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The chromatin reader eleven–nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9–mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line– and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. Significance: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483

The kappa agonist structure-activity relationship around the novel, pyrrolidinyl substituted pyranopiperazine scaffold was developed. More specifically, the dichloroPhenylAcetamide-Pyrrolidinyl-PyranoPiperazine (PAPPP) core A was the focus of our work. The modulation of kappa receptor potency/G-protein activation and arrestin recruitment with respect to changes of the piperazine R group in A was demonstrated. Reduced β

With over 200 million cases and close to half a million deaths each year, malaria is a threat to global health, particularly in developing countries.

Supplementary Data from Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia

The chromatin reader eleven–nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9–mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line– and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach.Significance:AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation.This article is highlighted in the In This Issue feature, p. 2483

In the hit identification stage of drug discovery, a diverse chemical space needs to be explored to identify initial hits. Contrary to empirical scoring functions, absolute protein-ligand binding free energy perturbation (ABFEP) provides a theoretically more rigorous and accurate description of protein-ligand binding thermodynamics and could in principle greatly improve the hit rates in virtual screening. In this work, we describe an implementation of an accurate and reliable ABFEP method in FEP+. We validated the ABFEP method on eight congeneric compound series binding to eight protein receptors including both neutral and charged ligands. For ligands with net charges, the alchemical ion approach is adopted to avoid artifacts in electrostatic potential energy calculations. The calculated binding free energies are highly correlated with experimental results with the weighted average of R2 of 0.55 for the entire dataset and an overall RMSE of 1.1 kcal/mol when protein reorganization effect upon ligand binding was accounted for. Through ABFEP calculations using apo versus holo protein structures, we demonstrated that the protein conformational and protonation state changes between the apo and holo proteins are the main physical factors contributing to the protein reorganization free energy manifested by the overestimation of raw ABFEP calculated binding free energies using the holo structures of the proteins. Furthermore, we performed ABFEP calculations in three virtual screening applications for hit enrichment. ABFEP greatly improves the hit rates as compared to docking scores or other methods like metadynamics. The highly accurate ABFEP results demonstrated in this work position it as a useful tool to improve the hit rates in virtual screening, thus facilitate hit discovery.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

[Image: see text] Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

The reaction of CO2 with H2O to form HCO3- and H+ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+ response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3- or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea.Statement of significanceAdenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H2O + CO2 ↔ HCO3- (bicarbonate) + H+. The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO3- to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.

Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received FDA approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain-penetrance limiting their use in patients with CNS malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound we discovered TDI-11904 (compound 21); a novel, highly-potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-impos...

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) are phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill non-replicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target, because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The co-crystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose-dependently led to accumulation of Pup-tagged GFP that is degradable but resistant to depupylation., and death of non-replicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

The oncoprotein and Parkinson's disease-associated enzyme DJ-1/PARK7 has emerged as a promiscuous deglycase that can remove methylglyoxal-induced glycation adducts from both proteins and nucleotides. However, dissecting its structural and enzymatic functions remains a challenge due to the lack of potent, specific, and pharmacokinetically stable inhibitors targeting its catalytic site (including Cys106). To evaluate potential drug-like leads against DJ-1, we leveraged its deglycase activity in an enzyme-coupled, fluorescence lactate-detection assay based on the recent understanding of its deglycation mechanism. In addition, we developed assays to directly evaluate DJ-1's esterase activity using both colorimetric and fluorescent substrates. The resulting optimized assay was used to evaluate a library of potential reversible and irreversible DJ-1 inhibitors. The deglycase activity-oriented screening strategy described herein establishes a new platform for the discovery of potential anti-cancer drugs.

[Image: see text] Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages - erythrocytic stages, gametocyte stages, liver stages and gamete activation, indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the b5 subunit of the Plasmodium falciparum proteasome, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum -infected mice.

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.

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.

Both dopamine D3 receptor (D3R) partial agonists and antagonists have been implicated as potential medications for substance use disorders. In contrast to antagonists, partial agonists may cause fewer side effects since they maintain some dopaminergic tone and may be less disruptive to normal neuronal functions. Here, we report three sets of 4-phenylpiperazine stereoisomers that differ considerably in efficacy: the (R)-enantiomers are antagonists/weak partial agonists, whereas the (S)-enantiomers are much more efficacious. To investigate the structural basis of partial agonism, we performed comparative microsecond-scale molecular dynamics simulations starting from the inactive state of D3R in complex with these enantiomers. Analysis of the simulation results reveals common structural rearrangements near the ligand binding site induced by the bound (S)-enantiomers, but not by the (R)-enantiomers, that are features of partially activated receptor conformations. These receptor models bound with partial agoni...

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Co-evolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders non-replicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure-activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases., Cyclic GMP-AMP synthase (cGAS) is involved in the modulation of inflammatory responses. Here, the authors present small-molecule inhibitors of human cGAS, characterize their interaction with the protein, and show that the compounds are active in interferon-producing cells including primary human macrophages.

Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD(+) pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC(50) assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.

Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Significance Protozoal proteasome is a validated target for antimalarial drug development, but species selectivity of reported inhibitors is suboptimal. Here we identify inhibitors with improved selectivity for malaria proteasome β5 subunit over each active subunit of human proteasomes. These compounds kill the parasite in each stage of its life cycle. They interact synergistically with a β2 inhibitor and with artemisinin. Resistance to the β5 inhibitor arose through a point mutation in the nonproteolytic β6 subunit. The same mutation made the mutant strain more sensitive to a β2 inhibitor and less fit to withstand irradiation. These findings reveal complex interplay among proteasome subunits and introduce the prospect that combined inhibition of β2 and β5 subunits can afford synergy and thwart resistance., We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) β5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The β5 inhibitors synergize with a β2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA β5 inhibitor surprisingly harbored a point mutation in the noncatalytic β6 subunit. The β6 mutant was resistant to the species-selective Pf20S β5 inhibitor but remained sensitive to the species-nonselective β5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S β5 inhibitor was accompanied by increased sensitivity to a Pf20S β2 inhibitor. Finally, the β5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S β5 and β2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Recently, we have demonstrated that N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (SB269652) (1) adopts a bitopic pose at one protomer of a dopamine D2 receptor (D2R) dimer to negatively modulate the binding of dopamine at the other protomer. The 1H-indole-2-carboxamide moiety of 1 extends into a secondary pocket between the extracellular ends of TM2 and TM7 within the D2R protomer. To target this putative allosteric site, we generated and characterized fragments that include and extend from the 1H-indole-2-carboxamide moiety of 1. N-Isopropyl-1H-indole-2-carboxamide (3) displayed allosteric pharmacology and sensitivity to mutations of the same residues at the top of TM2 as was observed for 1. Using 3 as an "allosteric lead", we designed and synthesized an extensive fragment library to generate novel SAR and identify N-butyl-1H-indole-2-carboxamide (11d), which displayed both increased negative cooperativity and affinity for the D2R. These data illustrate that fragmentation of extended compounds can expose fragments with purely allosteric pharmacology.

G protein-coupled receptors (GPCRs) are integral membrane proteins that represent an important class of drug targets. In particular, aminergic GPCRs interact with a significant portion of drugs currently on the market. However, most drugs that target these receptors are associated with undesirable side effects, which are due in part to promiscuous interactions with close homologs of the intended target receptors. Here, based on a systematic analysis of all 37 of the currently available high-resolution crystal structures of aminergic GPCRs, we review structural elements that contribute to and can be exploited for designing subtype-selective compounds. We describe the roles of secondary binding pockets (SBPs), as well as differences in ligand entry pathways to the orthosteric binding site, in determining selectivity. In addition, using the available crystal structures, we have identified conformational changes in the SBPs that are associated with receptor activation and explore the implications of these changes for the rational development of selective ligands with tailored efficacy.

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.

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.

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.

Novel 1-, 5-, and 8-substituted analogues of sumanirole (1), a dopamine D2/D3 receptor (D2R/D3R) agonist, were synthesized. Binding affinities at both D2R and D3R were higher when determined in competition with the agonist radioligand [(3)H]7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT) than with the antagonist radioligand [(3)H]N-methylspiperone. Although 1 was confirmed as a D2R-preferential agonist, its selectivity in binding and functional studies was lower than previously reported. All analogues were determined to be D2R/D3R agonists in both GoBRET and mitogenesis functional assays. Loss of efficacy was detected for the N-1-substituted analogues at D3R. In contrast, the N-5-alkyl-substituted analogues, and notably the n-butyl-arylamides (22b and 22c), all showed improved affinity at D2R over 1 with neither a loss of efficacy nor an increase in selectivity. Computational modeling provided a structural basis for the D2R selectivity of 1, illustrating how subtle differences in the highly homologous orthosteric binding site (OBS) differentially affect D2R/D3R affinity and functional efficacy.

The dopamine D3 receptor (D3R) has been implicated in substance abuse and other neuropsychiatric disorders. The high sequence homology between the D3R and D2R, especially within the orthosteric binding site (OBS) that binds dopamine, has made the development of D3R-selective compounds challenging. Here, we deconstruct into pharmacophoric elements a series of D3R-selective substituted-4-phenylpiperazine compounds, and use computational simulations and binding and activation studies to dissect the structural bases for D3R selectivity and efficacy. We find that selectivity arises from divergent interactions within a second binding pocket (SBP) separate from the OBS, whereas efficacy depends on the binding mode in the OBS. Our findings reveal structural features of the receptor that are critical to selectivity and efficacy that can be used to design highly D3R-selective ligands with targeted efficacies. These findings are generalizable to other GPCRs in which the SBP can be targeted by bitopic or allosteric ligands.

The prediction of intramolecular contacts has a useful application in predicting the three-dimensional structures of proteins. The accuracy of the template-based contact prediction methods depends on the quality of the template structures. To reduce the false positive predictions associated with using the entire set of template-derived contacts, we develop selection filters that use sequence conservation information to predict subsets of contacts more likely to be structurally conserved between the template and the target. The method is developed specifically for protein families with few available templates such as the G protein-coupled receptor (GPCR) family. It is validated on a test set of 342 template-target pairs from three protein families, and applied to one template-target pair from the GPCR family. We find that the filter selection method increases the accuracy of contact prediction with sufficient coverage for structure prediction.

To understand the structural basis for the Na(+)-sensitivity of ligand binding to dopamine D2-like receptors, using computational analysis in combination with binding assays, we identified interactions critical in propagating the impact of Na(+) on receptor conformations and on the ligand-binding site. Our findings expand the pharmacologically-relevant conformational spectrum of these receptors.

The transcription factor NFAT5/TonEBP is evolutionarily the oldest member of the NFAT/Rel family of transcription factors. We show that NFAT5 is uniquely related to NF-κB and is the only member of the Rel/NFAT family to be activated by osmotic stress. Like Rel/NF-κB proteins but unlike the calcium-regulated NFAT proteins, NFAT5 is constitutively dimeric, and dimerization is essential for DNA binding and transcriptional activity. Using dominant-negative proteins that inhibit NFAT5 dimerization, we show that NFAT5 regulates expression of the TNFα and lymphotoxin-β genes in osmotically stressed T cells. Chromatin immunoprecipitation experiments confirm that NFAT5 binds to the TNFα promoter in vivo. We suggest that NFAT5 participates in specific aspects of host defense by upregulating TNF family genes and other target genes in T cells.

Building reliable structural models of G protein-coupled receptors (GPCRs) is a difficult task because of the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template-based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1-3 A C alpha RMSD of the native structure remains a major challenge. Here, we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter-residue contacts between the template and target, and exploits an all-atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor-ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human beta(2)-adrenergic receptor (beta(2)AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison with the X-ray crystal structure of the beta(2)AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix-helix packing interactions that distinguish rhodopsin from beta(2)AR.

A three-dimensional model of the human Calcium-sensing receptor (CaSR) seven transmembrane domain was built via a novel sequence alignment method based on the conserved contacts in proteins using the crystal structure of bovine rhodopsin as the template. This model was tested by docking NPS 2143, the first identified allosteric antagonist of CaSR. In our model, Glu837 plays a critical role in anchoring the protonated nitrogen atom and hydroxy oxygen atom of NPS 2143. The phenyl moiety of the ligand contacts residues Phe668, Pro672, and Ile841. The naphthalene moiety is surrounded by several hydrophobic residues, including Phe684, Phe688, and Phe821. Our model appears to be consistent with all six residues that have been demonstrated to be critical for NPS 2143 binding, in contrast with existing homology models based on traditional sequence alignment of CaSR to rhodopsin. This provides validation of our sequence alignment method and the use of the rhodopsin backbone as the initial structure in homology modeling of other G protein-coupled receptors that are not members of the rhodopsin family. Proteins 2008. © 2007 Wiley-Liss, Inc.

The dopamine D2-like receptors (D2R, D3R, and D4R), which belong to class A G protein-coupled receptors, are important targets for antipsychotics. In the development of novel antipsychotics to avoid adverse side effects, subtype-selective dopamine receptor ligands that bind divergent secondary binding pockets (SBP) in addition to the conserved orthosteric binding site (OBS) have been discovered. SB269652, an allosteric modulator of D2R, consists of a tetrahydroisoquinoline pharmacophore bound in the OBS and an indole-2-carboxamide moiety bound in a SBP between TM2 and TM7. This ligand was recently characterized to act allosterically by a novel mechanism involving a D2R homodimer whereby the bitopic binding mode of SB269652 in one protomer negatively modulates the binding of an orthosteric ligand, either an agonist or an inverse agonist, to the second protomer (Lane et al., Nature Chemical Biology, 2014).To characterize the structural basis for the allosteric effect of SB269652, we combined ligand modifications, complementary receptor mutagenesis, and computational analysis, to identify the key interactions between SB269652 and D2R and the correspondingly induced conformational changes that are critical to propagate the allosteric impact across the dimer interface. We specifically considered three pairs of SB269652 derivatives that are modified in the orthosteric or secondary pharmacophore, or the linker region - in each pair, the modifications either retain/improve or eliminate the allosteric property. Using molecular docking and dynamics simulations, we characterized the binding modes of these derivatives in D2R, either in a protomer alone or in a dimer context with an orthosteric ligand bound in the other protomer. The results of our comparative modeling and analysis highlight the importance of the receptor-ligand interaction formed in the SBP between Glu95(2.65) and the indole-2-carboxamide moiety, in modulating the conformational rearrangements of the dimer interface(s).