A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5'-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

A Homogeneous Bioluminescent System to Monitor Cyclic Guanosine Monophosphate.

Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is mediated through highly regulated phosphodiesterase activities. cGMP metabolism has been targeted for the treatment of several diseases, including erectile dysfunction, hypertension, and heart failure. As more drugs are being sought, it will be critical to develop assays that accurately determine cGMP levels. Here, we present cGMP Lumit, a sensitive and specific bioluminescent assay to detect cGMP. We demonstrate the utility of the detection system in enzyme assays, cell-based assays, and high-throughput screening formats. It is anticipated that this assay will be of significant value to aid in further understanding the role of cGMP in physiology and support further drug discovery efforts toward the treatment of human disease.

Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae.

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

Small-molecule inhibition of the archetypal UbiB protein COQ8.

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

2-Propylphenol Allosterically Modulates COQ8A to Enhance ATPase Activity.

COQ8A is an atypical kinase-like protein that aids the biosynthesis of coenzyme Q, an essential cellular cofactor and antioxidant. COQ8A's mode of action remains unclear, in part due to the lack of small molecule tools to probe its function. Here, we blend NMR and hydrogen-deuterium exchange mass spectrometry to help determine how a small CoQ precursor mimetic, 2-propylphenol, modulates COQ8A activity. We identify a likely 2-propylphenol binding site and reveal that this compound modulates a conserved COQ8A domain to increase nucleotide affinity and ATPase activity. Our findings promise to aid further investigations into COQ8A's precise enzymatic function and the design of compounds capable of boosting endogenous CoQ production for therapeutic gain.

Clinico-Genetic, Imaging and Molecular Delineation of COQ8A-Ataxia: A Multicenter Study of 59 Patients.

OBJECTIVE: To foster trial-readiness of coenzyme Q8A (COQ8A)-ataxia, we map the clinicogenetic, molecular, and neuroimaging spectrum of COQ8A-ataxia in a large worldwide cohort, and provide first progression data, including treatment response to coenzyme Q10 (CoQ10). METHODS: Cross-modal analysis of a multicenter cohort of 59 COQ8A patients, including genotype-phenotype correlations, 3D-protein modeling, in vitro mutation analyses, magnetic resonance imaging (MRI) markers, disease progression, and CoQ10 response data. RESULTS: Fifty-nine patients (39 novel) with 44 pathogenic COQ8A variants (18 novel) were identified. Missense variants demonstrated a pleiotropic range of detrimental effects upon protein modeling and in vitro analysis of purified variants. COQ8A-ataxia presented as variable multisystemic, early-onset cerebellar ataxia, with complicating features ranging from epilepsy (32%) and cognitive impairment (49%) to exercise intolerance (25%) and hyperkinetic movement disorders (41%), including dystonia and myoclonus as presenting symptoms. Multisystemic involvement was more prevalent in missense than biallelic loss-of-function variants (82-93% vs 53%; p = 0.029). Cerebellar atrophy was universal on MRI (100%), with cerebral atrophy or dentate and pontine T2 hyperintensities observed in 28%. Cross-sectional (n = 34) and longitudinal (n = 7) assessments consistently indicated mild-to-moderate progression of ataxia (SARA: 0.45/year). CoQ10 treatment led to improvement by clinical report in 14 of 30 patients, and by quantitative longitudinal assessments in 8 of 11 patients (SARA: -0.81/year). Explorative sample size calculations indicate that ≥48 patients per arm may suffice to demonstrate efficacy for interventions that reduce progression by 50%. INTERPRETATION: This study provides a deeper understanding of the disease, and paves the way toward large-scale natural history studies and treatment trials in COQ8A-ataxia. ANN NEUROL 2020;88:251-263.

Crystal structure of the WFIKKN2 follistatin domain reveals insight into how it inhibits growth differentiation factor 8 (GDF8) and GDF11.

Growth differentiation factor 8 (GDF8; also known as myostatin) and GDF11 are closely related members of the transforming growth factor ß (TGF-ß) family. GDF8 strongly and negatively regulates skeletal muscle growth, and GDF11 has been implicated in various age-related pathologies such as cardiac hypertrophy. GDF8 and GDF11 signaling activities are controlled by the extracellular protein antagonists follistatin; follistatin-like 3 (FSTL3); and WAP, follistatin/kazal, immunoglobulin, Kunitz, and netrin domain-containing (WFIKKN). All of these proteins contain a follistatin domain (FSD) important for ligand binding and antagonism. Here, we investigated the structure and function of the FSD from murine WFIKKN2 and compared it with the FSDs of follistatin and FSTL3. Using native gel shift and surface plasmon resonance analyses, we determined that the WFIKKN2 FSD can interact with both GDF8 and GDF11 and block their interactions with the type II receptor activin A receptor type 2B (ActRIIB). Further, we solved the crystal structure of the WFIKKN2 FSD to 1.39 Å resolution and identified surface-exposed residues that, when substituted with alanine, reduce antagonism of GDF8 in full-length WFIKKN2. Comparison of the WFIKKN2 FSD with those of follistatin and FSTL3 revealed differences in both the FSD structure and position of residues within the domain that are important for ligand antagonism. Taken together, our results indicate that both WFIKKN and follistatin utilize their FSDs to block the type II receptor but do so via different binding interactions.

Crystal structure of [propane-1,3-diylbis(piperidine-4,1-di-yl)]bis-[(pyridin-4-yl)methanone]-isophthalic acid (1/1).

In the crystal structure of the title co-crystal, C25H32N4O2·C8H6O4, isophthalic acid and [propane-1,3-diylbis(piperidine-4,1-di-yl)]bis-(pyridin-4-yl-methanone) mol-ecules are connected into supra-molecular chains aligned along the c axis by O-H⋯N hydrogen bonding. These aggregate into supra-molecular layers oriented parallel to the ac plane by C-H⋯O inter-actions. These layers then stack in an ABCD pattern along the b-axis direction by additional C-H⋯O inter-actions to give the full three-dimensional crystal structure. The central chain in the di-pyridyl-amide molecule has an anti-gauche conformation.