Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2.

The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from -4.9 (2) and -7.5 (3) to -6.2 (4) and -14.5 (5), but also a less favorable binding entropy (-TΔS, kcal/mol) from -2.3 (2) and -1.3 (3) to -0.5 (4) and 4.8 (5). Thus, the dissociation constants (Kd, µM) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 µM L-glutamate at homomeric rat GluA2(Q)i receptors with EC50 values of 67.3 and 2.45 µM, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.

Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4ß2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity.

Neuronal α4ß2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two α4 subunits in the (α4)(3)(ß2)(2) receptor subpopulation was discovered. In particular, three residues, H142, Q150, and T152, were demonstrated to be involved in the distinct pharmacology of the α4-α4 versus α4-ß2 binding sites. To obtain insight into the three-dimensional structure of the α4-α4 binding site, a surrogate protein reproducing α4-α4 binding characteristics was constructed by introduction of three point mutations, R104H, L112Q, and M114T, into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP). Cocrystallization with two agonists possessing distinct pharmacologic profiles, NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the α4-α4 interface. Confirmed by mutational studies, our structures suggest a unique ligand-specific role of residue H142 on the α4 subunit. In the cocrystal structure of the mutated Ls-AChBP with the high-efficacy ligand NS3920, the corresponding histidine forms an intersubunit bridge that reinforces the ligand-mediated interactions between subunits. The structures further reveal that the binding site residues gain different and ligand-dependent interactions that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting specific nicotinic acetylcholine receptor interfaces.

Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4ß2 receptors: unique role of halogen bonding revealed.

The α4ß2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4ß2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4ß2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4ß2 receptors. Crystal structures of five agonists with efficacies at α4ß2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

Structural analysis of the positive AMPA receptor modulators CX516 and Me-CX516 in complex with the GluA2 ligand-binding domain.

Positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) can serve as lead compounds for the development of cognitive enhancers. Several benzamide-type (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor modulators such as aniracetam, CX516 and CX614 have been shown to inhibit the deactivation of AMPA receptors with a less pronounced effect on desensitization. Despite CX516 being an extensively investigated AMPA receptor modulator and one of the few clinically evaluated compounds, the binding mode of CX516 to AMPA receptors has not been reported. Here, the structures of a GluA2 ligand-binding domain mutant in complex with CX516 and the 3-methylpiperidine analogue of CX516 (Me-CX516) are reported. The structures show that the binding modes of CX516 and Me-CX516 are similar to those of aniracetam and CX614 and that there is limited space for substitution at the piperidine ring of CX516. The results therefore support that CX516, like aniracetam and CX614, modulates deactivation of AMPA receptors.

Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2.

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6 µM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1 kcal≈4.187 kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46 mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.

Immunomodulation-A Molecular Solution to Treating Patients with Severe Bladder Pain Syndrome?

BACKGROUND: Patients with bladder pain syndrome experience debilitating pain and extreme frequency of urination. Numerous therapeutic approaches have been tested, but as the molecular basis of disease has remained unclear, specific therapies are not available. OBJECTIVE: Recently, a systematic gene deletion strategy identified interleukin-1 (IL-1) hyperactivation as a cause of severe cystitis in a murine model. Treatment with an IL-1 receptor antagonist (IL-1RA) restored health in genetically susceptible mice, linking IL-1-dependent inflammation to pain and pathology in the bladder mucosa. The study objective was to investigate whether IL-1RA treatment might be beneficial in patients with bladder pain syndrome. DESIGN SETTING AND PARTICIPANTS: Patients diagnosed with bladder pain syndrome were invited to participate and subjected to daily IL-1RA injections for 1 wk, followed by a treatment break. Patients with other urological disorders accompanied by pain were included as controls. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: When symptoms returned, treatment was resumed and responding patients were maintained on treatment long term, with individualized dosing regimens. Symptom scores were recorded and molecular effects were quantified by neuropeptide and gene expression analysis. DNA samples were subjected to exome genotyping. RESULTS AND LIMITATIONS: IL-1RA treatment reduced bladder pain and the frequency of urination in 13/17 patients (p < 0.001). Substance P levels in urine were lowered, and responders returned to a more normal lifestyle. Neuroinflammatory-dependent and IL-1-dependent gene networks were inhibited, as well as regulators of innate immunity. Genotyping revealed disease-associated IL1R1, NLRP3, and IL1RN DNA sequence variants in the responders. Controls did not benefit from IL-1RA treatment, except for one patent with cystitis cystica. CONCLUSIONS: In this clinical study, IL-1RA treatment is proposed to reduce chronic bladder pain, immediately and in the long term. Despite the limited number of study patients, the potent acute effect and lasting symptom relief indicate that this therapeutic approach may be worth exploring in controlled clinical trials. PATIENT SUMMARY: Treatment with an interleukin-1 (IL-1) receptor antagonist is proposed for treating bladder pain syndrome, as it can result in symptom relief and increase quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects.

Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2.

Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e., negative allosteric modulators; NAMs) used for treatment of epilepsy, and uncompetitive antagonists. We here report a 4.65 Å resolution X-ray structure of GluA2, revealing that four molecules of the competitive antagonist ZK200775 and four molecules of the NAM GYKI53655 are capable of binding at the same time. Using negative stain electron microscopy, we show that GYKI53655 alone or ZK200775/GYKI53655 in combination predominantly results in compact receptor forms. The agonist AMPA provides a mixed population of compact and bulgy shapes of GluA2 not impacted by addition of GYKI53655. Taken together, this suggests that the two different mechanisms of antagonism that lead to channel closure are independent and that the distribution between bulgy and compact receptors primarily depends on the ligand bound in the glutamate binding site. DATABASE: The atomic coordinates and structure factors from the crystal structure determination have been deposited in the Protein Data Bank under accession code https://doi.org/10.2210/pdb6RUQ/pdb. The electron microscopy 3D reconstruction volumes have been deposited in EMDB (EMD-4875: Apo; EMD-4920: ZK200775/GYKI53655; EMD-4921: AMPA compact; EMD-4922: AMPA/GYKI53655 bulgy; EMD-4923: GYKI53655; EMD-4924: AMPA bulgy; EMD-4925: AMPA/GYKI53655 compact).

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

Studies on Aryl-Substituted Phenylalanines: Synthesis, Activity, and Different Binding Modes at AMPA Receptors.

A series of racemic aryl-substituted phenylalanines was synthesized and evaluated in vitro at recombinant rat GluA1-3, at GluK1-3, and at native AMPA receptors. The individual enantiomers of two target compounds, (RS)-2-amino-3-(3,4-dichloro-5-(5-hydroxypyridin-3-yl)phenyl)propanoic acid 37 and (RS)-2-amino-3-(3'-hydroxybiphenyl-3-yl)propanoic acid 38, were characterized. (S)-37 and (R)-38 were identified as the only biologically active isomers, both being antagonists at GluA2 receptors with Kb of 1.80 and 3.90 µM, respectively. To address this difference in enantiopharmacology, not previously seen for amino acid-based AMPA receptor antagonists, X-ray crystal structures of both eutomers in complex with the GluA2 ligand binding domain were solved. The cocrystal structures of (S)-37 and (R)-38 showed similar interactions of the amino acid parts but unexpected and different orientations and interactions of the biaromatic parts of the ligands inside the binding site, with (R)-38 having a binding mode not previously identified for amino acid-based antagonists.

Perilipin 1 binds to aquaporin 7 in human adipocytes and controls its mobility via protein kinase A mediated phosphorylation.

Accumulating evidence suggests that dysregulated glycerol metabolism contributes to the pathophysiology of obesity and type 2 diabetes. Glycerol efflux from adipocytes is regulated by the aquaglyceroporin AQP7, which is translocated upon hormone stimulation. Here, we propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Biochemical analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation. Together, these findings are indicative of how glycerol release is controlled in adipocytes, and may pave the way for the future design of drugs against human metabolic pathologies.

L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain.

UNLABELLED: In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because L-Glu is difficult to displace, despite extensive dialysis. Here, we show that L-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mM) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mM), and we use differential scanning fluorimetry to show that L-Asp is able to stabilize the isolated GluA2 LBD. We also show that L-Asp can replace L-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mM L-Asp. Surprisingly, with 7.5 mM L-Asp, the GluA2 LBD crystallized as a mixed dimer, with L-Glu being present in one subunit, and neither L-Asp nor L-Glu being present in the other subunit. Thus, residual L-Glu is retained from the expression medium. On the other hand, only L-Asp was found at the binding site when 50 or 250 mM L-Asp was used for crystallization. The binding mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taking our findings together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. DATABASE: Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mM L-Asp), 4O3C (50 mM L-Asp), and 4O3A (250 mM L-Asp).

Synthesis, pharmacological and structural characterization, and thermodynamic aspects of GluA2-positive allosteric modulators with a 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide scaffold.

Positive allosteric modulators of ionotropic glutamate receptors are potential compounds for treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind within the dimer interface of the ligand-binding domain (LBD) and stabilize the agonist-bound conformation, thereby slowing receptor desensitization and/or deactivation. Here we describe the synthesis and pharmacological testing at GluA2 of a new generation of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. The most potent modulator 3 in complex with GluA2-LBD-L483Y-N754S was subjected to structural analysis by X-ray crystallography, and the thermodynamics of binding was studied by isothermal titration calorimetry. Compound 3 binds to GluA2-LBD-L483Y-N754S with a Kd of 0.35 µM (ΔH = -7.5 kcal/mol and -TΔS = -1.3 kcal/mol). This is the first time that submicromolar binding affinity has been achieved for this type of positive allosteric modulator. The major structural factor increasing the binding affinity of 3 seems to be interactions between the cyclopropyl group of 3 and the backbone of Phe495 and Met496.

Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL) isoforms.

BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependent manner, where the predominant 84 kDa form in adipocytes is the most extensively studied. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed negative stain electron microscopy (EM) to analyze the quarternary structure of the different HSL isoforms. The results show that all three isoforms adopt a head-to-head homodimeric organization, where each monomer contains two structural domains. We also used enzymatic assays to show that despite the variation in the size of the N-terminal domain all three isoforms exhibit similar enzymological properties with regard to psychrotolerance and protein kinase A (PKA)-mediated phosphorylation and activation. CONCLUSIONS/SIGNIFICANCE: We present the first data on the quaternary structure and domain organization of the three HSL isoforms. We conclude that despite large differences in the size of the N-terminal, non-catalytic domain all three HSL isoforms exhibit the same three-dimensional architecture. Furthermore, the three HSL isoforms are very similar with regard to two unique enzymological characteristics of HSL, i.e., cold adaptation and PKA-mediated activation.

Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area.

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL. Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA. This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate.

Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates.

BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate. CONCLUSIONS: Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region.