Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics.

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

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.

Small molecule 1a reduces FMRpolyG-mediated toxicity in in vitro and in vivo models for FMR1 premutation.

Fragile X-associated tremor and ataxia syndrome (FXTAS) is a late-onset, progressive neurodegenerative disorder characterized by tremors, ataxia and neuropsychological problems. This disease is quite common in the general population with approximately 20 million carriers worldwide. The risk of developing FXTAS increases dramatically with age, with about 45% of male carriers over the age of 50 being affected. FXTAS is caused by a CGG-repeat expansion (CGGexp) in the fragile X mental retardation 1 (FMR1) gene. CGGexp RNA is translated into the FMRpolyG protein by a mechanism called RAN translation. Although both gene and pathogenic trigger are known, no therapeutic interventions are available at this moment. Here, we present, for the first time, primary hippocampal neurons derived from the ubiquitous inducible mouse model which is used as a screening tool for targeted interventions. A promising candidate is the repeat binding, RAN translation blocking, small molecule 1a. Small molecule 1a shields the disease-causing CGGexp from being translated into the toxic FMRpolyG protein. Primary hippocampal neurons formed FMRpolyG-positive inclusions, and upon treatment with 1a, the numbers of FMRpolyG-positive inclusions are reduced. We also describe for the first time the formation of FMRpolyG-positive inclusions in the liver of this mouse model. Treatment with 1a reduced the insoluble FMRpolyG protein fraction in the liver but not the number of inclusions. Moreover, 1a treatment had a reducing effect on the number of Rad23b-positive inclusions and insoluble Rad23b protein levels. These data suggest that targeted small molecule therapy is effective in an FXTAS mouse model and has the potential to treat CGGexp-mediated diseases, including FXTAS.

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.

Chemical systems biology reveals mechanisms of glucocorticoid receptor signaling.

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, ligand class analysis, to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle-sparing or anabolic for protein balance and mitochondrial potential. Ligand class analysis defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.

Probing a distinct druggable tubulin binding site with gatorbulins 1-7, their metabolic and physicochemical properties, and pharmacological consequences.

Microtubules, consisting of α/ß-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2-7 (1b-1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and ß-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

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.

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.

Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that recognizes the structure of r(CUG)exp and cleaves it in both DM1 patient-derived myotubes and a DM1 mouse model, leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects in vivo without detectable off-targets. Thus, small molecules that bind and cleave RNA have utility as lead chemical probes and medicines and can selectively target disease-causing RNA structures to broadly improve defects in preclinical animal models.

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.

Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-κB activation.

Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.

M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer's disease genes and improving memory.

Alzheimer's disease (AD) comprises multifactorial ailments for which current therapeutic strategies remain insufficient to broadly address the underlying pathophysiology. Epigenetic gene regulation relies upon multifactorial processes that regulate multiple gene and protein pathways, including those involved in AD. We therefore took an epigenetic approach where a single drug would simultaneously affect the expression of a number of defined AD-related targets. We show that the small-molecule histone deacetylase inhibitor M344 reduces beta-amyloid (Aß), reduces tau Ser396 phosphorylation, and decreases both ß-secretase (BACE) and APOEε4 gene expression. M344 increases the expression of AD-relevant genes: BDNF, α-secretase (ADAM10), MINT2, FE65, REST, SIRT1, BIN1, and ABCA7, among others. M344 increases sAPPα and CTFα APP metabolite production, both cleavage products of ADAM10, concordant with increased ADAM10 gene expression. M344 also increases levels of immature APP, supporting an effect on APP trafficking, concurrent with the observed increase in MINT2 and FE65, both shown to increase immature APP in the early secretory pathway. Chronic i.p. treatment of the triple transgenic (APPsw/PS1M146V/TauP301L) mice with M344, at doses as low as 3 mg/kg, significantly prevented cognitive decline evaluated by Y-maze spontaneous alternation, novel object recognition, and Barnes maze spatial memory tests. M344 displays short brain exposure, indicating that brief pulses of daily drug treatment may be sufficient for long-term efficacy. Together, these data show that M344 normalizes several disparate pathogenic pathways related to AD. M344 therefore serves as an example of how a multitargeting compound could be used to address the polygenic nature of multifactorial diseases.

Precise small-molecule recognition of a toxic CUG RNA repeat expansion.

Excluding the ribosome and riboswitches, developing small molecules that selectively target RNA is a longstanding problem in chemical biology. A typical cellular RNA is difficult to target because it has little tertiary, but abundant secondary structure. We designed allele-selective compounds that target such an RNA, the toxic noncoding repeat expansion (r(CUG)exp) that causes myotonic dystrophy type 1 (DM1). We developed several strategies to generate allele-selective small molecules, including non-covalent binding, covalent binding, cleavage and on-site probe synthesis. Covalent binding and cleavage enabled target profiling in cells derived from individuals with DM1, showing precise recognition of r(CUG)exp. In the on-site probe synthesis approach, small molecules bound adjacent sites in r(CUG)exp and reacted to afford picomolar inhibitors via a proximity-based click reaction only in DM1-affected cells. We expanded this approach to image r(CUG)exp in its natural context.

Evaluation of Platensimycin and Platensimycin-Inspired Thioether Analogues against Methicillin-Resistant Staphylococcus aureus in Topical and Systemic Infection Mouse Models.

Staphylococcus aureus is one of the most common pathogens causing hospital-acquired and community-acquired infections. Methicillin-resistant S. aureus (MRSA)-formed biofilms in wounds are difficult to treat with conventional antibiotics. By targeting FabB/FabF of bacterial fatty acid synthases, platensimycin (PTM) was discovered to act as a promising natural antibiotic against MRSA infections. In this study, PTM and its previously synthesized sulfur-Michael derivative PTM-2t could reduce over 95% biofilm formation by S. aureus ATCC 29213 when used at 2 µg/mL in vitro. Topical application of ointments containing PTM or PTM-2t (2 × 4 mg/day/mouse) was successfully used to treat MRSA infections in a BABL/c mouse burn wound model. As a potential prodrug lead, PTM-2t showed improved in vivo efficacy in a mouse peritonitis model compared with PTM. Our study suggests that PTM and its analogue may be used topically or locally to treat bacterial infections. In addition, the use of prodrug strategies might be instrumental to improve the poor pharmacokinetic properties of PTM.

Design of a small molecule against an oncogenic noncoding RNA.

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

Design, synthesis, and evaluation of simple phenol amides as ERRγ agonists.

Herein we report the design and synthesis of a series of simple phenol amide ERRγ agonists based on a hydrazone lead molecule. Our structure activity relationship studies in this series revealed the phenol portion of the molecule to be required for activity. Attempts to replace the hydrazone with more suitable chemotypes led to a simple amide as a viable alternative. Differential hydrogen-deuterium exchange experiments were used to help understand the structural basis for binding to ERRγ and aid in the development of more potent ligands.

Identification of an aminothiazole series of RORß modulators.

Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind RORß, however, none of these molecules represent good starting points to develop optimized small molecule modulators. Recently, Compound 1 was identified as a potent dual RORß and RORγ inverse agonist with no activity towards RORα (Fig. 1). To our knowledge, this is one of only two small molecule RORß inverse agonists identified in the primary literature from a tractable chemical series and represents an ideal starting point from which to design RORß-selective modulators. Herein we describe our SAR optimization efforts that led to a series of potent neutral antagonists of RORß.

Development of matrix metalloproteinase-13 inhibitors - A structure-activity/structure-property relationship study.

A structure-activity/structure-property relationship study based on the physicochemical as well as in vitro pharmacokinetic properties of a first generation matrix metalloproteinase (MMP)-13 inhibitor (2) was undertaken. After systematic variation of inhibitor 2, compound 31 was identified which exhibited microsomal half-life higher than 20 min, kinetic solubility higher than 20 µM, and a permeability coefficient greater than 20 × 10-6 cm/s. Compound 31 also showed excellent in vivo PK properties after IV dosing (Cmax = 56.8 µM, T1/2 (plasma) = 3.0 h, Cl = 0.23 mL/min/kg) and thus is a suitable candidate for in vivo efficacy studies in an OA animal model.

Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists.

Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-ß have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.