MT-125 Inhibits Non-Muscle Myosin IIA and IIB, Synergizes with Oncogenic Kinase Inhibitors, and Prolongs Survival in Glioblastoma.

We have identified a NMIIA and IIB-specific small molecule inhibitor, MT-125, and have studied its effects in GBM. MT-125 has high brain penetrance and retention and an excellent safety profile; blocks GBM invasion and cytokinesis, consistent with the known roles of NMII; and prolongs survival as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, and it synergizes with FDA-approved PDGFR and mTOR inhibitors in vitro . Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor significantly improves survival in orthotopic GBM models over either drug alone, and in the case of sunitinib, markedly prolongs survival in ∼40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM. Highlights: MT-125 is a highly specific small molecule inhibitor of non-muscle myosin IIA and IIB, is well-tolerated, and achieves therapeutic concentrations in the brain with systemic dosing.Treating preclinical models of glioblastoma with MT-125 produces durable improvements in survival.MT-125 stimulates PDGFR- and MAPK-driven signaling in glioblastoma and increases dependency on these pathways.Combining MT-125 with an FDA-approved PDGFR inhibitor in a mouse GBM model synergizes to improve median survival over either drug alone, and produces tumor free, prolonged survival in over 40% of mice.

HDAC specificity and kinase off-targeting by purine-benzohydroxamate anti-hematological tumor agents.

A series of hybrid inhibitors, combining pharmacophores of known kinase inhibitors bearing anilino-purines (ruxolitinib, ibrutinib) and benzohydroxamate HDAC inhibitors (nexturastat A), were generated in the present study. The compounds have been synthesized and tested against solid and hematological tumor cell lines. Compounds 4d-f were the most promising in cytotoxicity assays (IC50 ≤ 50 nM) vs. hematological cells and displayed moderate activity in solid tumor models (EC50 = 9.3-21.7 µM). Compound 4d potently inhibited multiple kinase targets of interest for anticancer effects, including JAK2, JAK3, HDAC1, and HDAC6. Molecular dynamics simulations showed that 4d has stable interactions with HDAC and members of the JAK family, with differences in the hinge binding energy conferring selectivity for JAK3 and JAK2 over JAK1. The kinase inhibition profile of compounds 4d-f allows selective cytotoxicity, with minimal effects on non-tumorigenic cells. Moreover, these compounds have favorable pharmacokinetic profiles, with high stability in human liver microsomes (e.g., see t1/2: >120 min for 4f), low intrinsic clearance, and lack of significant inhibition of four major CYP450 isoforms.

1,3-Diphenylureido hydroxamate as a promising scaffold for generation of potent antimalarial histone deacetylase inhibitors.

We report a series of 1,3-diphenylureido hydroxamate HDAC inhibitors evaluated against sensitive and drug-resistant P. falciparum strains. Compounds 8a-d show potent antiplasmodial activity, indicating that a phenyl spacer allows improved potency relative to cinnamyl and di-hydrocinnamyl linkers. In vitro, mechanistic studies demonstrated target activity for PfHDAC1 on a recombinant level, which agreed with cell quantification of the acetylated histone levels. Compounds 6c, 7c, and 8c, identified as the most active in phenotypic assays and PfHDAC1 enzymatic inhibition. Compound 8c stands out as a remarkable inhibitor, displaying an impressive 85% inhibition of PfHDAC1, with an IC50 value of 0.74 µM in the phenotypic screening on Pf3D7 and 0.8 µM against multidrug-resistant PfDd2 parasites. Despite its potent inhibition of PfHDAC1, 8c remains the least active on human HDAC1, displaying remarkable selectivity. In silico studies suggest that the phenyl linker has an ideal length in the series for permitting effective interactions of the hydroxamate with PfHDAC1 and that this compound series could bind as well as in HsHDAC1. Taken together, these results highlight the potential of diphenylurea hydroxamates as a privileged scaffold for the generation of potent antimalarial HDAC inhibitors with improved selectivity over human HDACs.

Basolateral amygdala corticotropin releasing factor receptor 2 interacts with nonmuscle myosin II to destabilize memory in males.

Preclinical studies show that inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory in male and female adult and adolescent rodents. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g., dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To understand the mechanisms responsible for drug specific selectivity we began by investigating, in male mice, the pharmacokinetic differences in METH and COC brain exposure . Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, we next assessed transcriptional differences. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotropin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility. Pretreatment with AS2B prevented the ability of Blebb to disrupt an established METH-associated memory. Alternatively, combining CRF2 overexpression and agonist treatment, urocortin 3 (UCN3), in the BLA during conditioning rendered COC-associated memory susceptible to disruption by NMII inhibition, mimicking the Blebb-induced, retrieval-independent memory disruption seen with METH. These results suggest that BLA CRF2 receptor activation during memory formation in male mice can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption by NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Structure-Activity Relationship and Biological Investigation of a REV-ERBα-Selective Agonist SR-29065 (34) for Autoimmune Disorders.

Autoimmune diseases affect 50 million Americans, predominantly women, and are thought to be one of the top 10 leading causes of death among women in age groups up to 65 years. A central role for TH17 cells has been highlighted by genome-wide association studies (GWAS) linking genes preferentially expressed in TH17 cells to several human autoimmune diseases. We and others have reported that the nuclear receptors REV-ERBα and ß are cell-intrinsic repressors of TH17 cell development and pathogenicity and might therefore be therapeutic targets for intervention. Herein, we describe detailed SAR studies of a novel REV-ERBα-selective scaffold. Metabolic stability of the ligands was optimized allowing for in vivo interrogation of the receptor in a mouse model of multiple sclerosis (EAE) with a ligand (34). Reduction in frequency and number of T-cells in the CNS as well as key REV-ERB target genes is a measure of target engagement in vivo.

Basolateral Amygdala Corticotrophin Releasing Factor Receptor 2 Interacts with Nonmuscle Myosin II to Destabilize Memory.

Inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g. dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To investigate a potential source of this specificity, pharmacokinetic differences in METH and COC brain exposure were examined. Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, transcriptional differences were next assessed. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotrophin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility after METH conditioning. Pretreatment with AS2B occluded the ability of Blebb to disrupt an established METH-associated memory. Alternatively, the Blebb-induced, retrieval-independent memory disruption seen with METH was mimicked for COC when combined with CRF2 overexpression in the BLA and its ligand, UCN3 during conditioning. These results indicate that BLA CRF2 receptor activation during learning can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption via NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Addressing the oxamniquine in vitro-in vivo paradox to facilitate a new generation of anti-schistosome treatments.

The antischistosomal drug oxamniquine, OXA, requires activation by a sulfotransferase within the parasitic worm to enable killing. Examination of the pharmacokinetic/pharmacodynamic (PK/PD) relationship for OXA identified an in vitro-in vivo paradox with the maximal clinical plasma concentrations five-to ten-times lower than the efficacious concentration for in vitro schistosomal killing. The parasite resides in the vasculature between the intestine and the liver, and modeling the PK data to determine portal concentrations fits with in vitro studies and explains the required human dose. In silico models were used to predict murine dosing to recapitulate human conditions for OXA portal concentration and time course. Follow-up PK studies verified in mice that a 50-100 mg/kg oral gavage dose of OXA formulated in acetate buffer recapitulates the 20-40 mg/kg dose common in patients. OXA was rapidly cleared through a combination of metabolism and excretion into bile. OXA absorbance and tissue distribution were similar in wild-type and P-gp efflux transporter knockout mice. The incorporation of in vitro efficacy data and portal concentration was demonstrated for an improved OXA-inspired analog that has been shown to kill S. mansoni, S. haematobium, and S. japonicum, whereas OXA is only effective against S. mansoni. Second-generation OXA analogs should optimize both in vitro killing and physiochemical properties to achieve high portal concentration via rapid oral absorption, facilitated by favorable solubility, permeability, and minimal intestinal metabolism.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Identification and Characterization of a Novel Indoleamine 2,3-Dioxygenase 1 Protein Degrader for Glioblastoma.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunosuppressive enzyme that inhibits the antitumor immune response through both tryptophan metabolism and non-enzymatic functions. To date, most IDO1-targeted approaches have focused on inhibiting tryptophan metabolism. However, this class of drugs has failed to improve the overall survival of patients with cancer. Here, we developed and characterized proteolysis targeting chimeras (PROTACs) that degrade the IDO1 protein. IDO1-PROTACs were tested for their effects on IDO1 enzyme and non-enzyme activities. After screening a library of IDO1-PROTAC derivatives, a compound was identified that potently degraded the IDO1 protein through cereblon-mediated proteasomal degradation. The IDO1-PROTAC: (i) inhibited IDO1 enzyme activity and IDO1-mediated NF-κB phosphorylation in cultured human glioblastoma (GBM) cells, (ii) degraded the IDO1 protein within intracranial brain tumors in vivo, and (iii) mediated a survival benefit in mice with well-established brain tumors. This study identified and characterized a new IDO1 protein degrader with therapeutic potential for patients with glioblastoma.

DNA-Encoded Library Screening To Inform Design of a Ribonuclease Targeting Chimera (RiboTAC).

Ribonuclease targeting chimeras (RiboTACs) induce degradation of an RNA target by facilitating an interaction between an RNA and a ribonuclease (RNase). We describe the screening of a DNA-encoded library (DEL) to identify binders of monomeric RNase L to provide a compound that induced dimerization of RNase L, activating its ribonuclease activity. This compound was incorporated into the design of a next-generation RiboTAC that targeted the microRNA-21 (miR-21) precursor and alleviated a miR-21-associated cellular phenotype in triple-negative breast cancer cells. The RNA-binding module in the RiboTAC is Dovitinib, a known receptor tyrosine kinase (RTK) inhibitor, which was previously identified to bind miR-21 as an off-target. Conversion of Dovitinib into this RiboTAC reprograms the known drug to selectively affect the RNA target. This work demonstrates that DEL can be used to identify compounds that bind and recruit proteins with effector functions in heterobifunctional compounds.

Dual Inhibitors of Main Protease (MPro) and Cathepsin L as Potent Antivirals against SARS-CoV2.

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.

Development of a putative Zn2+-chelating but highly selective MMP-13 inhibitor.

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.

Morphing Natural Product Platensimycin via Heck, Sonogashira, and One-Pot Sonogashira/Cycloaddition Reactions to Produce Antibiotics with In Vivo Activity.

Type II fatty acid synthases are promising drug targets against major bacterial pathogens. Platensimycin (PTM) is a potent inhibitor against ß-ketoacyl-[acyl carrier protein] synthase II (FabF) and ß-ketoacyl-[acyl carrier protein] synthase I (FabB), while the poor pharmacokinetics has prevented its further development. In this work, thirty-two PTM derivatives were rapidly prepared via Heck, Sonogashira, and one-pot Sonogashira/cycloaddition cascade reactions based on the Gram-scale synthesis of 6-iodo PTM (4). About half of the synthesized compounds were approximately equipotent to PTM against the tested Staphylococcus aureus strains. Among them, the representative compounds 4, A4, and B8 exhibited different plasma protein binding affinity or stability in the human hepatic microsome assay and showed improved in vivo efficacy over PTM in a mouse peritonitis model. In addition, A4 was also effective in an S. aureus-infected skin mouse model. Our study not only significantly expands the known PTM derivatives with improved antibacterial activities in vivo, but showcased that C-C cross-coupling reactions are useful tools to functionalize natural product drug leads.

An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

Advances in Sulfonamide Kappa Opioid Receptor Antagonists: Structural Refinement and Evaluation of CNS Clearance.

Focused modification of a sulfonamide-based kappa opioid receptor (KOR) antagonist series previously reported by this laboratory was investigated. A total of 32 analogues were prepared to explore linker replacement, constraint manipulation, and aryl group or amine substitution. All analogues were assayed for KOR antagonist activity, and the initial lead compound was assessed for in vivo CNS penetration. The most improved analogue possessed a 4-fold increase of potency (IC50 = 18.9 ± 4.4 nM) compared with the lead compound (IC50 = 83.5 ± 20 nM) from an earlier work. The initial lead compound was found to attain suitable brain levels and to possess a shorter clearance time than canonical KOR antagonists such as JDTic.

A Novel HER2-Selective Kinase Inhibitor Is Effective in HER2 Mutant and Amplified Non-Small Cell Lung Cancer.

In-frame insertions in exon 20 of HER2 are the most common HER2 mutations in patients with non-small cell lung cancer (NSCLC), a disease in which approved EGFR/HER2 tyrosine kinase inhibitors (TKI) display poor efficiency and undesirable side effects due to their strong inhibition of wild-type (WT) EGFR. Here, we report a HER2-selective covalent TKI, JBJ-08-178-01, that targets multiple HER2 activating mutations, including exon 20 insertions as well as amplification. JBJ-08-178-01 displayed strong selectivity toward HER2 mutants over WT EGFR compared with other EGFR/HER2 TKIs. Determination of the crystal structure of HER2 in complex with JBJ-08-178-01 suggests that an interaction between the inhibitor and Ser783 may be responsible for HER2 selectivity. The compound showed strong antitumoral activity in HER2-mutant or amplified cancers in vitro and in vivo. Treatment with JBJ-08-178-01 also led to a reduction in total HER2 by promoting proteasomal degradation of the receptor. Taken together, the dual activity of JBJ-08-178-01 as a selective inhibitor and destabilizer of HER2 represents a combination that may lead to better efficacy and tolerance in patients with NSCLC harboring HER2 genetic alterations or amplification. SIGNIFICANCE: This study describes unique mechanisms of action of a new mutant-selective HER2 kinase inhibitor that reduces both kinase activity and protein levels of HER2 in lung cancer.

Oxidative Metabolism as a Modulator of Kratom's Biological Actions.

The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom's major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

Discovery of Selective Inhibitors for In Vitro and In Vivo Interrogation of Skeletal Myosin II.

Myosin IIs, actin-based motors that utilize the chemical energy of adenosine 5'-triphosphate (ATP) to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite the therapeutic potential for muscle disorders, SkMII-specific inhibitors have not been reported and characterized. Here, we present the discovery, synthesis, and characterization of "skeletostatins," novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity 40- to 170-fold for SkMII over all other myosin II family members. In addition, the skeletostatins bear improved potency, solubility, and photostability, without cytotoxicity. Based on its optimal in vitro profile, MT-134's in vivo tolerability, efficacy, and pharmacokinetics were determined. MT-134 was well-tolerated in mice, impaired motor performance, and had excellent exposure in muscles. Skeletostatins are useful probes for basic research and a strong starting point for drug development.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

A promising chemical series of positive allosteric modulators of the µ-opioid receptor that enhance the antinociceptive efficacy of opioids but not their adverse effects.

Positive allosteric modulators (PAMs) of the µ-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.