Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

Characterization of PCS1055, a novel muscarinic M4 receptor antagonist.

Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M4 receptor antagonist. PCS1055 inhibited radioligand [(3)H]-NMS binding to the M4 receptor with a Ki=6.5nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M4-selective reagents such as the muscarinic-peptide toxins (Karlsson et al., 1994; Santiago and Potter, 2001a) at the M1 subtype, and benzoxazine ligand PD102807 at the M3-subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [(3)H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[(35)S] binding. These GTP-γ-[(35)S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M4 versus the M1-, M2(-), M3-or M5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72nM. Therefore, PCS1055 represents a new M4-preferring antagonist that may be useful in elucidating the roles of M4 receptor signaling.

Design and synthesis of N-[6-(Substituted Aminoethylideneamino)-2-Hydroxyindan-1-yl]arylamides as selective and potent muscarinic M1 agonists.

The observation that cholinergic deafferentation of circuits projecting from forebrain basal nuclei to frontal and hippocampal circuits occurs in Alzheimer's disease has led to drug-targeting of muscarinic M1 receptors to alleviate cognitive symptoms. The high homology within the acetylcholine binding domain of this family however has made receptor-selective ligand development challenging. This work presents the synthesis scheme, pharmacokinetic and structure-activity-relationship study findings for M1-selective ligand, LY593093. Pharmacologically the compound acts as an orthosteric ligand. The homology modeling work presented however will illustrate that compound binding spans from the acetylcholine pocket to the extracellular loops of the receptor, a common allosteric vestibule for the muscarinic protein family. Altogether LY593093 represents a growing class of multi-topic ligands which interact with the receptors in both the ortho- and allosteric binding sites, but which exert their activation mechanism as an orthosteric ligand.

Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.

The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(ß,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved (Kruse et al., 2013), a structure that provides crucial insights into the mechanisms of orthosteric activation and allosteric modulation of muscarinic receptors.

Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites.

In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dim​ethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-ß-hydroxy-17-α-ethynyl-δ-4-androstano[3,2-b]pyrimido[1,2-a]benzimidazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.

Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

Functional proteomics identifies targets of phosphorylation by B-Raf signaling in melanoma.

Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.

Solvent accessibility of protein surfaces by amide H/2H exchange MALDI-TOF mass spectrometry.

One advantage of detecting amide H/2H exchange by mass spectrometry instead of NMR is that the more rapidly exchanging surface amides are still detectable. In this study, we present quench-flow amide H/2H exchange experiments to probe how rapidly the surfaces of two different proteins exchange. We compared the amide H/2H exchange behavior of thrombin, a globular protein, and IkappaBalpha, a nonglobular protein, to explore any differences in the determinants of amide H/2H exchange rates for each class of protein. The rates of exchange of only a few of the surface amides were as rapid as the "intrinsic" exchange rates measured for amides in unstructured peptides. Most of the surface amides exchanged at a slower rate, despite the fact that they were not seen to be hydrogen bonded to another protein group in the crystal structure. To elucidate the influence of the surface environment on amide H/2H exchange, we compared exchange data with the number of amides participating in hydrogen bonds with other protein groups and with the solvent accessible surface area. The best correlation with amide H/2H exchange was found with the total solvent accessible surface area, including side chains. In the case of the globular protein, the correlation was modest, whereas it was well correlated for the nonglobular protein. The nonglobular protein also showed a correlation between amide exchange and hydrogen bonding. These data suggest that other factors, such as complex dynamic behavior and surface burial, may alter the expected exchange rates in globular proteins more than in nonglobular proteins where all of the residues are near the surface.

Thermodynamics reveal that helix four in the NLS of NF-kappaB p65 anchors IkappaBalpha, forming a very stable complex.

IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Application of amide proton exchange mass spectrometry for the study of protein-protein interactions.

This protocol describes amide proton exchange experiments that probe for changes in solvent accessibility at protein-protein interfaces. The simplest version of the protocol, termed the "on-exchange" experiment, detects protein-protein interfaces by taking advantage of the fact that solvent deuterium oxide (D2O) molecules are excluded from the surface of a protein to which another protein is bound. A more complete version of the experiment can also be performed in which the rate of surface deuteration is initially measured separately for each of the proteins involved in the interaction, after which the deuterated proteins are allowed to complex and the rate of "off-exchange" (i.e., replacement of surface deuterons by protons from solvent H2O molecules) at the resulting protein-protein interface is measured. This version of the experiment yields additional kinetic information that can help to define the solvent-inaccessible "core" of the interface.