Identification Of Inhibitors And Structural Analysis Of Complexes Against PDZ Family Proteins MDA-9 And PARD3

PDZ is an acronym for the three proteins PSD95/SAP90,DLGA,and ZO-1.It plays a key role as the most common protein-protein interaction module in regulating several cellular pathways,such as signal transduction,cell polarity,and adhesion.However,dysfunction of PDZ domains often leads to the occurrence of tumors and neurodegenerative diseases.Therefore,the PDZ domain family has become a potential target of great interest.Currently,inhibiting PDZ domain function has become an important pharmacological strategy for treating specific diseases.In recent years,significant progress has been made in the development of PDZ domain inhibitors.For example,in the field of neurological disorders,the PSD-95 inhibitors ZL006 and IC87250 can effectively inhibit the interaction between PSD-95 PDZ2/nNOS and have effective neuroprotective activity.However,developing high-affinity small molecule inhibitors against PDZ domains still faces challenges.This paper aims to screen and study inhibitors targeting the PDZ domains of melanoma differentiation-associated gene-9(MDA-9)and partition-defective 3(PARD3),the two members of the PDZ protein family.MDA-9 is a scaffolding protein with tandem PDZ1 and PDZ2 domains that plays a promoting role in the occurrence and metastasis of various human cancers.However,the narrow substrate binding groove of the PDZ domain of MDA-9 makes it difficult to develop high-affinity drug-like compounds.Here,we used a protein-observed NMR fragment screening method to successfully discover four hits targeting the PDZ1 and PDZ2 domains of MDA-9,named PI1A,PIIB,PI2A,and PI2B,respectively.In order to further explore the interaction modes between the hits and the PDZ domains,and provide a basis for the subsequent evolution of the compounds,we solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B,and found that PI1B binds to the natural substrate binding groove of the PDZ1 domain,with the carbonyl group of the molecule forming hydrogen bonds with the main chain amino groups of residues I125 and G126 of MDA-9 PDZ1 domain.Meanwhile,through molecular docking guided by paramagnetic relaxation enhancement NMR restraints,we characterized the best binding modes of PI1A with MDA-9 PDZ1 and PI2A with PDZ2,respectively.At the natural substrate binding groove,the PI1A molecule forms a direct hydrogen bond with the L127 residue of the PDZ1 domain;the NH of the pyrazole ring in PI2A forms a direct hydrogen bond with the F211 residue of the PDZ2 domain,which makes the small molecule more stable at the binding groove.Furthermore,the protein-ligand interaction mode was cross-validated by mutating key residues of the PDZ domains.Further competitive fluorescence polarization experiments revealed that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains,respectively.Moreover,these inhibitors exhibited low cellular toxicity and effectively suppressed the migration of MDA-MB-231 breast cancer cells,which recapitulated the phenotype of MDA-9 knockdown.When PI1A and PI2A were used in combination,the inhibitory effect was more significant.This work has paved the way for the development of more effective bivalent inhibitors targeting MDA-9 based on structure-guided fragment linking in the future.PARD3 is a member of the PDZ protein family,which as a polarity protein,plays a critical role in maintaining tissue organization and cell-cell interactions.Aberrant expression of PARD3 can lead to tissue disorganization,induce epithelialmesenchymal transition,and increase the risk of tumorigenesis.In this study,we used fragment-based nuclear magnetic resonance screening methods to identify three hits targeting the PARD3 PDZ2 domain and four hits targeting the PARD3 PDZ3 domain.Additionally,we determined the binding affinities and interaction sites of these compounds with the corresponding PDZ domains using chemical shift perturbation experiments.Furthermore,we obtained the best binding modes of each lead compound with the corresponding PDZ domain using molecular docking guided by chemical shift perturbation.Our work provides a foundation for structure-guided discovery of lead compounds,as well as further development of drug candidates.PDZ proteins are considered promising drug targets for treating various diseases.In this study,we focused on the PDZ domains of MDA-9 and PARD3,members of the PDZ protein family,and discovered multiple inhibitors using fragment-based nuclear magnetic resonance screening methods.We also revealed the binding patterns of these compounds to the corresponding PDZ proteins.Based on the binding modes of these molecules,we laid the groundwork for further evolution of the compounds into lead compounds and provided support for developing drugs targeting the PDZ domains.