Dataset on Galanin Receptor 3 mutants that improve recombinant receptor expression and stability in an agonist and antagonist bound form.

Galanin Receptor 3 (GALR3) is a G-protein-coupled receptor with a widespread distribution in the brain and plays a role in a variety of physiologic processes including cognition/memory, sensory/pain processing, hormone secretion, and feeding behavior. Therefore, GALR3 is considered an attractive CNS drug target (Freimann et al., 2015) [1]. This dataset contains GALR3 point mutants that improve recombinant protein expression and thermal stability of the receptor contained in virus-like particles (VLPs) or obtained by detergent-purification of baculovirus-infected insect cells. The mutations listed can be grouped in those that improve the stability of the agonist-bound and the antagonist-bound form of the receptor. Protein characteristics in terms of protein expression and thermal stability were comparable between GPCR-VLP and GPCR overexpressing Sf9 cultures. The further analysis and detailed results of these mutants as well as their impact on biophysical assay development for drug discovery can be found in "Method for Rapid Optimization of Recombinant GPCR Protein Expression and Stability using Virus-Like Particles" (Ho et al., 2017) [2].

Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles.

Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay.

Reversible and irreversible small molecule inhibitors of monoamine oxidase B (MAO-B) investigated by biophysical techniques.

Monoamine oxidase B (MAO-B) plays a key role in the metabolism of dopamine, a neurotransmitter critical for the maintenance of cognitive function. Consequently, MAO-B is an important therapeutic target for disorders characterized by a decline in dopaminergic neurotransmission, including Parkinson's disease (PD). An emerging strategy in drug discovery is to utilize the biophysical approaches of thermal shift and isothermal titration calorimetry (ITC) to gain insight into binding modality and identify thermodynamically privileged chemical scaffolds. Described here is the development of such approaches for reversible and irreversible small molecule inhibitors of MAO-B. Investigation of soluble recombinant MAO-B revealed mechanism-based differences in the thermal shift and binding thermodynamic profiles of MAO-B inhibitors. Irreversible inhibitors demonstrated biphasic protein melt curves, large enthalpically favorable and entropically unfavorable binding, in contrast to reversible compounds, which were characterized by a dose-dependent increase in thermal stability and enthalpically-driven binding. The biophysical approaches described here aim to facilitate the discovery of next-generation MAO-B inhibitors.

Identification and SAR of selective inducible nitric oxide synthase (iNOS) dimerization inhibitors.

We have identified and synthesized a series of imidazole containing dimerization inhibitors of inducible nitric oxide synthase (iNOS). The necessity of key imidazole and piperonyl functionality was demonstrated and SAR studies led to the identification of compound 35, which showed a dose dependant inhibition in multiple pain models, including tactile allodynia induced by spinal nerve ligation (Chung model).

Pharmacological characterization of KLYP961, a dual inhibitor of inducible and neuronal nitric-oxide synthases.

Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (≤ 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states.

KLYP956 is a non-imidazole-based orally active inhibitor of nitric-oxide synthase dimerization.

Nitric-oxide synthases (NOS) generate nitric oxide (NO) through the oxidation of l-arginine. Inappropriate or excessive production of NO by NOS is associated with the pathophysiology of various disease states. Efforts to treat these disorders by developing arginine mimetic, substrate-competitive NOS inhibitors as drugs have met with little success. Small-molecule-mediated inhibition of NOS dimerization represents an intriguing alternative to substrate-competitive inhibition. An ultra-high-throughput cell-based screen of 880,000 small molecules identified a novel quinolinone with inducible NOS (iNOS) inhibitory activity. Exploratory chemistry based on this initial screening hit resulted in the synthesis of KLYP956, which inhibits iNOS at low nanomolar concentrations. The iNOS inhibitory potency of KLYP956 is insensitive to changes in concentrations of the substrate arginine, or the cofactor tetrahydrobiopterin. Mechanistic analysis suggests that KLYP956 binds the oxygenase domain in the vicinity of the active site heme and inhibits iNOS and neuronal NOS (nNOS) by preventing the formation of enzymatically active dimers. Oral administration of KLYP956 [N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide] inhibits iNOS activity in a murine model of endotoxemia and blocks pain behaviors in a formalin model of nociception. KLYP956 thus represents the first nonimidazole-based inhibitor of iNOS and nNOS dimerization and provides a novel pharmaceutical alternative to previously described substrate competitive inhibitors.

Back to basics: label-free technologies for small molecule screening.

Small molecule high-throughput screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.

differential roles for the E2A activation domains in B lymphocytes and macrophages.

The E2A gene encodes two E protein/class I basic helix-loop-helix transcription factors, E12 and E47, that are essential for B lymphopoiesis. In addition to the DNA-binding and protein dimerization domain, the E proteins share two highly conserved transcription activation domains. In this study, we show that both activation domains are required for optimal E2A-dependent transcription. Surprisingly, however, neither activation domain is required for E2A to rescue B lymphopoiesis from E2A(-/-) hemopoietic progenitors, although the N terminus of E2A, which harbors some transcription capacity, is required. Therefore, the E protein activation domains function redundantly in promoting B cell development. In contrast, the N-terminal activation domain, AD1, is required for a newly described ability of E2A to suppress macrophage development in vitro. Our findings demonstrate distinct functionalities for the E protein activation domains in B lymphocytes and macrophages.

Inhibition of inducible nitric oxide synthase expression by a novel small molecule activator of the unfolded protein response.

The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.