrast to the improvement seen with hematological parameters, inflammatory markers remained either unchanged or slightly elevated after 15 days of treatment with Tucaresol. Plasma levels of sICAM-1 and sVCAM-1 were unchanged while sE-Selectin was elevated. In summary, in this short course of treatment of SCD mice, Tucaresol had an impact on the hematological markers that was consistent with effects observed in the clinic, but did not modulate the inflammation response.All experiments were within guidelines and were reviewed and approved by Pfizer institutional animal care and use committee. DisclosuresKnee:Pfizer Inc: Employment. Jasuja:Pfizer: Employment. Barakat:Pfizer: Employment. Kelly:Pfizer: Employment. Singh:Pfizer Inc: Employment. Janz:Pfizer: Employment.

Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase

Allostery is a phenomenon observed in many proteins where binding of a macromolecular partner or a small-molecule ligand at one location leads to specific perturbations at a site not in direct contact with the region where the binding occurs. The list of proteins under allosteric regulation includes AGC protein kinases. AGC kinases have a conserved allosteric site, the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF) pocket, which regulates protein ATP-binding, activity, and interaction with substrates. In this study, we identify small molecules that bind to the ATP-binding site andaffect the PIF pocket of AGC kinase family members, PDK1 and Aurora kinase. We describe the mechanistic details and show that although PDK1 and Aurora kinase inhibitors bind to the conserved ATP-binding site, they differentially modulate physiological interactions at the PIF-pocket site. Our work outlines a strategy for developing bidirectional small-molecule allosteric modulators of protein kinases and other signaling proteins.

Discovery of Small-Molecule Modulators of the Human Y4 Receptor.

The human neuropeptide Y4 receptor (Y4R) and its native ligand, pancreatic polypeptide, are critically involved in the regulation of human metabolism by signaling satiety and regulating food intake, as well as increasing energy expenditure. Thus, this receptor represents a putative target for treatment of obesity. With respect to new approaches to treat complex metabolic disorders, especially in multi-receptor systems, small molecule allosteric modulators have been in the focus of research in the last years. However, no positive allosteric modulators or agonists of the Y4R have been described so far. In this study, small molecule compounds derived from the Niclosamide scaffold were identified by high-throughput screening to increase Y4R activity. Compounds were characterized for their potency and their effects at the human Y4R and as well as their selectivity towards Y1R, Y2R and Y5R. These compounds provide a structure-activity relationship profile around this common scaffold and lay the groundwork for hit-to-lead optimization and characterization of positive allosteric modulators of the Y4R.

Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators.

G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively "tuning" GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand-GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu5 NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu5 via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu5-bound probe via tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu5 and negative cooperativity with glutamate, interacted with the "common allosteric-binding site," displayed slow binding kinetics, and could irreversibly label mGlu5 following UV exposure. We depleted the number of functional mGlu5 receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes via click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu5 allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily.

Abstract B132: Discovery of small molecule allosteric modulators of Abl kinase activity and function

Abstract The Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and forms oncogenic fusion proteins that drive several cancer types. The kinase activity of Abl is repressed by intramolecular interactions involving its regulatory SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the back of the C-lobe of the kinase domain. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate Abl kinase activity through its non-catalytic domains may represent selective probes of Abl function. In this study, we developed a high-throughput screening (HTS) assay to identify chemical modulators of Abl kinase activity that target the regulatory SH3:linker interaction. The fluorescence polarization (FP) assay employs a purified recombinant Abl protein comprised of the regulatory N-cap, SH3 and SH2 domains plus the SH2-kinase linker (Abl N32L protein) and a short fluorescein-labeled peptide probe that selectively binds to the Abl SH3 domain. During assay development, we found that the peptide probe binds to the recombinant Abl N32L protein in vitro, producing a robust FP signal that can be competitively displaced by excess unlabeled peptide. The FP signal is not observed in control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved compounds in the FP assay identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays demonstrated that one of the hits, the antithrombotic drug dipyridamole, enhances Abl kinase activity in vitro to a greater extent than the previously described Abl agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates Abl by disrupting this regulatory interaction. Interestingly, this compound does not significantly affect the kinase activity of Hck, a Src family member that is regulated by similar SH3:linker interactions. Treatment with dipyrimadole in combination with DPH led to strong enhancement of Abl kinase activity in transfected cells. The Abl N32L FP assay has been implemented in a fully automated format and used to screen diversity libraries totaling 60,000 compounds. The Z-factor coefficients from this HTS campaign indicated that the FP assay performed well and several additional scaffolds were identified for future development. These results demonstrate that screening assays that incorporate the non-catalytic domains of Abl can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. Citation Format: Prerna Grover, Haibin Shi, Matthew Baumgartner, David Close, Paul A. Johnston, Carlos J. Camacho, Thomas E. Smithgall. Discovery of small molecule allosteric modulators of Abl kinase activity and function. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B132.

Small-Molecule Allosteric Modulators of the Protein Kinase PDK1 from Structure-Based Docking.

Finding small molecules that target allosteric sites remains a grand challenge for ligand discovery. In the protein kinase field, only a handful of highly selective allosteric modulators have been found. Thus, more general methods are needed to discover allosteric modulators for additional kinases. Here, we use virtual screening against an ensemble of both crystal structures and comparative models to identify ligands for an allosteric peptide-binding site on the protein kinase PDK1 (the PIF pocket). We optimized these ligands through an analog-by-catalog search that yielded compound 4, which binds to PDK1 with 8 μM affinity. We confirmed the docking poses by determining a crystal structure of PDK1 in complex with 4. Because the PIF pocket appears to be a recurring structural feature of the kinase fold, known generally as the helix αC patch, this approach may enable the discovery of allosteric modulators for other kinases.

Discovery and Development of Small Molecule Allosteric Modulators of Glycoprotein Hormone Receptors.

Glycoprotein hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are heterodimeric proteins with a common α-subunit and hormone-specific β-subunit. These hormones are dominant regulators of reproduction and metabolic processes. Receptors for the glycoprotein hormones belong to the family of G protein-coupled receptors. FSH receptor (FSHR) and LH receptor are primarily expressed in somatic cells in ovary and testis to promote egg and sperm production in women and men, respectively. TSH receptor is expressed in thyroid cells and regulates the secretion of T3 and T4. Glycoprotein hormones bind to the large extracellular domain of the receptor and cause a conformational change in the receptor that leads to activation of more than one intracellular signaling pathway. Several small molecules have been described to activate/inhibit glycoprotein hormone receptors through allosteric sites of the receptor. Small molecule allosteric modulators have the potential to be administered orally to patients, thus improving the convenience of treatment. It has been a challenge to develop a small molecule allosteric agonist for glycoprotein hormones that can mimic the agonistic effects of the large natural ligand to activate similar signaling pathways. However, in the past few years, there have been several promising reports describing distinct chemical series with improved potency in preclinical models. In parallel, proposal of new structural model for FSHR and in silico docking studies of small molecule ligands to glycoprotein hormone receptors provide a giant leap on the understanding of the mechanism of action of the natural ligands and new chemical entities on the receptors. This review will focus on the current status of small molecule allosteric modulators of glycoprotein hormone receptors, their effects on common signaling pathways in cells, their utility for clinical application as demonstrated in preclinical models, and use of these molecules as novel tools to dissect the molecular signaling pathways of these receptors.

Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.

CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [(3)H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased "fingerprints" that may ultimately allow the development of improved CB1R-targeted therapies.

Targeting the serotonin (5-HT) 5-HT2c receptor: Rational design of small molecule allosteric modulators to treat psychostimulant use disorders

Targeting the serotonin (5-HT) 5-HT2c receptor: Rational design of small molecule allosteric modulators to treat psychostimulant use disorders

Allosteric targeting of receptor tyrosine kinases.

The drug discovery landscape has been transformed over the past decade by the discovery of allosteric modulators of all major mammalian receptor superfamilies. Allosteric ligands are a rich potential source of drugs and drug targets with clear therapeutic advantages. G protein-coupled receptors, ligand-gated ion channels and intracellular nuclear hormone receptors have all been targeted by allosteric modulators. More recently, a receptor tyrosine kinase (RTK) has been targeted by an extracellular small-molecule allosteric modulator. Allosteric mechanisms of structurally distinct molecules that target the various receptor families are more alike than originally anticipated and include selectivity, orthosteric probe dependence and pathway-biased signaling.

International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

Activation and allosteric modulation of a muscarinic acetylcholine receptor

Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.

Determination of cGMP levels in rodent tissues following oral dosing of a soluble guanylate cyclase stimulator

Background In the vasculature, nitric oxide (NO) binds and activates smooth muscle soluble guanylate cyclase (sGC), leading to increased intracellular cGMP, which triggers smooth muscle relaxation and vasodilation. sGC stimulators are a class of small molecule allosteric modulators, which stimulate cGMP production independently of NO but also act in synergy with NO. Evidence to date suggests that sGC stimulators may be balanced vasodilators, meaning that they elicit vasorelaxation in both the arterial and venous vasculature; however, there have been conflicting reports [1,2]. Our approach to developing a better understanding of the

Multidomain integration in the structure of the HNF-4α nuclear receptor complex

The hepatocyte nuclear factor 4 alpha (HNF4α, NR2A1) is a member of the nuclear receptor (NR) family of transcription factors that use conserved DNA binding domains (DBDs) and ligand binding domains (LBDs)1,2. HNF4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF4α response element 1,3,4. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism3,5,6. In the pancreas too, HNF4α is a master regulator controlling an estimated 11% of islet genes7. HNF4α protein mutations are linked to Maturity Onset of Diabetes in Young 1 (MODY1) and hyperinsulinemic hypoglycemia (HH)8–11. Prior structural analyses of NRs, while productive with individual domains, have lagged in revealing the connectivity patterns of NR domains. Here, we describe the 2.9 Å crystal structure of the multi-domain HNF4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain-domain interactions. These post-translational modifications manifest into changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the LBD and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the inter-junctional surfaces of the complex. The overall domain representation of the HNF4α homodimer is different from that of the PPARγ-RXRα heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and inter-domain connections in NRs could be exploited by small-molecule allosteric modulators that impact distal functions in these polypeptides.

Fluorescence Polarization Screening for Allosteric Small Molecule Ligands of the Cholecystokinin Receptor

The success in screening for drug candidates is highly dependent on the power of the strategy implemented. In this work, we report and characterize a novel fluorescent benzodiazepine antagonist of the type 1 cholecystokinin receptor (3-(3-(7-fluoro-1-(2-isopropyl(4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]-diazepin-3-yl)ureido)benzoic acid) that can be used as a receptor ligand in a fluorescence polarization assay, which is ideally suited for the identification of small molecule allosteric modulators of this physiologically important receptor. By binding directly to the small molecule-docking region within the helical bundle of this receptor, this indicator can be displaced by many small molecule candidate drugs, even those that might not affect the binding of an orthosteric cholecystokinin-like peptide ligand. The biological, pharmacological, and fluorescence properties of this reagent are described, and proof-of-concept is provided in a fluorescence polarization assay utilizing this fluorescent benzodiazepine ligand.

Morpheeins – A New Pathway For Allosteric Drug Discovery~!2010-02-12~!2010-05-21~!2010-06-08~!

The morpheein model of allosteric regulation can be applied as a novel approach to the discovery of small molecule allosteric modulators of protein function. Morpheeins are homo-oligomeric proteins where, under physiological conditions, the oligomer can dissociate, the dissociated units can change conformation, and the altered conformational state can reassociate to a structurally and functionally distinct oligomer. This phenomenon serves as a basis for allostery, as a basis for conformational diseases, as a basis for drug discovery, and may be applicable to personalized medicine such as in the prediction of drug side effects. Each of these relationships has been established for the prototype morpheein, porphobilinogen synthase, where the conformational disease is a porphyria and the drug application is in antimicrobial discovery. These data are presented along with a discussion of other drug targets for which the morpheein model of allostery may apply. Such targets include HIV integrase, TNFα, β-tryptase, and p53.