Genome-wide CRISPR-Cas9 knockout screens identify DNMT1 as a druggable dependency in sonic hedgehog medulloblastoma.

Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.

Development of Nurr1 agonists from amodiaquine by scaffold hopping and fragment growing.

The neuroprotective transcription factor nuclear receptor-related 1 (Nurr1) has shown great promise as a therapeutic target in Parkinson's and Alzheimer's disease as well as multiple sclerosis but high-quality chemical tools for pharmacological target validation of Nurr1 are rare. We have employed the weak Nurr1 modulator amodiaquine (AQ) and AQ-derived fragments as templates to design a new Nurr1 agonist chemotype by scaffold hopping and fragment growing strategies. Systematic structural optimization of this scaffold yielded Nurr1 agonists with nanomolar potency and binding affinity. Comprehensive in vitro profiling revealed efficient cellular target engagement and compliance with the highest probe criteria. In human midbrain organoids bearing a Parkinson-driving LRRK2 mutation, a novel Nurr1 agonist rescued tyrosine hydroxylase expression highlighting the potential of the new Nurr1 modulator chemotype as lead and as a chemical tool for biological studies.

Structural Optimization of Oxaprozin for Selective Inverse Nurr1 Agonism.

Nuclear receptor related 1 (Nurr1, NR4A2) is a ligand-sensing transcription factor with neuroprotective and anti-inflammatory roles widely distributed in the CNS. Pharmacological Nurr1 modulation is considered a promising experimental strategy in Parkinson's and Alzheimer's disease but target validation is incomplete. While significant progress has been made in Nurr1 agonist development, inverse agonists blocking the receptor's constitutive activity are lacking. Here we report comprehensive structure-activity relationship elucidation of oxaprozin which acts as moderately potent and nonselective inverse Nurr1 agonist and RXR agonist. We identified structural determinants selectively driving RXR agonism or inverse Nurr1 agonism of the scaffold enabling the development of selective inverse Nurr1 agonists with enhanced potency and strong efficacy.

Chemogenomics for NR1 nuclear hormone receptors.

Nuclear receptors (NRs) regulate transcription in response to ligand binding and NR modulation allows pharmacological control of gene expression. Although some NRs are relevant as drug targets, the NR1 family, which comprises 19 NRs binding to hormones, vitamins, and lipid metabolites, has only been partially explored from a translational perspective. To enable systematic target identification and validation for this protein family in phenotypic settings, we present an NR1 chemogenomic (CG) compound set optimized for complementary activity/selectivity profiles and chemical diversity. Based on broad profiling of candidates for specificity, toxicity, and off-target liabilities, sixty-nine comprehensively annotated NR1 agonists, antagonists and inverse agonists covering all members of the NR1 family and meeting potency and selectivity standards are included in the final NR1 CG set. Proof-of-concept application of this set reveals effects of NR1 members in autophagy, neuroinflammation and cancer cell death, and confirms the suitability of the set for target identification and validation.

CRISPR-Cas9 screens reveal common essential miRNAs in human cancer cell lines.

BACKGROUND: Genome-wide functional screening using the CRISPR-Cas9 system is a powerful tool to uncover tumor-specific and common genetic dependencies across cancer cell lines. Current CRISPR-Cas9 knockout libraries, however, primarily target protein-coding genes. This limits functional genomics-based investigations of miRNA function. METHODS: We designed a novel CRISPR-Cas9 knockout library (lentiG-miR) of 8107 distinct sgRNAs targeting a total of 1769 human miRNAs and benchmarked its single guide RNA (sgRNA) composition, predicted on- and off-target activity, and screening performance against previous libraries. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Fitness miRNAs in each cell line were scored using a combination of supervised and unsupervised essentiality classifiers. Common essential miRNAs across distinct cancer cell lines were determined using the 90th percentile method. For subsequent validation, we performed knockout experiments for selected common essential miRNAs in distinct cancer cell lines and gene expression profiling. RESULTS: We found significantly lower off-target activity for protein-coding genes and a higher miRNA gene coverage for lentiG-miR as compared to previously described miRNA-targeting libraries, while preserving high on-target activity. A minor fraction of miRNAs displayed robust depletion of targeting sgRNAs, and we observed a high level of consistency between redundant sgRNAs targeting the same miRNA gene. Across 45 human cancer cell lines, only 217 (12%) of all targeted human miRNAs scored as a fitness gene in at least one model, and fitness effects for most miRNAs were confined to small subsets of cell lines. In contrast, we identified 49 common essential miRNAs with a homogenous fitness profile across the vast majority of all cell lines. Transcriptional profiling verified highly consistent gene expression changes in response to knockout of individual common essential miRNAs across a diverse set of cancer cell lines. CONCLUSIONS: Our study presents a miRNA-targeting CRISPR-Cas9 knockout library with high gene coverage and optimized on- and off-target activities. Taking advantage of the lentiG-miR library, we define a catalogue of miRNA fitness genes in human cancer cell lines, providing the foundation for further investigation of miRNAs in human cancer.

Development of a Potent and Selective G2A (GPR132) Agonist.

G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.

Azologs of the Fatty Acid Mimetic Drug Cinalukast Enable Light-Induced PPARα Activation.

Photo-switchable nuclear receptor modulators ("photohormones") enable spatial and temporal control over transcription factor activity and are valuable precision tools for biological studies. We have developed a new photohormone chemotype by incorporating a light-switchable motif in the scaffold of a cinalukast-derived PPARα ligand and tuned light-controlled activity by systematic structural variation. An optimized photohormone exhibited PPARα agonism in its light-induced (Z)-configuration and strong selectivity over related lipid-activated transcription factors representing a valuable addition to the collection of light-controlled tools to study nuclear receptor activity.

Prospective de novo drug design with deep interactome learning.

De novo drug design aims to generate molecules from scratch that possess specific chemical and pharmacological properties. We present a computational approach utilizing interactome-based deep learning for ligand- and structure-based generation of drug-like molecules. This method capitalizes on the unique strengths of both graph neural networks and chemical language models, offering an alternative to the need for application-specific reinforcement, transfer, or few-shot learning. It enables the "zero-shot" construction of compound libraries tailored to possess specific bioactivity, synthesizability, and structural novelty. In order to proactively evaluate the deep interactome learning framework for protein structure-based drug design, potential new ligands targeting the binding site of the human peroxisome proliferator-activated receptor (PPAR) subtype gamma are generated. The top-ranking designs are chemically synthesized and computationally, biophysically, and biochemically characterized. Potent PPAR partial agonists are identified, demonstrating favorable activity and the desired selectivity profiles for both nuclear receptors and off-target interactions. Crystal structure determination of the ligand-receptor complex confirms the anticipated binding mode. This successful outcome positively advocates interactome-based de novo design for application in bioorganic and medicinal chemistry, enabling the creation of innovative bioactive molecules.

Functionally-instructed modifiers of response to ATR inhibition in experimental glioma.

BACKGROUND: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. METHODS: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. RESULTS: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. CONCLUSION: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.

Rational design and virtual screening identify mimetics of the RXR agonist valerenic acid.

The ligand-sensing transcription factor retinoid X receptor (RXR) is the universal heterodimer partner of nuclear receptors and involved in multiple physiological processes. Its pharmacological modulation holds therapeutic potential in cancer and neurodegeneration but many available RXR ligands lack specificity. The sesquiterpenoid valerenic acid has been identified as RXR agonist with unprecedented subtype and homodimer preference. Here, we identified simplified mimetics of the complex natural product by rational design and virtual screening that exhibited similar activity profiles on RXR and informed about structural elements contributing to the favorable activity.

Structure-Guided Design of a Highly Potent Partial RXR Agonist with Superior Physicochemical Properties.

Retinoid X receptors (RXRs, NR2B1-3) hold therapeutic potential in oncology, neurodegeneration, and metabolic diseases, but traditional RXR agonists mimicking the natural ligand 9-cis retinoic acid exhibit poor physicochemical properties, pharmacokinetics, and safety profiles. Improved RXR ligands are needed to exploit RXR modulation as a promising therapeutic concept in various indications beyond its current role in second-line cancer treatment. Here, we report the co-crystal structure of RXR in complex with a novel pyrimidine-based ligand and the structure-informed optimization of this scaffold to highly potent and highly soluble RXR agonists. Focused structure-activity relationship elucidation and rigidization resulted in a substantially optimized partial RXR agonist with low nanomolar potency, no cytotoxic activity, and very favorable physicochemical properties highlighting this promising scaffold for the development of next-generation RXR targeting drugs.

Dual soluble epoxide hydrolase inhibitor - farnesoid X receptor agonist interventional treatment attenuates renal inflammation and fibrosis.

Introduction: Renal fibrosis associated with inflammation is a critical pathophysiological event in chronic kidney disease (CKD). We have developed DM509 which acts concurrently as a farnesoid X receptor agonist and a soluble epoxide hydrolase inhibitor and investigated DM509 efficacy as an interventional treatment using the unilateral ureteral obstruction (UUO) mouse model. Methods: Male mice went through either UUO or sham surgery. Interventional DM509 treatment (10mg/kg/d) was started three days after UUO induction and continued for 7 days. Plasma and kidney tissue were collected at the end of the experimental protocol. Results: UUO mice demonstrated marked renal fibrosis with higher kidney hydroxyproline content and collagen positive area. Interventional DM509 treatment reduced hydroxyproline content by 41% and collagen positive area by 65%. Renal inflammation was evident in UUO mice with elevated MCP-1, CD45-positive immune cell positive infiltration, and profibrotic inflammatory gene expression. DM509 treatment reduced renal inflammation in UUO mice. Renal fibrosis in UUO was associated with epithelial-to-mesenchymal transition (EMT) and DM509 treatment reduced EMT. UUO mice also had tubular epithelial barrier injury with increased renal KIM-1, NGAL expression. DM509 reduced tubular injury markers by 25-50% and maintained tubular epithelial integrity in UUO mice. Vascular inflammation was evident in UUO mice with 9 to 20-fold higher ICAM and VCAM gene expression which was reduced by 40-50% with DM509 treatment. Peritubular vascular density was reduced by 35% in UUO mice and DM509 prevented vascular loss. Discussion: Interventional treatment with DM509 reduced renal fibrosis and inflammation in UUO mice demonstrating that DM509 is a promising drug that combats renal epithelial and vascular pathological events associated with progression of CKD.