{"doi":"10.1101/2024.01.05.574351","title":"Enabling Systemic Identification and Functionality Profiling for Cdc42 Homeostatic Modulators","abstract":"Abstract Homeostatic modulation is pivotal in modern therapeutics. However, the discovery of bioactive materials to achieve this functionality is often random and unpredictive. Here, we enabled a systemic identification and functional classification of chemicals that elicit homeostatic modulation of signaling through Cdc42, a classical small GTPase of Ras superfamily. Rationally designed for high throughput screening, the capture of homeostatic modulators (HMs) along with molecular re-docking uncovered at least five functionally distinct classes of small molecules. This enabling led to partial agonists, hormetic agonists, bona fide activators and inhibitors, and ligand-enhanced agonists. Novel HMs exerted striking functionality in bradykinin-Cdc42 activation of actin remodelingand modified Alzheimer’s disease-like behavior in mouse model. This concurrent computer-aided and experimentally empowered HM profiling highlights a model path for predicting HM landscape. One Sentence Summary With concurrent experimental biochemical profiling and in silico computer-aided drug discovery (CADD) analysis, this study enabled a systemic identification and holistic classification of Cdc42 homeostatic modulators (HMs) and demonstrated the power of CADD to predict HM classes that can mimic the pharmacological functionality of interests. Introduction Maintainingbody homeostasisis the ultimate keyto health. Thereare rich resources of bioactive materials for this functionality from both natural and synthetic chemical repertories including partial agonists (PAs) and various allosteric modulators. These homeostatic modulators (HMs) play a unique role in modern therapeutics for human diseases such as mental disorders and drug addiction. Buspirone, for example, acts as a PA for serotonin 5-HT 1A receptor but is an antagonist of the dopamine D 2 receptor. Such medical useto treat general anxietydisorders (GADs) has become one of the most-commonly prescribed medications. However, most HMs in current uses target membrane proteins and are often derived from random discoveries. HMs as therapeutics targeting cytoplasmic proteins are even more rare despite that they are in paramount needs (e. g. targeting Ras superfamily small GTPases). Rationale Cdc42, a classical member of small GTPases of Ras superfamily, regulates PI3K-AKT and Raf-MEK-ERK pathways and has been implicated in various neuropsychiatric and mental disorders as well as addictive diseases and cancer. We previously reported the high-throughput in-silico screening followed by biological characterization of novel small molecule modulators (SMMs) of Cdc42-intersectin (ITSN) protein-protein interactions (PPIs). Based on a serendipitously discovered SMM ZCL278 with PA profile as a model compound, we hypothesized that there are more varieties of such HMs of Cdc42 signaling, and the model HMs can be defined by their distinct Cdc42-ITSN binding mechanisms using computer-aided drug discovery (CADD) analysis. We further reasoned that molecular modeling coupled with experimental profiling can predict HM spectrum and thus open the door for the holistic identification and classification of multifunctional cytoplasmic target-dependent HMs as therapeutics. Results The originally discovered Cdc42 inhibitor ZCL278 displaying PA properties prompted the inquiry whether this finding represented a random encounter of PAs or whether biologically significant PAs can be widely present. The top ranked compounds were initially defined by structural fitness and binding scores to Cdc42. Because higher binding scores do not necessarily translate to higher functionality, we performed exhaustive experimentations with over 2,500 independent Cdc42-GEF (guanine nucleotide exchange factor) assays to profile the GTP loading activities on all 44 top ranked compounds derived from the SMM library. The N-MAR-GTP fluorophore-based Cdc42-GEF assay platform provided the first glimpse of the breadth of HMs. A spectrum of Cdc42 HMs was unc","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":493033,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9519,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1273858,"name":"Fereshteh Azimian","orcid":"0000-0002-8248-1607","position":1,"is_corresponding":false},{"id":876469,"name":"Yanhua Chen","orcid":"0000-0002-4514-9410","position":2,"is_corresponding":false},{"id":331997,"name":"Jeffery L. Twiss","orcid":"0000-0001-7875-6682","position":3,"is_corresponding":false},{"id":664208,"name":"Christi Boykin","orcid":null,"position":4,"is_corresponding":false},{"id":326762,"name":"Shayan Nik Akhtar","orcid":"0009-0001-5435-9241","position":5,"is_corresponding":false},{"id":663524,"name":"Qun Lu","orcid":"0000-0003-3545-7142","position":6,"is_corresponding":false},{"id":1273857,"name":"Satyaveni Malasala","orcid":"0000-0002-5084-6376","position":0,"is_corresponding":true}],"reference_count":50,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:00.148120Z","pmid":"38260445","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}