Pollari, Sirkku

Abstract Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in many cancer cells to meet elevated energy demands, which is associated with poor prognosis. This suggests that MtCK may be a promising target for cancer therapeutics. We sought to discover first-in-class selective inhibitors of MtCK with diverse mechanisms of action using high-throughput screening, biochemical characterization and cryo-EM studies. Through these studies, we identified diverse types of compounds that modulate activity of MtCK in vitro , including fast-equilibrium and time-dependent orthosteric and allosteric inhibitors. Select hits were subjected to in vitro enzymatic and binding assays to assess MtCK inhibition and binding. A subset of inhibitors was advanced into structural studies using cryo-EM resulting in the molecular structure of MtCK with and without bound substrates in complex with an allosteric uncompetitive inhibitor that we discovered. These studies identified compound’s unique binding pocket on MtCK and established the molecular steps manifesting in the apparent uncompetitive mode of inhibition. We demonstrate that the compound stabilizes an active site loop in a closed conformation restricting access of the creatine substrate to and the release of phosphocreatine product from its binding pocket. These findings establish chemical tools suitable for validation of MtCK as a promising breast cancer target with high therapeutic potential and build a foundation for future structure-guided optimization of the hit compounds of MtCK we identified and de novo rational design of novel MtCK inhibitors. Abstract Figure

Class B G protein‐coupled receptors (GPCRs) are a highly important group of therapeutic targets with strong physiologic implications in cardiovascular, metabolic, or gastrointestinal systems. Endogenously activated by moderate‐length peptide hormones, class B GPCRs entertain a complex binding mechanism to induce stimulatory signaling processes. The secretin receptor (SCTR) is a prototypical member of this receptor family, having clinical relevance in multiple diseases including heart failure, digestive disorders, diabetes and obesity. [1‐4] However, its endogenous ligand secretin and peptide‐based analogs are the only ligands known to interact with SCTRs, likely due to challenges in class B GPCR drug discovery [5]. To find orally available therapeutic leads targeting this “undruggable” receptor, we focused on identifying positive allosteric modulators (PAMs), which act via structurally distinct binding pockets and potentiate hormonal signaling of native agonists. Thus, we developed a testing funnel tailored to discover SCTR small molecule modulators. After screening a 12,000‐compound library with a unique primary assay that used a mixture of stimulators, we identified three structurally related scaffolds (A‐C) enhancing peptidic SCTR activation [5]. Applying a comprehensive toolbox with binding and functional assays, we studied structure‐activity relationships of a well‐considered set of commercially available analogs incorporating these three scaffolds. Our investigations afforded an optimized analog B2, which showed substantial positive cooperativity to low‐potent peptide agonists and significantly decelerated secretin dissociation. Due to the innate electrophilicity of scaffold set A, we recognized a structural similarity to PAMs acting on glucagon‐like peptide‐1 receptor (GLP‐1R), a prominent member of class B GPCR family. We further demonstrated that a subset of SCTR PAMs was able to augment not only SCTR signaling but also GLP‐1R functions. A recent study described that dual activation of SCTRs and GLP‐1Rs was benefiting glucose homeostasis and appetite regulation while avoiding proliferative effects on pancreas mass [6]. Determining cooperativity between PAMs and selected orthosteric peptides, we established distinct selectivity and probe dependency profiles. To decipher reversible and covalent mechanisms‐of‐action as well as potential binding sites of novel PAMs, we conducted glutathione reactivity, cAMP‐washout and receptor mutational studies. [7] To conclude, we discovered a unique class of SCTR modulators providing useful pharmacologic tools to study SCTR‐ or GLP‐1R‐specific activation pathways and explore novel allosteric binding sites with potential to instigate the development of first‐in‐class therapeutics for treating comorbid conditions obesity and diabetes. [7] References [1] Annu Rev Pharmacol Toxicol, 2020 , 60 , 89‐107; [2] Trends in Biochemical Sciences, 2017 , 12 , 946‐960; [3] Experimental Physiology, 2013 , 5 , 973‐987; [4] Current Neuropharmacology, 2007 , 3 , 168‐179; [5] SLAS Discovery, 2021 , 26 , 1‐16; [6] Journal of Peptide Science, 2017 , 12 , 845‐854; [7] submitted to Biochemical Pharmacology 2021 .

The Secretin receptor (SCTR) has been cloned as the first member of class B G protein‐coupled receptors (GPCRs) , giving rise to this highly important group of therapeutic targets, which are crucial for hormonal homeostasis . Despite the wide recognition of their functional importance, the development of potent small molecule ligands for Class B GPCRs remained challenging . To date, no ligands other than secretin peptide and its close analogs are known to interact with SCTRs, despite its therapeutic relevance in multiple diseases, such as heart failure, obesity, diabetes and cancer . In order to enhance our chances for discovering orally bioavailable drug candidates targeting an ‚undruggable’ class B GPCR, we focused our efforts on the identification of positive allosteric modulators (PAMs), which are likely to interact via structurally distinct binding sites and potentiate effects of native hormones and neurotransmitters . We developed a comprehensive tool box with robust high‐throughput screening and lead characterization/optimization assays capable of identifying and characterizing biologically relevant SCTR small molecule modulators. The dominant physiological coupling of SCTRs occurs via Gs proteins. Hence, we utilized this signaling mechanism to establish diverse cAMP accumulation assays suitable for simultaneous detection of PAMs modulating secretin and its potential metabolites. We also established a TR‐FRET based binding assay to further characterize potential PAMs with respect to their kinetic properties. Since functional selectivity has been proven to be therapeutically relevant for this family of membrane proteins, we additionally designed assays to investigate signaling mechanisms beyond Gs protein coupling, such as β‐arrestin recruitment, receptor internalization and calcium flux. High‐throughput screening (HTS) of a 360,000 compound library provided promising sets of hits, which have been further investigated regarding their allosteric activity and mechanism of action. We identified three related scaffolds with significant PAM activity at SCTRs. Selectivity studies revealed that this specific set of compounds demonstrated positive modulation not only on SCTRs but also on GLP‐1Rs (glucagon‐like peptide 1 receptors). Interestingly, dual activation of SCTRs and GLP‐1Rs has been shown to be beneficial for appetite regulation and glucose homeostasis without having proliferative effects on exocrine or total pancreas mass . To conclude, HTS and the development of a diverse panel of binding and functional assays has led to the discovery of small molecules with a unique dual‐acting pharmacological profile and potential to serve as starting points for developing first‐in‐class drug candidates for the treatment of comorbid conditions obesity and diabetes. Support or Funding Information Funding: NHLBI award 5R01HL133501