Automated design of multi-target ligands by generative deep learning.

Generative deep learning models enable data-driven de novo design of molecules with tailored features. Chemical language models (CLM) trained on string representations of molecules such as SMILES have been successfully employed to design new chemical entities with experimentally confirmed activity on intended targets. Here, we probe the application of CLM to generate multi-target ligands for designed polypharmacology. We capitalize on the ability of CLM to learn from small fine-tuning sets of molecules and successfully bias the model towards designing drug-like molecules with similarity to known ligands of target pairs of interest. Designs obtained from CLM after pooled fine-tuning are predicted active on both proteins of interest and comprise pharmacophore elements of ligands for both targets in one molecule. Synthesis and testing of twelve computationally favored CLM designs for six target pairs reveals modulation of at least one intended protein by all selected designs with up to double-digit nanomolar potency and confirms seven compounds as designed dual ligands. These results corroborate CLM for multi-target de novo design as source of innovation in drug discovery.

Development of Tailless Homologue Receptor (TLX) Agonist Chemical Tools.

The human tailless homologue receptor (TLX) is a ligand-activated transcription factor acting as a master regulator of neural stem cell homeostasis. Despite its promising potential in neurodegenerative disease treatment, TLX ligands are rare but required to explore phenotypic effects of TLX modulation and for target validation. We have systematically studied and optimized a TLX agonist scaffold obtained by fragment fusion. Structural modification enabled the development of two TLX agonists endowed with nanomolar potency and binding affinity. Both exhibited favorable chemical tool characteristics including high selectivity and low toxicity. Most notably, the TLX agonists comprise different scaffolds and display high chemical diversity, enabling their use as a set for target identification and validation studies.

A High-Quality Photoswitchable Probe that Selectively and Potently Regulates the Transcription Factor RORγ.

Retinoic acid receptor-related orphan receptor γ (RORγ) is a nuclear hormone receptor with multiple biological functions. As an experimental therapeutic target in inflammation and immunity, there is great interest in spatially-localised RORγ inhibition; and its cyclic temporal role in circadian rhythms also makes it an intriguing target for time-resolved pharmacology. To create tools that can study RORγ biology with appropriate spatial and temporal resolution, we designed light-dependent inverse RORγ agonists by building azobenzene photoswitches into ligand consensus structures. Optimizations gave photoswitchable RORγ inhibitors with a large degree of potency photocontrol, plus remarkable on-target potency, plus excellent selectivity over related off-target receptors. This still-rare, but urgently-needed combination of performance features, distinguishes them as high quality photopharmaceutical probes; and they can now serve as high precision tools to study the spatial and dynamic intricacies of RORγ action in signaling and in inflammatory disorders.

Tuning RXR Modulators for PGC1α Recruitment.

The molecular activation mechanism of the nuclear retinoid X receptors (RXRs) crucially involves ligand-induced corepressor release and coactivator recruitment which mediate transcriptional repression or activation. The ability of RXR to bind diverse coactivators suggests that a coregulator-selective modulation by ligands may open an avenue to tissue- or gene-selective RXR activation. Here, we identified strong induction of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) binding to RXR by a synthetic agonist but not by the endogenous ligand 9-cis retinoic acid. Structure-guided diversification of this lead resulted in a set of three structurally related RXR agonists with different ability to promote PGC1α recruitment in cell-free and cellular context. These results demonstrate that selective modulation of coregulator recruitment to RXR can be achieved with molecular glues and potentially open new therapeutic opportunities by targeting the ligand-induced RXR-PGC1α interaction.

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.

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.

Structural Fusion of Natural and Synthetic Ligand Features Boosts RXR Agonist Potency.

The retinoid X receptors (RXRs) are ligand-activated transcription factors involved in, for example, differentiation and apoptosis regulation. Currently used reference RXR agonists suffer from insufficient specificity and poor physicochemical properties, and improved tools are needed to capture the unexplored therapeutic potential of RXR. Endogenous vitamin A-derived RXR ligands and the natural product RXR agonist valerenic acid comprise acrylic acid residues with varying substitution patterns to engage the critical ionic contact with the binding site arginine. To mimic and exploit this natural ligand motif, we probed its structural fusion with synthetic RXR modulator scaffolds, which had profound effects on agonist activity and remarkably boosted potency of an oxaprozin-derived RXR agonist chemotype. Bioisosteric replacement of the acrylic acid to overcome its pan-assay interference compounds (PAINS) character enabled the development of a highly optimized RXR agonist chemical probe.

Structural Modification of the Natural Product Valerenic Acid Tunes RXR Homodimer Agonism.

Retinoid X receptors (RXR) are ligand-sensing transcription factors with a unique role in nuclear receptor signaling as universal heterodimer partners. RXR modulation holds potential in cancer, neurodegeneration and metabolic diseases but adverse effects of RXR activation and lack of selective modulators prevent further exploration as therapeutic target. The natural product valerenic acid has been discovered as RXR agonist with unprecedented preference for RXR subtype and homodimer activation. To capture structural determinants of this activity profile and identify potential for optimization, we have studied effects of structural modification of the natural product on RXR modulation and identified an analogue with enhanced RXR homodimer agonism.

No NLRP3 inflammasome activity in kidney epithelial cells, not even when the NLRP3-A350V Muckle-Wells variant is expressed in podocytes of diabetic mice.

Background: The NLRP3 inflammasome integrates several danger signals into the activation of innate immunity and inflammation by secreting IL-1ß and IL-18. Most published data relate to the NLRP3 inflammasome in immune cells, but some reports claim similar roles in parenchymal, namely epithelial, cells. For example, podocytes, epithelial cells critical for the maintenance of kidney filtration, have been reported to express NLRP3 and to release IL-ß in diabetic kidney disease, contributing to filtration barrier dysfunction and kidney injury. We questioned this and hence performed independent verification experiments. Methods: We studied the expression of inflammasome components in human and mouse kidneys and human podocytes using single-cell transcriptome analysis. Human podocytes were exposed to NLRP3 inflammasome agonists in vitro and we induced diabetes in mice with a podocyte-specific expression of the Muckle-Wells variant of NLRP3, leading to overactivation of the Nlrp3 inflammasome (Nphs2Cre;Nlrp3A350V) versus wildtype controls. Phenotype analysis included deep learning-based glomerular and podocyte morphometry, tissue clearing, and STED microscopy of the glomerular filtration barrier. The Nlrp3 inflammasome was blocked by feeding ß-hydroxy-butyrate. Results: Single-cell transcriptome analysis did not support relevant NLRP3 expression in parenchymal cells of the kidney. The same applied to primary human podocytes in which NLRP3 agonists did not induce IL-1ß or IL-18 secretion. Diabetes induced identical glomerulomegaly in wildtype and Nphs2Cre;Nlrp3A350V mice but hyperfiltration-induced podocyte loss was attenuated and podocytes were larger in Nphs2Cre;Nlrp3A350V mice, an effect reversible with feeding the NLRP3 inflammasome antagonist ß-hydroxy-butyrate. Ultrastructural analysis of the slit diaphragm was genotype-independent hence albuminuria was identical. Conclusion: Podocytes express low amounts of the NLRP3 inflammasome, if at all, and do not produce IL-1ß and IL-18, not even upon introduction of the A350V Muckle-Wells NLRP3 variant and upon induction of podocyte stress. NLRP3-mediated glomerular inflammation is limited to immune cells.

Structure-Guided Design of Nurr1 Agonists Derived from the Natural Ligand Dihydroxyindole.

The neuroprotective transcription factor Nurr1 was recently found to bind the dopamine metabolite 5,6-dihydroxyindole (DHI) providing access to Nurr1 ligand design from a natural template. We screened a custom set of 14 k extended DHI analogues in silico for optimized descendants to select 24 candidates for microscale synthesis and in vitro testing. Three out of six primary hits were validated as novel Nurr1 agonists with up to sub-micromolar binding affinity, highlighting the druggability of the Nurr1 surface region lining helix 12. In vitro profiling confirmed cellular target engagement of DHI descendants and demonstrated remarkable additive effects of combined Nurr1 agonist treatment, indicating diverse binding sites mediating Nurr1 activation, which may open new avenues in Nurr1 modulation.

A "Kidney-on-the-Chip" Model Composed of Primary Human Tubular, Endothelial, and White Blood Cells.

State-of-the-art cell culture systems may enlist a variety of features to push the significance of in vitro models beyond classical 2D single cell culture; among them are the 3D scaffolds of organic or artificial materials, multi-cell setups, and the use of primary cells as source materials. Obviously, operational complexity increases with each additional feature and feasibility, whereas reproducibility may suffer.We report a multicellular setup using primary human cells and the Mimetas scaffold that aims to increase pathophysiological significance of in vitro culture and simultaneously allows for relatively high-throughput and easy handling.

Targeting the Alternative Vitamin E Metabolite Binding Site Enables Noncanonical PPARγ Modulation.

The lipid-sensing transcription factor PPARγ is the target of antidiabetic thiazolidinediones (TZD). At two sites within its ligand binding domain, it also binds oxidized vitamin E metabolites and the vitamin E mimetic garcinoic acid. While the canonical interaction within the TZD binding site mediates classical PPARγ activation, the effects of the second binding on PPARγ activity remain elusive. Here, we identified an agonist mimicking dual binding of vitamin E metabolites and developed a selective ligand of the second site, unveiling potential noncanonical regulation of PPARγ activities. We found that this alternative binding event can simultaneously occur with orthosteric ligands and it exerted different effects on PPARγ-cofactor interactions compared to both orthosteric PPARγ agonists and antagonists, indicating the diverse roles of the two binding sites. Alternative site binding lacked the pro-adipogenic effect of TZD and mediated no classical PPAR signaling in differential gene expression analysis but markedly diminished FOXO signaling, suggesting potential therapeutic applications.

Development of a Potent Nurr1 Agonist Tool for In Vivo Applications.

Nuclear receptor related 1 (Nurr1) is a neuroprotective transcription factor and an emerging target in neurodegenerative diseases. Despite strong evidence for a role in Parkinson's and Alzheimer's disease, pharmacological control and validation of Nurr1 are hindered by a lack of suitable ligands. We have discovered considerable Nurr1 activation by the clinically studied dihydroorotate dehydrogenase (DHODH) inhibitor vidofludimus calcium and systematically optimized this scaffold to a Nurr1 agonist with nanomolar potency, strong activation efficacy, and pronounced preference over the highly related receptors Nur77 and NOR1. The optimized compound induced Nurr1-regulated gene expression in astrocytes and exhibited favorable pharmacokinetics in rats, thus emerging as a superior chemical tool to study Nurr1 activation in vitro and in vivo.

De Novo Design of Nurr1 Agonists via Fragment-Augmented Generative Deep Learning in Low-Data Regime.

Generative neural networks trained on SMILES can design innovative bioactive molecules de novo. These so-called chemical language models (CLMs) have typically been trained on tens of template molecules for fine-tuning. However, it is challenging to apply CLM to orphan targets with few known ligands. We have fine-tuned a CLM with a single potent Nurr1 agonist as template in a fragment-augmented fashion and obtained novel Nurr1 agonists using sampling frequency for design prioritization. Nanomolar potency and binding affinity of the top-ranking design and its structural novelty compared to available Nurr1 ligands highlight its value as an early chemical tool and as a lead for Nurr1 agonist development, as well as the applicability of CLM in very low-data scenarios.

Tubular Epithelial Cell HMGB1 Promotes AKI-CKD Transition by Sensitizing Cycling Tubular Cells to Oxidative Stress: A Rationale for Targeting HMGB1 during AKI Recovery.

SIGNIFICANCE STATEMENT: Cells undergoing necrosis release extracellular high mobility group box (HMGB)-1, which triggers sterile inflammation upon AKI in mice. Neither deletion of HMGB1 from tubular epithelial cells, nor HMGB1 antagonism with small molecules, affects initial ischemic tubular necrosis and immediate GFR loss upon unilateral ischemia/reperfusion injury (IRI). On the contrary, tubular cell-specific HMGB1 deficiency, and even late-onset pharmacological HMGB1 inhibition, increased functional and structural recovery from AKI, indicating that intracellular HMGB1 partially counters the effects of extracellular HMGB1. In vitro studies indicate that intracellular HMGB1 decreases resilience of tubular cells from prolonged ischemic stress, as in unilateral IRI. Intracellular HMGB1 is a potential target to enhance kidney regeneration and to improve long-term prognosis in AKI. BACKGROUND: Late diagnosis is a hurdle for treatment of AKI, but targeting AKI-CKD transition may improve outcomes. High mobility group box-1 (HMGB1) is a nuclear regulator of transcription and a driver of necroinflammation in AKI. We hypothesized that HMGB1 would also modulate AKI-CKD transition in other ways. METHODS: We conducted single-cell transcriptome analysis of human and mouse AKI and mouse in vivo and in vitro studies with tubular cell-specific depletion of Hmgb1 and HMGB1 antagonists. RESULTS: HMGB1 was ubiquitously expressed in kidney cells. Preemptive HMGB1 antagonism with glycyrrhizic acid (Gly) and ethyl pyruvate (EP) did not affect postischemic AKI but attenuated AKI-CKD transition in a model of persistent kidney hypoxia. Consistently, tubular Hmgb1 depletion in Pax8 rtTA, TetO Cre, Hmgb1fl/fl mice did not protect from AKI, but from AKI-CKD transition. In vitro studies confirmed that absence of HMGB1 or HMGB1 inhibition with Gly and EP does not affect ischemic necrosis of growth-arrested differentiated tubular cells but increased the resilience of cycling tubular cells that survived the acute injury to oxidative stress. This effect persisted when neutralizing extracellular HMGB1 with 2G7. Consistently, late-onset HMGB1 blockade with EP started after the peak of ischemic AKI in mice prevented AKI-CKD transition, even when 2G7 blocked extracellular HMGB1. CONCLUSION: Treatment of AKI could become feasible when ( 1 ) focusing on long-term outcomes of AKI; ( 2 ) targeting AKI-CKD transition with drugs initiated after the AKI peak; and ( 3 ) targeting with drugs that block HMGB1 in intracellular and extracellular compartments.

Rational Design of a New RXR Agonist Scaffold Enabling Single-Subtype Preference for RXRα, RXRß, and RXRγ.

The three retinoid X receptor subtypes (RXRα, RXRß, RXRγ) exhibit critical regulatory roles in cell proliferation and differentiation, metabolism, and inflammation. Due to their importance in nuclear receptor signaling, RXRs are widely distributed and pan-RXR agonists cause adverse effects, but the three highly conserved RXR ligand binding sites render the development of subtype-selective ligands a major challenge. We have fused elements of known RXR ligands to obtain a new RXR agonist chemotype on which minor structural modifications enabled the development of tools with single-subtype preference for RXRα, RXRß, and RXRγ. Molecular modeling indicated different binding conformations and interaction patterns with the RXR LBDs as factors of preferential binding. In a phenotypic adipocyte differentiation experiment, only the RXRα preferential tool enhanced the adipogenic effects of pioglitazone, suggesting this subtype as particularly relevant in adipogenesis and highlighting the set of subtype-preferential RXR agonist tools as suitable for functional cellular studies.

Fragment-based discovery of orphan nuclear receptor Nur77/NGFI-B ligands.

The transcription factor nerve growth factor-induced clone B (NGFI-B, Nur77, NR4A1) is an orphan nuclear receptor playing a role in cell survival and apoptosis regulation. Pharmacological Nur77 modulation holds promise for cancer and (neuro-)inflammatory disease treatment. The available Nur77 ligand scaffolds based on highly lipophilic natural products cytosporone B, celastrol and isoalantolactone are inadequate for the development of potent Nur77 modulators with favorable properties as chemical tools and future drugs. By fragment library screening and subsequent modeling for fragment extension, we have obtained a set of new Nur77 ligands offering alternative chemotypes for the development of Nur77 agonists and inverse agonists. Computer-aided fragment extension in a second stage screening yielded a Nur77 agonist with significant activation efficacy and preference over the related NR4A receptors.

Druggability Evaluation of the Neuron Derived Orphan Receptor (NOR-1) Reveals Inverse NOR-1 Agonists.

The neuron derived orphan receptor (NOR-1, NR4A3) is among the least studied nuclear receptors. Its physiological role and therapeutic potential remain widely elusive which is in part due to the lack of chemical tools that can directly modulate NOR-1 activity. To probe the possibility of pharmacological NOR-1 modulation, we have tested a drug fragment library for NOR-1 activation and repression. Despite low hit-rate (<1 %), we have obtained three NOR-1 ligand chemotypes one of which could be rapidly expanded to an analogue comprising low micromolar inverse NOR-1 agonist potency and altering NOR-1 regulated gene expression in a cellular setting. It confirms druggability of the transcription factor and may serve as an early tool to assess the role and potential of NOR-1.