Secondary Binding Pocket Research Articles

Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access.

D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.

Correction to "Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs".

Correction to "Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs".

Synthesis of 3-(3-hydroxyphenyl)pyrrolidine dopamine D3 receptor ligands with extended functionality for probing the secondary binding pocket

A series of 3-(3-hydroxyphenyl)pyrrolidine analogues which incorporate N-alkyl groups and N-butylamide-linked benzamide functionality have been synthesized and their in vitro binding affinities at human dopamine receptors have been evaluated. Our ligand design strategy was to take the 3-(3-hydroxyphenyl)pyrrolidine scaffold and extend functionality from the orthosteric binding site to the secondary binding pocket for enhancing affinity and selectivity for the D3 receptor. The N-alkyl analogues constitute a homologous series from N-pentyl to N-decyl to probe the length/bulk tolerance of the secondary binding pocket of the D3 receptor. Enantiomeric 3-(3-hydroxyphenyl)pyrrolidine analogues were also prepared in order to test the chirality preference of the orthosteric binding site for this scaffold. Benzamide analogues were prepared to enhance affinity and/or selectivity based upon the results of the homologous series.

Evidence for a Stereoselective Mechanism of Action for Non‐competitive Antagonism of the D3 Dopamine Receptor by Extended‐Length Bitopic Ligands

Dopamine receptors (DRs) are comprised of two subfamilies based on structure and pharmacology: D1‐like (D1R and D5R) and D2‐like (D2R, D3R, and D4R). The D3R expression is more restricted to the limbic system, as compared to the D2R, suggesting that modulation of the D3R may be useful for treating substance abuse or psychosis‐related disorders without the motor side effects associated with D2R blockade. Extensive structure‐activity relationship investigations indicate that D3R‐selective antagonists with extended‐length structures engage two distinct sites on the receptor. The extended‐length ligand's primary pharmacophore (PP) binds to the orthosteric site whereas the ligand's secondary pharmacophore (SP) interacts with a unique secondary binding pocket (SBP). Bivalent compounds that concurrently engage both the orthosteric site and a SBP that has allosteric properties, have been termed “bitopic”. We previously synthesized and characterized several extended‐length compounds with a conformationally restricted trans‐cyclopropyl linking chain based on the structure of SB269,652, a potent D3R‐preferring bitopic antagonist that acts in a noncompetitive/allosteric manner at both the D3R and the D2R. We found that relatively subtle changes in both the linker and the SP can affect the competitive/noncompetitive nature of the compounds, and identified two novel ligands that behave as noncompetitive antagonists at the D3R. We resolved the optical isomers of one of these compounds (VK04‐87) to gain a better understanding of the molecular basis of their allosteric activity. We found that the (+)‐isomer has higher affinity and is more D3R>D2R selective than the (−)‐isomer. Strikingly, using beta‐arrestin recruitment assays as a functional output, we found that (+)‐VK04‐87 inhibits the D3R in a non‐competitive manner, while (−)‐VK04‐87 behaves as a competitive antagonist. These data suggest that the bitopic interactions of VK04‐87 with the D3R are stereoselective in nature with the allosteric properties residing with the (+)‐isomer. To further investigate, we made a mutation (E90A) in the secondary binding pocket of the D3R that was previously found to decrease the affinity and negative cooperativity of SB269,652 for the D2R. We found that this mutation decreased the affinity of extended‐length ligands, including SB269,652, for the D3R, but had no effect on the affinities of smaller, competitive antagonists consisting of PPs. Further analyses of the interactions of the extended‐length ligands with the D3R E90A mutant are ongoing. These studies could help to determine how ligand interactions with the SBP can be utilized to engender allosterism, potentially producing compounds with unique pharmacological effects.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The action of a negative allosteric modulator at the dopamine D2 receptor is dependent upon sodium ions

Sodium ions (Na+) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D2 receptor (D2R). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na+ to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the D2R that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na+ within the conserved Na+-binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na+ is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na+-sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na+ to modulate the binding of orthosteric ligands at the D2R, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.

The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors.

The dopamine D2 and D3 receptors (D2R and D3R) are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a secondary binding pocket (SBP) in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond) between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 μs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties.

The structural determinants of the bitopic binding mode of a negative allosteric modulator of the dopamine D2 receptor

SB269652 is a negative allosteric modulator of the dopamine D2 receptor (D2R) yet possesses structural similarity to ligands with a competitive mode of interaction. In this study, we aimed to understand the ligand-receptor interactions that confer its allosteric action. We combined site-directed mutagenesis with molecular dynamics simulations using both SB269652 and derivatives from our previous structure activity studies. We identify residues within the conserved orthosteric binding site (OBS) and a secondary binding pocket (SBP) that determine affinity and cooperativity. Our results indicate that interaction with the SBP is a requirement for allosteric pharmacology, but that both competitive and allosteric derivatives of SB269652 can display sensitivity to the mutation of a glutamate residue (E952.65) within the SBP. Our findings provide the molecular basis for the differences in affinity between SB269652 derivatives, and reveal how changes to interactions made by the primary pharmacophore of SB269652 in the orthosteric pocket can confer changes in the interactions made by the secondary pharmacophore in the SBP. Our insights provide a structure-activity framework towards rational optimization of bitopic ligands for D2R with tailored competitive versus allosteric properties.

Abstract B130: Differentiated pharmacology of the imipridone ONC201: the first selective DRD2/3 antagonist in clinical oncology

Abstract Background: ONC201, founding member of the imipridone class of compounds, has demonstrated biologic activity and exceptional safety in advanced cancer patients. In this study, we identified and characterized a previously unknown binding target of ONC201. Methods: Binding targets for ONC201 were predicted by BANDIT–a machine learning-based drug target identification platform that uses clinical, genomics, chemical, and structural datasets for small molecules with validated binding targets. GPCR antagonism was evaluated using beta-arrestin recruitment and cAMP reporter assays in Chinese hamster ovary cells. KB values were determined with Schild analyses and DRD2 binding was confirmed with radio-labeled ligand competition assays. Shotgun mutagenesis across 350 amino acids of DRD2 was performed and dopamine-induced calcium flux assays were conducted in HEK-293T cells. Results: BANDIT predicted that ONC201 would bind with high selectivity to the G-protein coupled receptors (GPCRs) dopamine receptor D2(DRD2) and D3 (DRD3). DRD2 is overexpressed in glioblastoma, controls prosurvival signaling pathways, and its antagonism causes proapoptotic and antiproliferative effects in malignant cells. β-arrestin recruitment and cAMP assays determined that ONC201 selectively antagonizes DRD2 and DRD3. Consistent with BANDIT predictions and in contrast to other DRD2 antagonist antipsychotics, ONC201 did not antagonize other dopamine receptors or other closely related GPCRs. Schild analyses and radiolabeled ligand competition assays revealed DRD2 affinities that were consistent with those identified for ONC201 anticancer activity. In accordance with superior selectivity, ONC201 exhibited a wider therapeutic window compared to other antipsychotics. In support of the hypothesis that selectively targeting the Gi-coupled D2-like receptors yields superior anticancer efficacy, combined inhibition of DRD2 and DRD1, a Gs-coupled D1-like receptor, was found to be inferior to DRD2 inhibition alone. ONC201 exhibited a very slow association rate for DRD2 relative to antipsychotics, whereas the dissociation rate was similar to atypical antipsychotics that are better tolerated. Shotgun mutagenesis of DRD2 identified 8 residues that are critical for ONC201-mediated antagonism of dopamine-induced calcium flux via DRD2. Some of the residues were specific to DRD2 and DRD3, suggesting a potential role of these residues in conferring ONC201 selectivity. Consistent with competitive inhibition, several mutated residues were within the orthosteric binding site (OBS); however, two distal residues were identified outside of the OBS. The latter suggesting a secondary binding pocket and concurrent noncompetitive antagonism of DRD2. Conclusion: The receptor pharmacology of ONC201, the first selective DRD2/3 antagonist in clinical oncology, may underpin its unique selectivity, safety, and anticancer activity that has been observed in clinical trials. Citation Format: Neel S. Madhukar, Varun Vijay Prabhu, Lakshmi Anantharaman, Chidananda Sulli, Edgar Davidson, Sean Deacon, Joseph Rucker, Neil Charter, Benjamin Doranz, Wolfgang Oster, Olivier Elemento, R. Benjamin Free, David Sibley, Joshua E. Allen. Differentiated pharmacology of the imipridone ONC201: the first selective DRD2/3 antagonist in clinical oncology [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B130.

EXTH-43. DIFFERENTIATED PHARMACOLOGY OF THE IMIPRIDONE ONC201, THE FIRST SELECTIVE DRD2/3 ANTAGONIST IN CLINICAL NEURO-ONCOLOGY

ONC201, founding member of the imipridone class of compounds, has demonstrated evidence of tumor shrinkage along with exceptional safety in recurrent glioblastoma patients. In this study, we identified and characterized a previously unknown binding target of ONC201. BANDIT – a machine learning-based drug target identification platform – predicted that ONC201 would bind with high selectivity to the G-protein coupled receptors (GPCRs) dopamine receptor D2(DRD2) and D3 (DRD3). DRD2 is overexpressed in glioblastoma, controls pro-survival mechanisms, and its antagonism causes pro-apoptotic effects in malignant cells. PathHunter® β-arrestin and cAMP assays determined that ONC201 selectively antagonizes DRD2 and DRD3. Consistent with BANDIT and in contrast to DRD2 blocking antipsychotics, ONC201 did not antagonize other dopamine receptors or other closely related GPCRs with identified endogenous ligands. Schild analyses and radioligand competition assays revealed DRD2 affinities that were consistent with those identified for ONC201 anticancer activity. In accordance with superior selectivity, ONC201 exhibited a wider therapeutic window compared to other antipsychotics. In support of the hypothesis that selectively targeting D2-like receptors yields superior anti-cancer efficacy, combined DRD2/DRD1 inhibition was found to be inferior to DRD2 inhibition alone. ONC201 exhibited a very slow association rate for DRD2 relative to antipsychotics, whereas the dissociation rate was similar to atypical antipsychotics that are better tolerated clinically. Shotgun mutagenesis across 350 amino acids of DRD2 identified 8 residues critical for ONC201-mediated antagonism of DRD2-induced calcium flux. Several residues were not conserved among other dopamine receptors, suggesting a potential role in conferring ONC201 selectivity. Consistent with competitive inhibition, several mutated residues were within the orthosteric binding site (OBS), however, two distal residues were identified outside of the OBS suggesting a secondary binding pocket. In summary, receptor pharmacology of ONC201, the first selective DRD2/3 antagonist in clinical neuro-oncology, may explain its unique selectivity, safety, and anti-cancer activity in clinical trials.

A Novel Class of Natural FXR Modulators with a Unique Mode of Selective Co-regulator Assembly.

The farnesoid X receptor (FXR) is an important target for drug discovery. Small molecules induce a conformational change in FXR that modulates its binding to co-regulators, thus resulting in distinct FXR functional profiles. However, the mechanisms for selectively recruiting co-regulators by FXR remain elusive, partly because of the lack of FXR-selective modulators. We report the identification of two natural terpenoids, tschimgine and feroline, as novel FXR modulators. Remarkably, their crystal structures uncovered a secondary binding pocket important for ligand binding. Further, tschimgine or feroline induced dynamic conformational changes in the activation function 2 (AF-2) surface, thus leading to differential co-regulator recruiting profiles, modulated by both hydrophobic and selective hydrogen-bond interactions unique to specific co-regulators. Our findings thus provide a novel structure template for optimization for FXR-selective modulators of clinical value.

Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity

The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational analysis of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications forunderstanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.

Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs.

DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic activity of DUSP5.

Synthesis and evaluation of C9 alkoxy analogues of (-)-stepholidine as dopamine receptor ligands

Tetrahydroprotoberberine alkaloids have shown interesting polypharmacological actions at dopamine receptors and are a unique template from which to mine novel molecules with dual selective actions at D1 and D3 receptors. Such compounds will be valuable to evaluate as anti-cocaine therapeutics. Towards that eventual goal, we engaged an SAR study in which a series of C9 alkoxy analogues of the D1/D2/D3 ligand (-)-stepholidine that possessed or lacked a C12 bromo functionality, were synthesized and evaluated for affinity at dopamine D1, D2 and D3 receptors. We found that the analogues are generally selective for the D1 receptor. Small n-alkoxy substituents (up to 4 carbons in length) were generally well tolerated for high D1 affinity but such groups reduced D3 affinity. In the case of C12 brominated analogues, C9 alkoxylation also had little effect on D1 affinity for the smaller alkoxy groups, but reduced D2 and D3 affinities significantly. C12 bromination tends to increase D1 receptor selectivity. A number of compounds were identified that retain affinity for D1 and D3 receptors but lack D2 receptor affinity. Among them, compound 22a was found to be a selective D1/D3 dual antagonist (Ki = 5.3 and 106 nM at D1 and D3 receptors). Docking studies performed on the analogues at the D3 receptor revealed a number of interactions that are important for affinity including a critical N - Asp110 salt bridge motif, H-bonds to Ser192 and Cys181 and hydrophobic interactions between the aryl rings and Phe106 and Phe345. The analogues adopt an orientation in which ring A is located in the orthosteric binding site while the C9 alkoxy substituents attached to ring D project into the secondary binding pocket of the D3 receptor.

Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis.

Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1.

Design, synthesis and evaluation of bitopic arylpiperazinephenyl-1,2,4-oxadiazoles as preferential dopamine D3 receptor ligands

The dopamine D3 receptor (D3R) was proposed as a therapeutic target for drug development to treat drug abuse and addiction and neuropsychiatric disorders. Several D3R-selective modulators over the dopamine D2 receptor (D2R) can avoid extrapyramidal symptoms (EPS) and hyperprolactinemia. However, few biased D3R ligands were identified or showed a narrow range of selectivity at the D3R over D2R because of their high sequence homology. Herein, we designed, synthesized and evaluated the binding affinity of a series of bitopic ligands: arypiperazine-phenyl-1,2,4-oxadiazoles. Compound 9e·HCl was the most potent and selective D3R modulator among these bitopic ligands. Molecular modeling revealed that D3R selectivity depends on the divergence of secondary binding pocket (SBP) in D3R and D2R. Specifically, non-conserved Tyr36, EL1 especially non-conserved Thr92 and Gly94, and EL2 Val180, Cys181 and Ser182 of D3R may contribute to D3R specificity over D2R.

Investigation of the binding and functional properties of extended length D3 dopamine receptor-selective antagonists

The D3 dopamine receptor represents an important target in drug addiction in that reducing receptor activity may attenuate the self-administration of drugs and/or disrupt drug or cue-induced relapse. Medicinal chemistry efforts have led to the development of D3 preferring antagonists and partial agonists that are >100-fold selective vs. the closely related D2 receptor, as best exemplified by extended-length 4-phenylpiperazine derivatives. Based on the D3 receptor crystal structure, these molecules are known to dock to two sites on the receptor where the 4-phenylpiperazine moiety binds to the orthosteric site and an extended aryl amide moiety docks to a secondary binding pocket. The bivalent nature of the receptor binding of these compounds is believed to contribute to their D3 selectivity. In this study, we examined if such compounds might also be “bitopic” such that their aryl amide moieties act as allosteric modulators to further enhance the affinities of the full-length molecules for the receptor. First, we deconstructed several extended-length D3-selective ligands into fragments, termed “synthons”, representing either orthosteric or secondary aryl amide pharmacophores and investigated their effects on D3 receptor binding and function. The orthosteric synthons were found to inhibit radioligand binding and to antagonize dopamine activation of the D3 receptor, albeit with lower affinities than the full-length compounds. Notably, the aryl amide-based synthons had no effect on the affinities or potencies of the orthosteric synthons, nor did they have any effect on receptor activation by dopamine. Additionally, pharmacological investigation of the full-length D3-selective antagonists revealed that these compounds interacted with the D3 receptor in a purely competitive manner. Our data further support that the 4-phenylpiperazine D3-selective antagonists are bivalent and that their enhanced affinity for the D3 receptor is due to binding at both the orthosteric site as well as a secondary binding pocket. Importantly, however, their interactions at the secondary site do not allosterically modulate their binding to the orthosteric site.

Structure-activity relationships of privileged structures lead to the discovery of novel biased ligands at the dopamine D₂ receptor.

Biased agonism at GPCRs highlights the potential for the discovery and design of pathway-selective ligands and may confer therapeutic advantages to ligands targeting the dopamine D2 receptor (D2R). We investigated the determinants of efficacy, affinity, and bias for three privileged structures for the D2R, exploring changes to linker length and incorporation of a heterocyclic unit. Profiling the compounds in two signaling assays (cAMP and pERK1/2) allowed us to identify and quantify determinants of biased agonism at the D2R. Substitution on the phenylpiperazine privileged structures (2-methoxy vs 2,3-dichloro) influenced bias when the thienopyridine heterocycle was absent. Upon inclusion of the thienopyridine unit, the substitution pattern (4,6-dimethyl vs 5-chloro-6-methoxy-4-methyl) had a significant effect on bias that overruled the effect of the phenylpiperazine substitution pattern. This latter observation could be reconciled with an extended binding mode for these compounds, whereby the interaction of the heterocycle with a secondary binding pocket may engender bias.

A Single Glycine in Extracellular Loop 1 Is the Critical Determinant for Pharmacological Specificity of Dopamine D2 and D3 Receptors

Subtype-selective agents for the dopamine D3 receptor (D3R) have been considered as potential medications for drug addiction and other neuropsychiatric disorders. Medicinal chemistry efforts have led to the discovery of 4-phenylpiperazine derivatives that are >100-fold selective for the dopamine D3 receptor over dopamine D2 receptor (D2R), despite high sequence identity (78% in the transmembrane domain). Based on the recent crystal structure of D3R, we demonstrated that the 4-phenylpiperazine moiety in this class of D3R-selective compounds binds to the conserved orthosteric binding site, whereas the extended aryl amide moiety is oriented toward a divergent secondary binding pocket (SBP). In an effort to further characterize molecular determinants of the selectivity of these compounds, we modeled their binding modes in D3R and D2R by comparative ligand docking and molecular dynamics simulations. We found that the aryl amide moiety in the SBP differentially induces conformational changes in transmembrane segment 2 and extracellular loop 1 (EL1), which amplify the divergence of the SBP in D3R and D2R. Receptor chimera and site-directed mutagenesis studies were used to validate these binding modes and to identify a divergent glycine in EL1 as critical to D3R over D2R subtype selectivity. A better understanding of drug-dependent receptor conformations such as these is key to the rational design of compounds targeting a specific receptor among closely related homologs, and may also lead to discovery of novel chemotypes that exploit subtle differences in protein conformations.

A Novel, “Double-Clamp” Binding Mode for Human Heme Oxygenase-1 Inhibition

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC50 = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC50 = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors.

Structure-Based Design of Ricin Inhibitors

Ricin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. Development of these lead compounds into potent drug candidates is a challenging prospect for numerous reasons, including poor solubility of pterins, the large and highly polar secondary binding pocket of RTA, as well as the enzyme’s near perfect catalytic efficiency and tight binding affinity for its natural substrate, the eukaryotic ribosome. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10−4 M range, leaving significant room for improvement. This review highlights the variety of techniques routinely employed in structure-based drug design projects, as well as the challenges faced in the design of RTA inhibitors.