The integrated stress response (ISR) is a highly conserved cellular pathway triggered by a variety of insults, reducing protein synthesis and inducing ATF4, leading to broadly remodeling the cellular transcriptome and metabolome. ISRIB, 1, the first identified eIF2B activator, attenuates the ISR restoring protein synthesis, but its poor solubility limits absorption and advancement. To improve drug-like properties, we explored replacements for both the cyclohexyl core and side chains of ISRIB. This effort initially led to truncated analogue, 2BAct, 13, which demonstrated improved solubility relative to 1; however, cardiovascular effects in higher species limited its progression into the clinic. Potent analogue 9 was identified with significantly improved solubility vs 1 but was still projected to have solubility-limited absorption. A prodrug campaign resulted in the identification of compound 26 (fosigotifator), which exhibited significantly improved solubility and is currently being investigated in the clinic.

Herein, we report the discovery and optimization of a series of cluster of differentiation 38 (CD38) inhibitors derived from a virtual ligand screening (VLS) campaign. VLS identified imidazopyridazine hit 9, which was optimized to a novel and potent CD38 inhibitor peripheral tool 25 (A-8531) with an excellent pharmacokinetic profile. A cocrystal structure in addition to biophysical and biochemical characterization of 25 is consistent with uncompetitive inhibition of CD38 via the formation of covalent adduct 28. Further structure- and property-based modifications of 25 afforded sulfuryl pyrazole 39 (A-3190) with improved CNS distribution properties. Brain-penetrant thienopyrimidine 39 shows robust rodent pharmacokinetics and in vivo target engagement across skin, lung, liver, and brain, making it an excellent CD38 tool for the study of indications requiring engagement of CD38 in the brain.

A novel class of selective and potent WRN helicase antagonists identified via a DNA-encoded library screen was rigorously validated by various biophysical assays including ASMS, TSA, and SPR. Preliminary structure-activity-relationship studies identified the key pharmacophores and advanced the biochemical potency to single digit nanomolar. Potent analogs demonstrated anti-proliferative activities specifically in cell lines with a WRN genetic dependency. A crystal structure of a ligand-WRN complex revealed an unexpected large shift of the helicase domain compared to the apo WRN structure with ADP. These structural insights led to the successful design of covalent inhibitors and the identification of compound resistant WRN mutants that confirmed on-mechanism cellular activity. The covalent interaction between the ligand and at WRN Cys727 was confirmed by intact mass spectrometry and a bound crystal structure. The discovery of these unique WRN inhibitors provides more insight into the field and offers an opportunity to further optimize such molecules.

Compounds that inhibit glutathione peroxidase 4 (GPX4) hold promise as cancer therapeutics in their ability to induce a form of nonapoptotic cell death called ferroptosis. Our research identified 24, a structural analog of the potent GPX4 inhibitor RSL3, that has much better plasma stability (t1/2 > 5 h in mouse plasma). The bioavailability of 24 provided efficacious plasma drug concentrations with IP dosing, thus enabling in vivo studies to assess tolerability and efficacy. An efficacy study in mouse using a GPX4-sensitive tumor model found that doses of 24 up to 50 mg/kg were tolerated for 20 days but had no effect on tumor growth, although partial target engagement was observed in tumor homogenate.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

TRPV3 is a nonselective cation channel activated by temperatures above 33°C and is reported to be localized in keratinocytes and nervous tissue. To investigate a role for TRPV3 in pain modulation, we conducted a series of in vivo electrophysiological studies on spinal and brain nociceptive neurons. Structurally diverse TRPV3 receptor antagonists reduced responses of spinal wide dynamic range (WDR) neurons to low-intensity mechanical stimulation in neuropathic rats, but only CNS-penetrant antagonists decreased elevated spontaneous firing. Injections of an antagonist into the neuronal receptive field, into the L5 dorsal root ganglion, or intracerebroventricularly (ICV) attenuated the evoked firing, but only ICV injections reduced spontaneous activity. Intraspinal injections did not affect either. Spinal transection blocked the effect on spontaneous but not evoked firing after systemic delivery of a TRPV3 antagonist. Systemic administration of an antagonist to neuropathic rats also impacted the firing of On- and Off-cells in the rostral ventromedial medulla in a manner consistent with dampening nociceptive signaling. An assessment of nonevoked "pain," an EEG-measured pain-induced sleep disturbance induced by hind paw injections of CFA, was also improved with CNS-penetrant TRPV3 antagonists but not by an antagonist with poor CNS penetration. Antagonism of TRPV3 receptors modulates activity of key classes of neurons in the pain pathway in a manner consistent with limiting pathological nociceptive signaling and was mediated by receptors in the periphery and brain. Blockade of TRPV3 receptors is likely an effective means to alleviate mechanical allodynia and nonevoked pain. However, the latter will only be obtained by blocking supraspinal TRPV3 receptors.NEW & NOTEWORTHY Recent studies have linked TRPV3 to pain modulation, and much of this work has focused on its role in the skin-primary afferent interface. In this electrophysiological study, we demonstrate that receptor antagonists modulate evoked signals through peripheral mechanisms but blockade of supraspinal TRPV3 receptors contributes to dampening both evoked and nonevoked "pain" through descending modulation. Thus, the full therapeutic potential of TRPV3 antagonists may only be realized with the ability to access receptors in the brain.

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

An efficient asymmetric synthesis of dipyridyl TRPV3 antagonist 1 is reported. The four-step route involves two C-C bond-forming steps, a highly diastereoselective alkene hydration, and asymmetric ketone hydrosilylation in 97% ee.

Transient receptor potential vanilloid 1 (TRPV1) is a multifunctional ion channel playing important roles in a numerous biological processes including the regulation of body temperature. Within distinct and tight chemical space of chromanyl ureas TRPV1 ligands were identified that exhibit distinctive pharmacology and a spectrum of thermoregulatory effects ranging from hypothermia to hyperthermia. The ability to manipulate these effects by subtle structural modifications of chromanyl ureas may serve as a productive approach in TRPV1 drug discovery programs addressing either side effect or desired target profiles of the compounds. Because chromanyl ureas in the TRPV1 context are generally antagonists, we verified observed partial agonist effects of a subset of compounds within that chemotype by comparing the in vitro profile of Compound 3 with known partial agonist 5'-I-RTX.

SAR studies on a series of thiophene amide derivatives provided CB(2) receptor agonists. The activity of the compounds was characterized by radioligand binding determination, multiple functional assays, ADME, and pharmacokinetic studies. A representative compound with selectivity for CB(2) over CB(1) effectively produced analgesia in behavioral models of neuropathic, inflammatory, and postsurgical pain. Control experiments using a CB(2) antagonist demonstrated the efficacy in the pain models resulted from CB(2) agonism.