Kappa opioid receptor agonists are clinically used to treat pruritus and have therapeutic potential for the treatment of pain and neuropsychiatric disorders. We have previously shown that triazole 1.1 is a G protein signaling-biased KOR agonist, that can suppress itch without producing signs of sedation in mice. This profile was recapitulated in rats and non-human primates; however, triazole 1.1 had limited potency as an antipruritic. Here we describe a more potent, G protein signaling-biased agonist, triazole 187. Triazole 187 is a potent antipruritic agent and does not decrease spontaneous locomotor activity; interestingly, it produces anxiolytic-like behaviors in mice, an effect not observed for triazole 1.1. In addition to curbing sedation, triazole 187 produces only mild diuresis, resulting in 30% of urine output induced by U50,488H at a dose that is more than 100-fold the antipruritic potency dose. Compounds like triazole 187 may present a means to treat anxiety that is independent of or accompanied by persistent chronic itch while avoiding sedation and diuresis accompanied by typical KOR agonists.

Kappa opioid receptor (KOR) agonists are used clinically to treat chronic itch and are promising candidates for the treatment of pain, mood disorders, and opioid use disorder. Unfortunately, adverse effects such as diuresis and sedation have hampered the widespread clinical development of KOR agonists. Preferentially activating the G protein activation pathway over the βarrestin2 recruitment pathway has been proposed as a method to minimize adverse effects while maintaining desirable effects such as antipruritic and antinociceptive properties. Through a structure–activity relationship (SAR) study of a bisamide-containing lead series, we have generated KOR agonists with sub-nanomolar potency for activating G proteins, as well as several with high bias for G protein activation over βarrestin2 recruitment. A representative member of the series is also brain-penetrant and detectable in mouse brain and plasma 60 minutes post-injection. Based on these in vitro and in vivo results, the bisamide KOR series may yield useful compounds for further development.

Non-muscle myosin II (NMII), a molecular motor that regulates critical processes such as cytokinesis and neuronal plasticity, has substantial therapeutic potential. However, translating this potential to in vivo use has been hampered by a lack of selective tools. The most prototypical non-selective inhibitor inactivates both NMII and cardiac muscle myosin II (CMII), a key regulator of heart function. Using rational drug design, we developed a series of NMII inhibitors that markedly improve tolerability by selectively targeting NMII over CMII, including MT-228 and clinical candidate MT-110. MT-228 and MT-110 have excellent properties, including high brain penetration and efficacy in preclinical models of methamphetamine use disorder (MUD), which has no current FDA-approved therapies. The structure of MT-228 bound to myosin II provides insight into its selectivity for NMII over CMII. The broad therapeutic windows of these NMII inhibitors provide valuable tools for the scientific community and a promising clinical candidate for the treatment of MUD.

Congenital Zika syndrome (CZS), the set of fetal and neonatal complications associated with Zika virus (ZIKV) infection in pregnancy, was first noted during the outbreak in the Americas in 2015-2016. However, there was an unequal distribution of ZIKV cases and severe outcomes in all areas where ZIKV emerged in the Americas, demonstrating that the risk of CZS varied over space and time. Recently, we demonstrated that phenotypic heterogeneity existed between closely related ZIKV strains. All ZIKV strains tested infected the placenta but varied in their capacity to cause overt fetal harm. Here, we further characterized the relative contributions of virus genotype and infecting dose of two phenotypically distinct ZIKV strains across multiple timepoints in gestation in pregnant mice that lack type-I interferon receptor function (Ifnar1-/-). To better understand the underlying causes of adverse fetal outcomes, we used RNA sequencing to compare ZIKV-infected and uninfected tissues. We found that ZIKV infection triggers retinoic acid-inducible gene I (RIG-I)-like receptor-mediated activation of the interferon response at the maternal-fetal interface. However, modest chemical inhibition of RIG-I activation in the decidua and placenta did not protect against fetal demise. Instead, the fetal interferon response was significantly associated with fetal demise. Together, these findings suggest that the response to ZIKV at the maternal-fetal interface can vary, depending on the infecting ZIKV genotype and dose, and that the fetal immune response is an important mediator of fetal harm. IMPORTANCE:Congenital Zika syndrome is a constellation of fetal abnormalities ranging from fetal demise and microcephaly to infants that are born apparently healthy only to develop neurocognitive impacts later. ZIKV is now endemic in many regions worldwide, but how ZIKV harms the developing fetus remains an outstanding question. Previously, we used a mouse model of ZIKV infection during pregnancy to assess the pathogenic potential to the fetus of a panel of five low-passage ZIKV strains representing the viral genetic diversity in the Americas. We found that phenotypic heterogeneity existed between these closely related ZIKV strains. Here, we show that this heterogeneity is driven by RIG-I-like receptor-mediated activation of the interferon response at the maternal-fetal interface. We used chemical inhibition of the RIG-I pathway and measured the transcriptional activity of interferon-stimulated genes in fetuses to demonstrate that the fetal immune response may contribute to fetal demise.

A ribonuclease-targeting chimera (RiboTAC) is a heterobifunctional compound that binds to an RNA target and recruits a ribonuclease to cleave the bound RNA. This study investigates the impact of linker length and composition on RiboTAC potency in leukemia and breast cancer cellular models. Increasing linker length from two polyethylene glycol units to eight increased RiboTAC potency, while longer linker lengths decreased potency. The optimized RiboTAC reduced the abundance of oncogenic LGALS1 transcripts in THP-1 leukemia cells (∼95%) and in MDA-MB-231 triple negative breast cancer cells (∼70%), leading to a subsequent decrease of galectin-1 protein and induction of apoptosis. A series of target engagement assays were developed to validate effector protein-small molecule engagement in vitro and in cells, demonstrating the broad potential of these assays to study recruitment of other effector proteins. Collectively, these findings underscore the importance of linker optimization for enhancing RiboTAC potency.

Schistosomiasis, a neglected tropical disease affecting over 250 million people worldwide, relies on praziquantel (PZQ) as its sole treatment. However, PZQ has significant limitations, including inactivity against juvenile worms, inability to prevent reinfection, and emerging drug resistance. In this review, we outline the development of CIDD-0150303, a novel oxamniquine (OXA) derivative with pan-species and pan-stage activity against Schistosoma mansoni, PZQ-resistant S. mansoni, and S. haematobium. Using a structure-guided design approach, over 350 OXA analogs were synthesized and screened to identify leading drug candidate CIDD-0150303. CIDD-0150303 demonstrates 100% lethality in vitro and up to 80% reduction in worm burden in vivo. CIDD-0150303 is effective against both juvenile and adult parasites as well as PZQ-resistant S. mansoni. This compound represents a promising advance in schistosomiasis treatment to address urgent gaps in control/elimination strategies and PZQ resistance. However, dedicated safety and toxicity studies are still ongoing, and additional in vivo validation is required.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by the aberrant alternative pre-mRNA splicing of microtubule-associated protein tau (MAPT) exon 10, the inclusion of which encodes a toxic tau protein harboring four microtubule domains (4R tau). Here, we describe the design of an RNA-targeted small molecule that thermodynamically stabilizes the structure of a pre-mRNA splicing regulator element in the MAPT pre-mRNA exon 10-intron 10 junction to reduce the inclusion of exon 10 and hence 4R tau abundance. Structure-guided drug design was used to obtain compounds that form a network of specific interactions to the RNA, including multiple interactions between a single nucleotide (nt) A-bulge and the Hoogsteen face of a closing GC base pair, the latter of which was enabled by the design of base triple interactions. A battery of assays revealed that the compound binds the target in vitro and in cells and affects pre-mRNA splicing in various cellular models, including primary neurons from a human tau (htau) knock-in mouse model. The orally bioavailable compound was administered per os (p.o.), where treatment diminished exon 10 inclusion and reduced the 4R tau protein isoform. Further, the molecule mitigated cellular pathologies and behavioral phenotypes observed in the htau transgenic mouse model. This study provides a potentially general pipeline to design compounds that target RNAs, affect disease pathways, and deliver compounds that have oral bioavailability and blood-brain barrier penetrance.

Glioblastoma (GBM) is the most lethal of primary brain tumors. Here, we report our studies of MT-125, a small-molecule inhibitor of non-muscle myosin II. MT-125 has high brain penetrance and an excellent safety profile, blocks GBM invasion and cytokinesis, and prolongs survival in murine GBM models. By impairing mitochondrial fission, MT-125 increases redox stress and consequent DNA damage, and it synergizes with radiotherapy. MT-125 also induces oncogene addiction to PDGFR signaling through a mechanism that is driven by redox stress, and it synergizes with FDA-approved PDGFR and mTOR inhibitors in vitro. Consistent with this, we find that combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor, significantly improves survival in orthotopic GBM models over either drug alone. Our results demonstrate that MT-125 is a first-in-class therapeutic that has strong clinical potential for the treatment of GBM.

Cancer cells are acutely dependent on nuclear transport due to elevated transcriptional activity, suggesting an unrealized opportunity for selective therapeutic inhibition of the nuclear pore complex (NPC). Through large-scale phenotypic profiling of cancer cell lines, genome-scale functional genomic modifier screens, and mass spectrometry-based proteomics, we discovered that the clinical drug PRLX-93936 is a molecular glue that binds and reprograms the TRIM21 ubiquitin ligase to degrade the NPC. Upon compound-induced TRIM21 recruitment, the nuclear pore is ubiquitylated and degraded, resulting in the loss of short-lived cytoplasmic mRNA transcripts and the induction of cancer cell apoptosis. Direct compound binding to TRIM21 was confirmed via surface plasmon resonance and X-ray crystallography, whereas compound-induced TRIM21-nucleoporin complex formation was demonstrated through multiple orthogonal approaches in cells and in vitro. Phenotype-guided optimization yielded compounds with 10-fold greater potency and drug-like properties, along with robust pharmacokinetics and efficacy against pancreatic cancer xenografts and patient-derived organoids. SIGNIFICANCE:This study establishes the cancer therapeutic potential of optimized TRIM21 molecular glues to degrade the NPC and underscores the value of reexamining drugs with previously unknown mechanisms using current technologies.

Non-muscle myosin II (NMII), a molecular motor that regulates critical processes such as cytokinesis and neuronal synaptic plasticity, has substantial therapeutic potential. However, translating this potential to in vivo use has been hampered by the lack of selective tools. The most prototypical non-selective inhibitor, blebbistatin inactivates both NMII and cardiac myosin II (CMII), a key regulator of heart function. Using rational drug design, we developed a series of NMII inhibitors that improve tolerability by selectively targeting NMII over CMII, including MT-228, which has excellent properties such as high brain penetration and efficacy in preclinical models of stimulant use disorder, which has no current FDA-approved therapies. The structure of MT-228 bound to myosin II provides insight into its 17-fold selectivity for NMII over CMII. MT-228's broad therapeutic window opens the door to new disease treatments and provides valuable tools for the scientific community, along with promising leads for future medication development. Highlights:Research suggests numerous indications, from axon regeneration and cancer, would benefit from a small molecule inhibitor of non-muscle myosin II, a molecular motor that regulates the actin cytoskeleton. Current chemical probe options are very limited and lack sufficient safety for in vivo studies, which we show is primarily due to potent inhibition of cardiac myosin II.Rational design that focused on improving target selectivity over the pan-myosin II inhibitor, blebbistatin, led to the identification of MT-228, a small molecule inhibitor with a wide therapeutic window.High-resolution structure of MT-228 bound to myosin II reveals that selectivity results from a different positioning compared to blebbistatin and an important sequence difference between cardiac and non-muscle myosin II in the inhibitor binding pocket.A single administration of MT-228 shows long-lasting efficacy in animal models of stimulant use disorder, a current unmet and rapidly escalating need with no FDA-approved treatments.

Kappa opioid receptor (KOR) agonists represent promising therapeutics for pain relief due to their analgesic properties along with lower abuse potential than opioids that act at the mu opioid receptor. However, typical KOR agonists produce sedation and dysphoria. Previous studies have shown that G protein signaling-biased KOR agonists may present a means to untangle the desired analgesic properties from undesired side effects. In this paper, we report a new series of G protein signaling-biased KOR agonists entailing -S- -> -CH2- replacement in a previously reported KOR agonist, triazole 1.1. With an optimized carbon linker in hand, further development of the scaffold was undertaken to investigate the appendages of the triazole core. The structure-activity relationship study of this series is described, including several analogues that display enhanced potency while maintaining G protein-signaling bias compared to triazole 1.1.

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 mu M). 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.

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)–activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037’s in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.

PDF file - 116K, Structure of the MCT1 inhibitors AR-C122982 (SR13800) and AR-C155858 (SR13801). Cell cycle analysis, viability, clonogenecity and lactate transport in the indicated cells treated with SR13800. Proliferation of MCF7 cells overexpressing MCT1 or MCT4.

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.

JBJ-04-125-02 as a single agent does not result in any toxicity issues associated with weight loss.

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.

Pharmacokinetic properties of JBJ-02-112-05 and JBJ-04-125-02.

Supplementary Data from A Novel HER2-Selective Kinase Inhibitor Is Effective in HER2 Mutant and Amplified Non–Small Cell Lung Cancer