Competitive Fluorescence Polarization Research Articles

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 μM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

Fluorescence polarization assay for screening FANCM-RMI inhibitors to target the alternative lengthening of telomeres.

Alternative Lengthening of Telomeres (ALT) is a mechanism used by 10-15% of all cancers to achieve replicative immortality, bypassing the DNA damage checkpoint associated with short telomeres that leads to cellular senescence or apoptosis. ALT does not occur in non-cancerous cells, presenting a potential therapeutic window for cancers where this mechanism is active. Disrupting the FANCM-RMI interaction has emerged as a promising therapeutic strategy that induces synthetic ALT lethality in genetic studies on cancer cell lines. There are currently no chemical inhibitors reported in the literature, in part due to the lack of reliable biophysical or biochemical assays to screen for FANCM-RMI disruption. Here we describe the development of a robust competitive fluorescence polarization (FP) assay that quantifies target binding at the FANCM-RMI interface. The assay employs a labeled peptide tracer TMR-RaMM2 derived from the native MM2 binding motif, which binds to recombinant RMI1-RMI2 and can be displaced by competitive inhibitors. We report the methods for recombinant production of RMI1-RMI2, design and evaluation of the tracer TMR-RaMM2, along with unlabeled peptide inhibitor controls to enable ALT-targeted drug discovery.

Discovery of a new inhibitor for YTH domain-containing m6A RNA readers.

N 6-methyladenosine (m6A) is an abundant modification in mammalian mRNAs and plays important regulatory roles in gene expression, primarily mediated through specific recognition by "reader" proteins. YTH family proteins are one major family of known m6A readers, which specifically recognize m6A-modified transcripts via the YTH domains. Despite the significant relevance of YTH-m6A recognition in biology and diseases, few small molecule inhibitors are available for specifically perturbing this interaction. Here we report the discovery of a new inhibitor ("N-7") for YTH-m6A RNA recognition, from the screening of a nucleoside analogue library against the YTH domain of the YTHDF1 protein. N-7 is characterized to be a pan-inhibitor in vitro against five YTH domains from human YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 proteins, with IC50 values in the range of 30-48 μM measured using a fluorescence polarization competition assay. We demonstrated that N-7 directly interacts with the YTH domain proteins via a thermal shift assay. N-7 expands the chemical structure landscape of the m6A YTH domain-containing reader inhibitors and potentiates future inhibitor development for reader functional studies and therapeutic efforts in targeting the epitranscriptome.

Design, synthesis, and biological evaluation of novel 8-substituted quercetin derivatives targeting the β‑catenin/B-cell lymphoma 9 interaction

The β-catenin/B-cell lymphoma 9 (BCL9) protein–protein interaction (PPI) is a potential target for aberrantly active Wnt/β-catenin signaling which actively participates in initiating and progressing of many cancers. Herein, we discovered novel 8-substituted quercetin derivatives with potential inhibitory activities targeting β-catenin/BCL9 PPI. Among all the derivatives, compound B4 displayed the most promising PPI inhibitory activity with an IC50 value of 2.25 μM in a competitive fluorescence polarization assay and a KD value of 1.44 μM for the β-catenin protein. Furthermore, B4 selectively inhibited the growth of colorectal cancer (CRC) cells, suppressed the transactivation of Wnt signaling, and downregulated the expression of oncogenic Wnt target gene. Especially, B4 showed potent anti-CRC activity in vivo with the tumor growth inhibition (TGI) of 75.99 % and regulated the tumor immune microenvironment.

Synthesis and interaction with growth factors of sulfated oligosaccharides containing an anomeric fluorinated tail

Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

Design and synthesis of a fluorescent probe to develop a fluorescence polarization assay for the E3 ligase FEM1C

A proteolysis targeting chimera (PROTAC) is a bivalent molecule consisting of an E3 ligase ligand and a protein of interest ligand, which promotes the degradation of specific proteins by recruiting the ubiquitin-proteasome system. Although VHL and CRBN ligands have been extensively used in PROTAC development, the availability of small molecule E3 ligase ligands remains limited. Therefore, identifying novel E3 ligase ligands would expand the repertoire for PROTAC development. FEM1C, an E3 ligase that recognizes proteins with an R/K-X-R or R/K-X-X-R motif at the C-terminus, is a promising candidate for this purpose. In this study, we present the design and synthesis of a fluorescent probe ES148, exhibiting a Ki value of 1.6 ± 0.1 µM for FEM1C. Utilizing this fluorescent probe, we have established a robust fluorescence polarization (FP) based competition assay to characterize FEM1C ligands, with a Z’ factor of 0.80 and a S/N ratio > 20 in a high-throughput format. Furthermore, we have validated binding affinities of FEM1C ligands using isothermal titration calorimetry, consistently corroborating the results from our FP assay. Thus, we anticipate that our FP competition assay will expedite the discovery of FEM1C ligands, offering new tools for PROTAC development.

Synthesis of Amino Acids Bearing Halodifluoromethyl Moieties and Their Application to p53-Derived Peptides Binding to Mdm2/Mdm4.

Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF2X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein-protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis. Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(εNHCOCF2X) exhibits an improved inhibition constant Ki compared to the unmodified peptide. Decreasing Ki values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(εNHCOCF2X) to Abu(γNHCOCF2X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

Discovery of Novel 3-Phenylpiperidine Derivatives Targeting the β-Catenin/B-Cell Lymphoma 9 Interaction as a Single Agent and in Combination with the Anti-PD-1 Antibody for the Treatment of Colorectal Cancer.

Direct disruption of the β-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential strategy for colorectal cancer (CRC) treatment through inhibiting oncogenic Wnt activity. Herein, a series of 3-phenylpiperidine derivatives were synthesized and evaluated as β-catenin/BCL9 PPI inhibitors. Among them, compound 41 showed the best IC50 (0.72 μM) in a competitive fluorescence polarization assay and a KD value of 0.26 μM for the β-catenin protein. This compound selectively inhibited the growth of CRC cells, suppressed Wnt signaling transactivation, and downregulated oncogenic Wnt target gene expression. In vivo, 41 showed potent anti-CRC activity and promoted the infiltration and function of cytotoxic T lymphocytes while decreasing the infiltration of regulatory T-cells (Tregs). Furthermore, the combination of 41 and the anti-PD-1 antibody (Ab) efficiently enhanced anti-CRC efficacy, first verifying the in vivo efficacy of the small-molecule β-catenin/BCL9 PPI inhibitor and anti-PD-1 Ab in combination.

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.

Engineering the Ligand Specificity of the Human Galectin-1 by Incorporation of Tryptophan Analogues.

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3'-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.

Discovery of DDO-2213 as a Potent and Orally Bioavailable Inhibitor of the WDR5-Mixed Lineage Leukemia 1 Protein-Protein Interaction for the Treatment of MLL Fusion Leukemia.

WD repeat-containing protein 5 (WDR5) is essential for the stability and methyltransferase activity of the mixed lineage leukemia 1 (MLL1) complex. Dysregulation of the MLL1 gene is associated with human acute leukemias, and the direct disruption of the WDR5-MLL1 protein-protein interaction (PPI) is emerging as an alternative strategy for MLL-rearranged cancers. Here, we represent a new aniline pyrimidine scaffold for WDR5-MLL1 inhibitors. A comprehensive structure-activity analysis identified a potent inhibitor 63 (DDO-2213), with an IC50 of 29 nM in a competitive fluorescence polarization assay and a Kd value of 72.9 nM for the WDR5 protein. Compound 63 selectively inhibited MLL histone methyltransferase activity and the proliferation of MLL translocation-harboring cells. Furthermore, 63 displayed good pharmacokinetic properties and suppressed the growth of MV4-11 xenograft tumors in mice after oral administration, first verifying the in vivo efficacy of targeting the WDR5-MLL1 PPI by small molecules.

Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL−1, respectively. The spiked recoveries were 78.3%−105.2% with relative standard deviations (RSDs) of 0.7%−15.4% for the competitive FPIA, while 78.7%−115.3% with RSDs of 0.7%−12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.

Binding affinity and synergy between agonists and peptides of the estrogen receptor‐beta (ERß) needed for arain‐selective SERB Activity

The estrogen receptor (ER) exist predominantly in two isoforms (ERα and ERß). Both are primarily activated by the naturally occurring hormone 17ß-Estradiol (E2) which regulates a variety of physiological processes. One of ER's major role is acting as key neuroprotective modulator for hippocampal plasticity in the brain. The ERß isoform is predominantly found in the brain, which makes it a promising drug target for memory consolidation. Selectivity for ERß over ERα is crucial to the development of drug leads, since ERα–induced cell proliferation results in risk of cancer. We have developed a selective and potent ERß agonist with in vivo efficacy for improving cognitive function within mice. A prior study determined that selectivity for ER is related to the ligand's ability to induce the conformational change necessary for the activation of transcription. Selective estrogen receptor modulators (SERMs) like raloxifene & tamoxifen are used to treat breast cancer by selectively targeting ERα in mammary tissue. This is done through the synergistic binding of coactivator proteins to the ER in the presence of an agonist. Similarly, it is predicted that selective estrogen beta agonists (SERBAs) have the ability to be tissue specific in the presence of specific coactivators. Through the use of computational modeling, a splice variant of SRC1 (steroid receptor coactivator-1), SRC1-4, was identified to be the most favorable brain-relevant peptide for ERß. This study determines the binding affinity of agonist for ERß and ERα in the presence and absence of SRC1-4. In addition we dive into the extent to which binding synergy between agonist and coactivator exist, which could lead to selective activity in the brain. The synergy associated with the conformational change is measured by ΔΔGsynergy based on binding affinity for both agonist and coactivator. The greater the synergy between the agonist and coactivator, the more promising the lead compound will be as a SERBA. Preliminary studies have been done for the binding of SRC1-4 using TR-FRET assays where the concentration of coactivator was varied. Fluorescence polarization (FP) studies were also utilized to determine a Kd. In these studies, FL-SRC1-4 remained constant while ERß-LBD concentrations varied in the presence of 1 μM (saturating) E2. The two methods are compared and contrasted. The same FP assay was completed again in the absence of E2 (no agonoism). This provides a binding affinity for SRC1-4 to the receptor when an agonist is not present. Computational modeling studies were performed to compare ER's affinity for SRC1-4 peptides over other coactivator peptides. 3 unlabeled peptides (SRC1-1, SRC1-3 and PGC1a) were used in a competitive FP binding assay with FL-SRC1-4. The data was consistent with prior data, indicating SRC1-4 has the highest affinity for ERß. The agonistic-synergy effect will be calculated through the measurement of E2 affinity for ERß in the presence and absence of SRC1-4. The comparison of ΔΔG(synergy) for ERß vs. ERα will ultimately provide a measurement of selectivity and synergy that is biologically relevant for SERB activity. Hanson, A. M. et al. A-C estrogens as potent and selective estrogen receptor-beta agonists (SERBAs) to enhance memory consolidation under low-estrogen conditions. J. Med. Chem.61, 4720–4738 (2018).

Identification of Phenazine-Based MEMO1 Small-Molecule Inhibitors: Virtual Screening, Fluorescence Polarization Validation, and Inhibition of Breast Cancer Migration.

Phosphorylation-dependent protein-protein interactions play a significant role in biological signaling pathways; therefore, small molecules that are capable of influencing these interactions can be valuable research tools and have potential as pharmaceutical agents. MEMO1 (mediator of ErbB2-cell driven motility) is a phosphotyrosine-binding protein that interacts with a variety of protein partners and has been found to be upregulated in breast cancer patients. Herein, we report the first small-molecule inhibitors of MEMO1 interactions identified through a virtual screening platform and validated in a competitive fluorescence polarization assay. Initial structure-activity relationships have been investigated for these phenazine-core inhibitors and the binding sites have been postulated using molecular dynamics simulations. The most potent biochemical inhibitor is capable of disrupting the large protein interface with a KI of 2.7 μm. In addition, the most promising phenazine core compounds slow the migration of breast cancer cell lines in a scratch assay.

CEREBROSPINAL FLUID SAMPLING IN A BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) UNDER GENERAL ANESTHESIA

Brucella ceti, associated with neurobrucellosis, has been isolated from cerebrospinal fluid (CSF) of postmortem cetaceans. A 106-kg, stranded female bottlenose dolphin (Tursiops truncatus) presented with serum antibodies to Brucella spp. via competitive enzyme-linked immunosorbent assay and fluorescence polarization assay. Polymerase chain reaction (PCR) and culture of whole blood, bronchoalveolar fluid, and rectal, nasal, and genital swabs for Brucella spp. were consistently negative. Serial computed tomography revealed mild focal dilatation of brain ventricles. CSF sampling was warranted to exclude neurobrucellosis. Sedation was achieved with 30 mg diazepam (0.28 mg/kg) orally 2.5 hours prior to arrival in hospital, followed by 5.3 mg midazolam (0.05 mg/kg) intramuscularly, and anesthetic induction with 2.5 mg midazolam (0.02 mg/kg) and 200 mg propofol (2 mg/kg) administered slowly intravenously, followed by intubation and maintenance on sevoflurane using controlled mechanical and apneustic anesthesia ventilation. The atlanto-occipital joint was opened by flexing the upper cervical region with the animal in left lateral recumbency. A 20-ga × 6-inch spinal needle was advanced into the cisterna magna using radiographic guidance. CSF was collected successfully with no neurological deficits appreciable on recovery. Brucella spp. was not identified via PCR or culture. This represents the first report of an antemortem CSF tap in a cetacean.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1.

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

Interactomic affinity profiling by holdup assay: Acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase.

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

Design, Synthesis, and Structure-Activity Relationships of Indoline-Based Kelch-like ECH-Associated Protein 1-Nuclear Factor (Erythroid-Derived 2)-Like 2 (Keap1-Nrf2) Protein-Protein Interaction Inhibitors.

The Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) pathway is the major defending mechanism against oxidative stresses, and directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has been an attractive strategy to target oxidative stress-related diseases, including cardiovascular diseases. Here, we describe the design, synthesis, and structure-activity relationships (SARs) of indoline-based compounds as potent Keap1-Nrf2 PPI inhibitors. Comprehensive SAR analysis and thermodynamics-guided optimization identified 19a as the most potent inhibitor in this series, with an IC50 of 22 nM in a competitive fluorescence polarization assay. Further evaluation indicated the proper drug-like properties of 19a. Compound 19a dose-dependently upregulated genes and protein level of Nrf2 as well as its downstream markers and showed protective effects against lipopolysaccharide-induced injury in both H9c2 cardiac cells and mouse models. Collectively, we reported here a novel indoline-based Keap1-Nrf2 PPI inhibitor as a potential cardioprotective agent.

Development of fluorescence polarization-based competition assay for nicotinamide N-methyltransferase.

Methylation-mediated pathways play important roles in the progression of various diseases. Thus, targeting methyltransferases has proven to be a promising strategy for developing novel therapies. Nicotinamide N-methyltransferase (NNMT) is a major metabolic enzyme involved in epigenetic regulation through catalysis of methyl transfer from the cofactor S-adenosyl-l-methionine onto nicotinamide and other pyridines. Accumulating evidence infers that NNMT is a novel therapeutic target for a variety of diseases such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. Therefore, there is an urgent need to discover potent and specific inhibitors for NNMT to assess its therapeutical potential. Herein, we reported the design and synthesis of a fluorescent probe II138, exhibiting a Kd value of 369±14nM for NNMT. We also established a fluorescence polarization (FP)-based competition assay for evaluation of NNMT inhibitors. Importantly, the unique feature of this FP competition assay is its capability to identify inhibitors that interfere with the interaction of the NNMT active site directly or allosterically. In addition, this assay performance is robust with a Z'factor of 0.76, indicating its applicability in high-throughput screening for NNMT inhibitors.