Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

A RhoA structure with switch II flipped outward revealed the conformational dynamics of switch II region.

Small GTPase RhoA switches from GTP-bound state to GDP-bound state by hydrolyzing GTP, which is accelerated by GTPases activating proteins (GAPs). However, less study of RhoA structural dynamic changes was conducted during this process, which is essential for understanding the molecular mechanism of GAP dissociation. Here, we solved a RhoA structure in GDP-bound state with switch II flipped outward. Because lacking the intermolecular interactions with guanine nucleotide, we proposed this conformation of RhoA could be an intermediate after GAP dissociation. Further molecular dynamics simulations found the conformational changes of switch regions are indeed existing in RhoA and involved in the regulation of GAP dissociation and GEF recognition. Besides, the guanine nucleotide binding pocket extended to switch II region, indicating a potential "druggable" cavity for RhoA. Taken together, our study provides a deeper understanding of the dynamic properties of RhoA switch regions and highlights the direction for future drug development.

Thimerosal, a competitive thioredoxin reductase 1 (TrxR1) inhibitor discovered via high-throughput screening.

Thioredoxin reductase 1 (TrxR1) is considered as an important anti-cancer drug target, inhibition of which can induce reactive oxygen species (ROS)-mediated apoptosis of human cancer cells. Here, we developed and optimized a high-throughput screening (HTS) assay based on enzyme kinetics for the discovery of TrxR1 inhibitors. By utilizing this assay, we performed a HTS for 2500 compounds from an in-house library against TrxR1. We found that a vaccine preservative, thimerosal, strongly inhibited TrxR1 in a competitive and reversible manner with an IC50 of 24.08 ± 0.86 nM. In addition, we determined that thiomersal has an inhibitory effect on the proliferation of A549 lung cancer cell line, with a GI50 of 6.81 ± 0.09 µM, slightly more potent than auranofin (GI50 = 11.85 ± 0.56 µM). Furthermore, we showed by flow cytometer that thimerosal effectively increased the content of ROS in A549 cells. Therefore, our work provided a high-throughput screening assay to quickly and effectively discover TrxR1 inhibitors, identifying thiomersal as a novel TrxR1 inhibitor and chemical probe.

The discovery of a non-competitive GOT1 inhibitor, hydralazine hydrochloride, via a coupling reaction-based high-throughput screening assay.

Glutamate oxaloacetate transaminase 1 (GOT1) plays a key role in aberrant glutamine metabolism. GOT1 suppression can arrest tumor growth and prevent the development of cancer, indicating GOT1 as a potential anticancer target. Reported GOT1 inhibitors, on the other hand, are quite restricted. Here, we developed and optimized a coupling reaction-based high-throughput screening assay for the discovery of GOT1 inhibitors. By using this screening assay, we found that the cardiovascular drug hydralazine hydrochloride inhibited GOT1 catalytic activity, with an IC50 of 26.62 ± 7.45 µM, in a non-competitive and partial-reversible manner. In addition, we determined the binding affinity of hydralazine hydrochloride to GOT1, with a Kd of 16.54 ± 8.59 µM, using a microscale thermophoresis assay. According to structure-activity relationship analysis, the inhibitory activity of hydralazine hydrochloride is mainly derived from its hydrazine group. Furthermore, it inhibits the proliferation of cancer cells MCF-7 and MDA-MB-468 with a slight inhibitory effect compared to other tested cancer cells, highlighting GOT1 as a promising therapeutic target for the treatment of breast cancer.

Discovery and characterization of a novel glucose-6-phosphate dehydrogenase (G6PD) inhibitor via high-throughput screening.

Altered glucose-6-phosphate dehydrogenase (G6PD) status is influential in many cellular pathophysiological processes and diseases, making G6PD a potential target for cancer therapy. However, the available G6PD inhibitors are very limited and restricted. Here we developed a reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) absorption photometry assay based on enzyme kinetics to characterize G6PD activity. In this way, we performed a high-throughput screening (HTS) to an in house library. And then we identified compound named Wedelolactone inhibiting G6PD strongly in a non-competitive, reversible way. In addition, we did the surface Plasmon Resonance (SPR) assay and indicated the KD between Wedelolactone and G6PD protein was 3.64 µM. Furthermore, our basic colony formation assay showed the inhibitory effect of Wedelolactone on the proliferation of ovarian cancer cells (IC50 ~ 10 µM). Thus, we provided a high-throughput screening assay to quickly and efficiently discover G6PD inhibitors, and identified Wedelolactone as a G6PD inhibitor, implying that Wedelolactone suppresses ovarian cancer partly through targeting G6PD.

Discovery of novel CBP bromodomain inhibitors through TR-FRET-based high-throughput screening.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 µM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 µM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.

Identification and Assessments of Novel and Potent Small-Molecule Inhibitors of EED-EZH2 Interaction of Polycomb Repressive Complex 2 by Computational Methods and Biological Evaluations.

Polycomb repressive complex 2 (PRC2) is an attractive drug target for anti-cancer treatment. Among the three core subunits (EZH2, EED and SUZ12) of PRC2, EZH2 is the catalytic subunit that methylates histone H3 lysine 27 (H3K27), while EED is the regulatory subunit. Besides the small-molecule inhibitors of EZH2, those targeting the protein-protein interaction (PPI) between EZH2 and EED have also been reported. Here, for the first time, we have identified the key residues that contributed most to the EED-EZH2 binding affinity by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations based on the 200 ns molecular dynamics simulation. Moreover, we report the identification of two novel and potent small-molecule inhibitors (35 and 49) of EZH2-EED interaction (bottom interaction surface) by virtual screening and biological evaluations. Binding modes of the two identified molecules with EED were probed by molecular docking. Additionally, 35 and 49 displayed cellular antiproliferative activity against diffuse large B-cell lymphoma (DLBCL) cancer cell line Toledo whose cell growth was driven by aberrant PRC2 activity. Our findings have provided structural insights for the design of novel EZH2-EED interaction inhibitors to regulate the activity of PRC2 complex.

Development of a high-throughput fluorescence polarization assay for the discovery of EZH2-EED interaction inhibitors.

Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.

Identification of novel EZH2 inhibitors through pharmacophore-based virtual screening and biological assays.

Polycomb repressive complex 2 (PRC2) acts as a primary writer for di- and tri-methylation of histone H3 at lysine 27. This protein plays an essential role in silencing gene expression. Enhancer of zeste 2 (EZH2), the catalytic subunit of PRC2, is considered as a promising therapeutic target for cancer. GSK126, a specific inhibitor of EZH2, is undergoing phase I trials for hypermethylation-related cancers. In addition, many derivatives of GSK126 are also commonly used in laboratory investigations. However, studies on the mechanism and drug development of EZH2 are limited by the absence of structural diversity of these inhibitors because they share similar SAM-like scaffolds. In this study, we generated a pharmacophore model based on reported EZH2 inhibitors and performed in silico screenings. Experimental validations led to the identification of two novel EZH2 inhibitors, DCE_42 and DCE_254, with IC50 values of 23 and 11µM, respectively. They also displayed significant anti-proliferation activity against lymphoma cell lines. Thus, we discovered potent EZH2 inhibitors with novel scaffold using combined in silico screening and experimental study. Results from this study can also guide further development of novel specific EZH2 inhibitors.

Structure-Based Design and Discovery of a Potent and Cell-Active LC3A/B Covalent Inhibitor.

Autophagy is a highly conserved cellular homeostasis maintenance mechanism in eukaryotes. Microtubule-associated protein light chain 3 (LC3) plays a crucial role in autophagy. It has multiple pairs of protein-protein interactions (PPIs) with other proteins, and these PPIs have an effect on the regulation of autophagosome formation and the recruitment of autophagic substrates. In our previous work, a small molecule covalent inhibitor DC-LC3in-D5 which could inhibit LC3A/B PPIs was identified, but a detailed study of structure-activity relationships (SARs) was lacking. Herein, a new molecule LC3in-C42 was discovered utilizing the hybridization of advantageous fragments, whose potency (IC50 = 7.6 nM) had been greatly improved compared with that of DC-LC3in-D5. LC3in-C42 inhibits autophagy at the cellular level and its efficacy far exceeds that of DC-LC3in-D5. Thus far, LC3in-C42 stands as the most potent LC3A/B small molecule inhibitor. LC3in-C42 could serve as a powerful tool for LC3A/B protein and autophagy research.

Uncovering PROTAC Sensitivity and Efficacy by Multidimensional Proteome Profiling: A Case for STAT3.

Proteolysis-targeting chimera (PROTAC) is a powerful technology that can effectively trigger the degradation of target proteins. The intricate interplay among various factors leads to a heterogeneous drug response, bringing about significant challenges in comprehending drug mechanisms. Our study applied data-independent acquisition-based mass spectrometry to multidimensional proteome profiling of PROTAC (DIA-MPP) to uncover the efficacy and sensitivity of the PROTAC compound. We profiled the signal transducer and activator of transcription 3 (STAT3) PROTAC degrader in six leukemia and lymphoma cell lines under multiple conditions, demonstrating the pharmacodynamic properties and downstream biological responses. Through comparison between sensitive and insensitive cell lines, we revealed that STAT1 can be regarded as a biomarker for STAT3 PROTAC degrader, which was validated in cells, patient-derived organoids, and mouse models. These results set an example for a comprehensive description of the multidimensional PROTAC pharmacodynamic response and PROTAC drug sensitivity biomarker exploration.

Role of tRNA amino acid-accepting end in aminoacylation and its quality control.

Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA76 end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA76 accepting end of tRNA(Leu) during the aminoacylation and editing reactions. The analysis showed that the terminal A76 is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C74 and C75 selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA76 binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA76 homogeneity of tRNAs.

Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase.

A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.

Computational and Structure-Based Development of High Potent Cell-Active Covalent Inhibitor Targeting the Peptidyl-Prolyl Isomerase NIMA-Interacting-1 (Pin1).

The unique proline isomerase peptidyl-prolyl isomerase NIMA-interacting-1 (Pin1) is reported to activate numerous cancer-driving pathways simultaneously, and aberrant Pin1 activation is present in many human cancers. Here, we identified a novel hit compound, ZL-Pin01, that covalently modified Pin1 at Cys113 with an half-maximal inhibitory concentration (IC50) of 1.33 ± 0.07 µM through screening an in-house library. Crystallographic study drove the process of structure-guided optimization and led to the potent inhibitor ZL-Pin13 with an IC50 of 0.067 ± 0.03 µM. We obtained four co-crystal structures of Pin1 complexed with inhibitors that elucidated the detailed binding mode of the derivatives with Pin1. Interestingly, the co-crystal of Pin1 with ZL-Pin13 obtained by co-crystallization revealed the conformational change of Gln129 induced by the inhibitor. Furthermore, ZL-Pin13 effectively inhibited the proliferation and downregulated the Pin1 substrates in MDA-MB-231 cells. Collectively, we developed a potent covalent inhibitor of Pin1, ZL-Pin13, which could be an effective probe for studying the functional roles of Pin1.

(-)-Epigallocatechin Gallate is a Noncompetitive Inhibitor of NAD Kinase.

Nicotinamide adenine dinucleotide kinase (NADK) controls the intracellular NADPH content and provides reducing power for the synthesis of macromolecules and anti-ROS. Moreover, NADK is considered to be a synthetic lethal gene for KRAS mutations. To discover NADK-targeted probes, a high-throughput screening assay was established and optimized with a Z factor of 0.71. The natural product (-)-epigallocatechin gallate (EGCG) was found to be a noncompetitive inhibitor of NADK with K i = 3.28 ± 0.32 µΜ. The direct binding of EGCG to NADK was determined by several biophysical methods, including NMR spectroscopy, surface plasmon resonance (SPR) assay, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The SPR assay showed a K d of 1.78 ± 1.15 µΜ. The HDX-MS experiment showed that EGCG was bound at the non-substrate-binding sites of NADK. Besides, binding mode prediction and derivative activity analysis revealed a potential structure-activity relationship between EGCG and NADK. Furthermore, EGCG can specifically inhibit the proliferation of KRAS-mutated lung cancer cell lines without affecting KRAS wild-type lung cancer cell lines.

Structure-Guided Development of Small-Molecule PRC2 Inhibitors Targeting EZH2-EED Interaction.

Disruption of EZH2-embryonic ectoderm development (EED) protein-protein interaction (PPI) is a new promising cancer therapeutic strategy. We have previously reported the discovery of astemizole, a small-molecule inhibitor targeting the EZH2-EED PPI. Herein, we report the cocrystal structure of EED in complex with astemizole at 2.15 Å. The structure elucidates the detailed binding mode of astemizole to EED and provides a structure-guided design for the discovery of a novel EZH2-EED interaction inhibitor, DC-PRC2in-01, with an affinity Kd of 4.56 µM. DC-PRC2in-01 destabilizes the PRC2 complex, thereby leading to the degradation of PRC2 core proteins and the decrease of global H3K27me3 levels in cancer cells. The proliferation of PRC2-driven lymphomas cells is effectively inhibited, and the cell cycle is arrested in the G0/G1 phase. Together, these data demonstrate that DC-PRC2in-01 could be an effective chemical probe for investigating the PRC2-related physiology and pathology and providing a promising chemical scaffold for further development.

Identification of catalytic and non-catalytic activity inhibitors against PRC2-EZH2 complex through multiple high-throughput screening campaigns.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) along with embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12), which implements transcriptional repression mainly by depositing trimethylation marks at lysine 27 of histone H3 (H3K27me3). Its catalytic activity is closely correlated with the stability of PRC2, and somatic activating mutation of EZH2 Y641F within the catalytic SET domain drives tumor aggressiveness, drug resistance, and poor prognosis. Here, we report two high-throughput screening (HTS) campaigns targeting EZH2 Y641F and EZH2-EED interaction, respectively. For the EZH2 Y641F mutant, the HTS campaign involved a library of 250,000 compounds using a homogenous time-resolved fluorescence (HTRF) assay and identified 162 hits, while 60,160 compounds were screened against EZH2-EED interaction with a fluorescence polarization (FP) assay resulting in 97 hits. Among the 162 EZH2 Y641F inhibitors, 38 also suppressed EZH2-EED interaction and 80 showed inhibitory effects on the wide-type (WT) EZH2. Meanwhile, 10 of the 97 EZH2-EED interaction inhibitors were active against WT EZH2. These hit compounds provide useful tools for the development of novel PRC2-EZH2 inhibitors targeting its catalytic and non-catalytic activities.

Discovery of 2-substituted-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide as potent and selective protein arginine methyltransferases 5 inhibitors: Design, synthesis and biological evaluation.

Protein arginine methyltransferases 5 (PRMT5) represents an attractive drug target in epigenetic field for the treatment of leukemia and lymphoma. Here, a series of N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amide derivatives targeting PRMT5 were designed with structure-based approach and synthesized. Among them, compound 46 showed potent and selective PRMT5 inhibition activity with an IC50 of 8.5 nM, which was approximately equivalent with the phase I clinical trial PRMT5 inhibitor GSK-3326595 (IC50 = 5.5 nM). Compound 46 also displayed pronounced anti-proliferative activity in MV4-11 cells (GI50 = 18 nM) and antitumor activity in MV4-11 mouse xenografts model. This molecule can serve as an excellent tool compound for probing the biological function of PRMT5.

Identification of novel inhibitors of histone acetyltransferase hMOF through high throughput screening.

The histone acetyltransferases (HATs) in mammals include GCN5 N-acetyltransferases, the MOZ, YBF2, SAS2, and TIP60 proteins, and the orphan HATs. The males absent on the first (MOF) is mainly related to acetylation of histone H4 Lys16 and has influence on downstream genes expression. However, the only inhibitor MG149 presented low activity against MOF. Besides, there was no high throughput screening platform on MOF, which limited the inhibitor discovery and functional study. In our study, we set up a high throughput screening platform based on amplified luminescent proximity homogeneous assay (ALPHA), which led us to a moderate inhibitor DC_M01. By chemical modification, we found DC_M01_7, which was the analog of DC_M01 with an IC50 value of 6 µM. DC_M01_7 significantly inhibited HCT116 cells proliferation and could also inhibit histone 4 lysine 16 acetylation in HCT116 cells. To sum up, our work will probably assist the further development of more potent MOF inhibitors and the functional study of hMOF.