Utilizing Designed Receptors Exclusively Activated by Designer Drug Chemogenetic Tools to Identify Beneficial G Protein-Coupled Receptor Signaling for Fibrosis.

Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFß-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFß To investigate endogenous GPCR modulating TGFß signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFß signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.

Macrocyclic α helical peptide therapeutic modality: A perspective of learnings and challenges.

Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development.

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein.

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2weeks, or from normal human subjects treated either placebo, 20mg, or 150mg anacetrapib daily for 2weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.

Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes.

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.

Discovery and Structure-Based Design of Macrocyclic Peptides Targeting STUB1.

Recent evidence suggests that deletion of STUB1─a pivotal negative regulator of interferon-γ sensing─may potentially clear malignant cells. However, current studies rely primarily on genetic approaches, as pharmacological inhibitors of STUB1 are lacking. Identifying a tool compound will be a step toward validating the target in a broader therapeutic sense. Herein, screening more than a billion macrocyclic peptides resulted in STUB1 binders, which were further optimized by a structure-enabled in silico design. The strategy to replace the macrocyclic peptides' hydrophilic and solvent-exposed region with a hydrophobic scaffold improved cellular permeability while maintaining the binding conformation. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to a certain extent while improving permeability, suggesting a path forward. Although not optimal for cellular study, the current lead provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes.

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling.

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.

NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis.

Fibrosis, or the accumulation of extracellular matrix, is a common feature of many chronic diseases. To interrogate core molecular pathways underlying fibrosis, we cross-examine human primary cells from various tissues treated with TGF-ß, as well as kidney and liver fibrosis models. Transcriptome analyses reveal that genes involved in fatty acid oxidation are significantly perturbed. Furthermore, mitochondrial dysfunction and acylcarnitine accumulation are found in fibrotic tissues. Substantial downregulation of the PGC1α gene is evident in both in vitro and in vivo fibrosis models, suggesting a common node of metabolic signature for tissue fibrosis. In order to identify suppressors of fibrosis, we carry out a compound library phenotypic screen and identify AMPK and PPAR as highly enriched targets. We further show that pharmacological treatment of MK-8722 (AMPK activator) and MK-4074 (ACC inhibitor) reduce fibrosis in vivo. Altogether, our work demonstrate that metabolic defect is integral to TGF-ß signaling and fibrosis.

Live Cell Membranome cDNA Screen: A Novel Homogenous Live Cell Binding Assay to Study Membrane Protein-Ligand Interaction.

Interactions between transmembrane receptors and their ligands play important roles in normal biological processes and pathological conditions. However, the binding partners for many transmembrane-like proteins remain elusive. To identify potential ligands of these orphan receptors, we developed a screening platform using a homogenous nonwash binding assay in live cells. A collection of ~1900 cDNA clones, encoding full-length membrane proteins, was assembled. As a proof of concept, cDNA clones were individually transfected into CHO-K1 cells in a high-throughput format, and soluble PD-L1-Fc fusion protein was used as bait. The interaction between the putative receptor and PD-L1-Fc was then detected by Alexa Fluor 647 conjugated anti-human immunoglobulin G Fc antibody and visualized using the Mirrorball fluorescence plate cytometer. As expected, PDCD1, the gene encoding programmed cell death protein 1 (PD-1), was revealed as the predominant hit. In addition, three genes that encode Fc receptors (FCGR1A, FCGR1B, and FCGR2A) were also identified as screen hits as the result of the Fc-tag fused to PD-L1, which has provided a reliable internal control for the screen. Furthermore, the potential of using a biotinylated ligand was explored and established to expand the versatility of the cDNA platform. This novel screening platform not only provides a powerful tool for the identification of ligands for orphan receptors but also has the potential for small-molecule target deconvolution.

Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions.

The human intestinal mucosa is a critical site for absorption, distribution, metabolism, and excretion (ADME)/Tox studies in drug development and is difficult to recapitulate in vitro. Using bioprinting, we generated three-dimensional (3D) intestinal tissue composed of human primary intestinal epithelial cells and myofibroblasts with architecture and function to model the native intestine. The 3D intestinal tissue demonstrates a polarized epithelium with tight junctions and specialized epithelial cell types and expresses functional and inducible CYP450 enzymes. The 3D intestinal tissues develop physiological barrier function, distinguish between high- and low-permeability compounds, and have functional P-gp and BCRP transporters. Biochemical and histological characterization demonstrate that 3D intestinal tissues can generate an injury response to compound-induced toxicity and inflammation. This model is compatible with existing preclinical assays and may be implemented as an additional bridge to clinical trials by enhancing safety and efficacy prediction in drug development.

A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition.

Mechanical hyperalgesia is a clinically-relevant form of pain sensitization that develops through largely unknown mechanisms. TRPA1, a Transient Receptor Potential ion channel, is a sensor of pungent chemicals that may play a role in acute noxious mechanosensation and cold thermosensation. We have developed a specific small molecule TRPA1 inhibitor (AP18) that can reduce cinnameldehyde-induced nociception in vivo. Interestingly, AP18 is capable of reversing CFA-induced mechanical hyperalgesia in mice. Although TRPA1-deficient mice develop normal CFA-induced hyperalgeisa, AP18 is ineffective in the knockout mice, consistent with an on-target mechanism. Therefore, TRPA1 plays a role in sensitization of nociception, and that compensation in TRPA1-deficient mice masks this requirement.

Effects of peripherally administered neuromedin U on energy and glucose homeostasis.

Neuromedin U (NMU) is a highly conserved peptide reported to modulate energy homeostasis. Pharmacological studies have shown that centrally administered NMU inhibits food intake, reduces body weight, and increases energy expenditure. NMU-deficient mice develop obesity, whereas transgenic mice overexpressing NMU become lean and hypophagic. Two high-affinity NMU receptors, NMUR1 and NMUR2, have been identified. NMUR1 is found primarily in the periphery and NMUR2 primarily in the brain, where it mediates the anorectic effects of centrally administered NMU. Given the broad expression pattern of NMU, we evaluated whether peripheral administration of NMU has effects on energy homeostasis. We observed that acute and chronic peripheral administration of NMU in rodents dose-dependently reduced food intake and body weight and that these effects required NMUR1. The anorectic effects of NMU appeared to be partly mediated by vagal afferents. NMU treatment also increased core body temperature and metabolic rate in mice, suggesting that peripheral NMU modulates energy expenditure. Additionally, peripheral administration of NMU significantly improved glucose excursion. Collectively, these data suggest that NMU functions as a peripheral regulator of energy and glucose homeostasis and the development of NMUR1 agonists may be an effective treatment for diabetes and obesity.

The antiobesity effects of centrally administered neuromedin U and neuromedin S are mediated predominantly by the neuromedin U receptor 2 (NMUR2).

Neuromedin U (NMU) and neuromedin S (NMS) are structurally related neuropeptides that have been reported to modulate energy homeostasis. Pharmacological data have shown that NMU and NMS inhibit food intake when administered centrally and that NMU increases energy expenditure. Additionally, NMU-deficient mice develop obesity, whereas transgenic mice overexpressing NMU are lean and hypophagic. Two high-affinity NMU/NMS receptors, NMUR1 and NMUR2, have been identified. NMUR1 is predominantly expressed in the periphery, whereas NMUR2 is predominantly expressed in the brain, suggesting that the effects of centrally administered NMU and NMS are mediated by NMUR2. To evaluate the role of NMUR2 in the regulation of energy homeostasis, we characterized NMUR2-deficient (Nmur2(-/-)) mice. Nmur2(-/-) mice exhibited a modest resistance to diet-induced obesity that was at least in part due to reduced food intake. Acute central administration of NMU and NMS reduced food intake in wild-type but not in Nmur2(-/-) mice. The effects on activity and core temperature induced by centrally administered NMU were also absent in Nmur2(-/-) mice. Moreover, chronic central administration of NMU and NMS evoked significant reductions in body weight and sustained reductions in food intake in mice. In contrast, Nmur2(-/-) mice were largely resistant to these effects. Collectively, these data demonstrate that the anorectic and weight-reducing actions of centrally administered NMU and NMS are mediated predominantly by NMUR2, suggesting that NMUR2-selective agonists may be useful for the treatment of obesity.

Instability of a premutation-sized CGG repeat in FMR1 YAC transgenic mice.

Fragile X syndrome results from the massive expansion of a CGG repeat in the 5' untranslated region of the gene FMR1. Data suggest that the hyperexpansion properties of FMR1 CGG repeats may depend on flanking cis-acting elements. We have therefore used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that carried repeats of varying lengths and amounts of flanking sequence. Length-dependent instability in the form of small expansions and contractions was observed in both male and female transmissions over five generations. No parent-of-origin effect or somatic instability was observed. Alterations in tract length were found to occur exclusively in the 3' uninterrupted CGG tract. Large expansion events indicative of a transition from a premutation to a full mutation were not observed. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and that in human.

A heat-sensitive TRP channel expressed in keratinocytes.

Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

A TRP channel that senses cold stimuli and menthol.

A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Here we describe the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. Our identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection.