Otilonium Bromide acts as a selective USP28 inhibitor and exhibits cytotoxic activity against multiple human cancer cell lines.

USP28 contributes to tumorigenesis through modulating the lifespan of oncogenic factors such as c-Myc and ΔNp63, and it has been identified as a potential target for anti-cancer drug development. Currently, although quite a number of USP28 inhibitors have been developed, they all are still in preclinical research stage. Besides, none of them exhibits satisfying inhibition selectivity against USP28 over its closest homologue USP25. Here in this manuscript, a high-throughput screening aiming to discover USP28 inhibitors with novel scaffold and enhanced inhibition selectivity were conducted. After the primary screening and the second round of validation, Otilonium Bromide, an approved drug for treating irritable bowel syndrome, was identified to inhibit USP28's activity with the IC50 value at 6.90 ± 0.90 µM. Besides, the drug exhibits a 3-4 folds inhibition selectivity against USP28 over USP25. According to the enzymatic kinetics analysis data and the hydrogen-deuterium exchange mass spectrometry results, Otilonium Bromide could bind to the allosteric pocket of USP28 and inhibit its activity in a reversible and non-competitive mode. The following performed cell-based assays revealed that the drug could cause cytotoxicity against human colorectal cancer cells and lung squamous carcinoma cells potentially through down-regulating USP28's oncogenic substrates c-Myc and/or ΔNp63. Meanwhile, since Otilonium Bromide has been found to preferentially distribute to gastrointestinal tissues, we then evaluated its potential in the combination treatment of colorectal cancer cells with Regorafenib, which is an approved drug for colorectal cancer therapy. As expected, Otilonium Bromide could significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.

Sequence-based drug design as a concept in computational drug design.

Drug development based on target proteins has been a successful approach in recent decades. However, the conventional structure-based drug design (SBDD) pipeline is a complex, human-engineered process with multiple independently optimized steps. Here, we propose a sequence-to-drug concept for computational drug design based on protein sequence information by end-to-end differentiable learning. We validate this concept in three stages. First, we design TransformerCPI2.0 as a core tool for the concept, which demonstrates generalization ability across proteins and compounds. Second, we interpret the binding knowledge that TransformerCPI2.0 learned. Finally, we use TransformerCPI2.0 to discover new hits for challenging drug targets, and identify new target for an existing drug based on an inverse application of the concept. Overall, this proof-of-concept study shows that the sequence-to-drug concept adds a perspective on drug design. It can serve as an alternative method to SBDD, particularly for proteins that do not yet have high-quality 3D structures available.

Spermine is a natural suppressor of AR signaling in castration-resistant prostate cancer.

In castration-resistant prostate cancer (CRPC), clinical response to androgen receptor (AR) antagonists is limited mainly due to AR-variants expression and restored AR signaling. The metabolite spermine is most abundant in prostate and it decreases as prostate cancer progresses, but its functions remain poorly understood. Here, we show spermine inhibits full-length androgen receptor (AR-FL) and androgen receptor splice variant 7 (AR-V7) signaling and suppresses CRPC cell proliferation by directly binding and inhibiting protein arginine methyltransferase PRMT1. Spermine reduces H4R3me2a modification at the AR locus and suppresses AR binding as well as H3K27ac modification levels at AR target genes. Spermine supplementation restrains CRPC growth in vivo. PRMT1 inhibition also suppresses AR-FL and AR-V7 signaling and reduces CRPC growth. Collectively, we demonstrate spermine as an anticancer metabolite by inhibiting PRMT1 to transcriptionally inhibit AR-FL and AR-V7 signaling in CRPC, and we indicate spermine and PRMT1 inhibition as powerful strategies overcoming limitations of current AR-based therapies in CRPC.

Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF).

O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC50 value of 6.56 ± 1.69 µM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (Kd) of approximately 1.7 µM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.

An ATG4B inhibitor blocks autophagy and sensitizes Sorafenib inhibition activities in HCC tumor cells.

Autophagy related 4B (ATG4B) which regulates autophagy by promoting the formation of autophagosome through reversible modification of LC3, is closely related to cancer cell growth and drug resistance, and therefore is an attractive therapeutic target. Recently, ATG4B inhibitors have been reported, yet with drawbacks including weak potency. To discover more promising ATG4B inhibitors, we developed a high-throughput screening (HTS) assay and identified a new ATG4B inhibitor named DC-ATG4in. DC-ATG4in directly binds to ATG4B and inhibits its enzyme activity with an IC50 of 3.08 ± 0.47 µM. We further confirmed that DC-ATG4in is an autophagy inhibitor and blocks autophagy induced by Sorafenib in Hepatocellular Carcinoma (HCC) cells. More importantly, combination of DC-ATG4in with Sorafenib synergized the cancer cell killing effect and proliferation inhibition activities on HCC cells. Our data suggested that inactivation of autophagy via ATG4B inhibition may be a viable strategy to sensitize existing targeted therapy such as Sorafenib in the future.

Discovery of new small molecule inhibitors of the BPTF bromodomain.

Chromatin remodeling regulates many basic cellular processes, such as gene transcription, DNA repair, and programmed cell death. As the largest member of nucleosome remodeling factor (NURF), BPTF plays a vital role in the occurrence and development of cancer. Currently, BPTF bromodomain inhibitors are still in development. In this study, by conducting homogenous time-resolved fluorescence resonance energy transfer (HTRF) assay, we identified a potential, novel BPTF inhibitor scaffold Sanguinarine chloride with the IC50 value of 344.2 ± 25.1 nM. Biochemical analysis revealed that compound Sanguinarine chloride exhibited high binding affinity to the BPTF bromodomain. Molecular docking predicted the binding mode of Sanguinarine chloride and elucidated the activities of its derivatives. Moreover, Sanguinarine chloride showed a potent anti-proliferative effect in MIAPaCa-2 cells and inhibited the expression of BPTF target gene c-Myc. Taken together, Sanguinarine chloride provides a qualified chemical tool for developing potent BPTF bromodomain inhibitors.

(-)-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.

Ribonucleotide reductase holoenzyme inhibitor COH29 interacts with deubiquitinase ubiquitin-specific protease 2 and downregulates its substrate protein cyclin D1.

USP2 contributes to the quality control of multiple oncogenic proteins including cyclin D1, Mdm2, Aurora-A, etc., and it is a potential target for anti-cancer drug development. However, currently only a few inhibitors with moderate inhibition activities against USP2 have been discovered. USP2-targeted active compounds with either new scaffolds or enhanced activities are in need. Here in this study, Ub-AMC hydrolysis assay-based screening against ~4000 commercially available drugs and drug candidates was performed to identify USP2-targeted inhibitors. COH29, which was originally developed as an anti-cancer agent by blocking the function of human ribonucleotide reductase (RNR, IC50  = 16 µM), was found to exhibit an inhibition activity against USP2 with the IC50 value at 2.02 ± 0.16 µM. The following conducted biophysical and biochemical experiments demonstrated that COH29 could specifically interact with USP2 and inhibit its enzymatic activity in a noncompetitive inhibition mode (Ki  = 1.73 ± 0.14 µM). Since COH29 shows similar inhibitory potencies against RNR (RRM2) and USP2, USP2 inhibition-dependent cellular consequences of COH29 are expected. The results of cellular assays confirmed that the application of COH29 could downregulate the level of cyclin D1 by enhancing its degradation via ubiquitin-proteasome system (UPS), and the modulation effect of COH29 on cyclin D1 is independent of RRM2. Since cyclin D1 acts as an oncogenic driver in human cancer, our findings suggest that USP2 might be a promising therapeutic target for cyclin D1-addicted cancers, and COH29 could serve as a starting compound for high selectivity inhibitor development against USP2.

Natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases.

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

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.

Aha1 Exhibits Distinctive Dynamics Behavior and Chaperone-Like Activity.

Aha1 is the only co-chaperone known to strongly stimulate the ATPase activity of Hsp90. Meanwhile, besides the well-studied co-chaperone function, human Aha1 has also been demonstrated to exhibit chaperoning activity against stress-denatured proteins. To provide structural insights for a better understanding of Aha1's co-chaperone and chaperone-like activities, nuclear magnetic resonance (NMR) techniques were used to reveal the unique structure and internal dynamics features of full-length human Aha1. We then found that, in solution, both the two domains of Aha1 presented distinctive thermal stabilities and dynamics behaviors defined by their primary sequences and three-dimensional structures. The low thermal stability (melting temperature of Aha128-162: 54.45 °C) and the internal dynamics featured with slow motions on the µs-ms time scale were detected for Aha1's N-terminal domain (Aha1N). The aforementioned experimental results suggest that Aha1N is in an energy-unfavorable state, which would therefore thermostatically favor the interaction of Aha1N with its partner proteins such as Hsp90's middle domain. Differently from Aha1N, Aha1C (Aha1's C-terminal domain) exhibited enhanced thermal stability (melting temperature of Aha1204-335: 72.41 °C) and the internal dynamics featured with intermediate motions on the ps-ns time scale. Aha1C's thermal and structural stabilities make it competent for the stabilization of the exposed hydrophobic groove of dimerized Hsp90's N-terminal domain. Of note, according to the NMR data and the thermal shift results, although the very N-terminal region (M1-W27) and the C-terminal relaxin-like factor (RLF) motif showed no tight contacts with the remaining parts of human Aha1, they were identified to play important roles in the recognition of intrinsically disordered pathological α-synuclein.

USP28 and USP25 are downregulated by Vismodegib in vitro and in colorectal cancer cell lines.

Deubiquitinase USP28 plays a crucial role in tumorigenesis by enhancing the stabilities of multiple cancer-related proteins including c-Myc, Notch1, and LSD1, and has become an attractive target for anticancer drug development. However, to date, only a few of USP28-targeted active compounds have been developed, and the active compound-binding pocket in USP28 has not been experimentally revealed yet. In this study, bioassay-based high-throughput screening was applied to discover USP28-targeted inhibitors from the commercially available drug library. Vismodegib, an inhibitor of Hedgehog signaling pathway and FDA-approved drug for the treatment of basal cell carcinoma, was found to exhibit inhibition activity against USP28 (IC50 : 4.41 ± 1.08 µm). Multiple biophysical and biochemical techniques including NMR, ITC, thermal shift assay, HDX-MS, and site-directed mutagenesis analysis were then used to characterize the interaction between Vismodegib and USP28. The binding pocket in USP28 for Vismodegib, which is mainly composed of two helical structures spanning D255-N278 and N286-Y293, was revealed. According to the possible binding pose generated by HDX-MS data-defined molecular docking, the binding cavity occupied by Vismodegib in USP28 aligns well with one of the reported-binding pockets in USP7 for its inhibitors. Furthermore, cellular assays were conducted to confirm that Vismodegib could interact with the evolutionarily related deubiquitinases USP28 and USP25 and downregulate the levels of the two enzymes' substrate proteins c-Myc, Notch1, and Tankyrase-1/2.

Structural and functional studies of USP20 ZnF-UBP domain by NMR.

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia-inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF-UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF-UBP domain and investigated its binding properties with mono-ubiquitin and poly-ubiquitin (K48-linked di-ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF-UBP domain forms a spherically shaped fold consisting of a central ß-sheet with either one α-helix or two α-helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF-UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF-UBP's ubiquitin binding capacity was revealed by NMR data-driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF-UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di-glycine motif and the conserved and non-conserved residues of USP20 ZnF-UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF-UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.

Structural and Functional Investigations of the N-Terminal Ubiquitin Binding Region of Usp25.

Ubiquitin-specific protease 25 (Usp25) is a deubiquitinase that is involved in multiple biological processes. The N-terminal ubiquitin-binding region (UBR) of Usp25 contains one ubiquitin-associated domain, one small ubiquitin-like modifier (SUMO)-interacting motif and two ubiquitin-interacting motifs. Previous studies suggest that the covalent sumoylation in the UBR of Usp25 impairs its enzymatic activity. Here, we raise the hypothesis that non-covalent binding of SUMO, a prerequisite for efficient sumoylation, will impair Usp25's catalytic activity as well. To test our hypothesis and elucidate the underlying molecular mechanism, we investigated the structure and function of the Usp25 N-terminal UBR. The solution structure of Usp251-146 is obtained, and the key residues responsible for recognition of ubiquitin and SUMO2 are identified. Our data suggest inhibition of Usp25's catalytic activity upon the non-covalent binding of SUMO2 to the Usp25 SUMO-interacting motif. We also find that SUMO2 can competitively block the interaction between the Usp25 UBR and its ubiquitin substrates. Based on our findings, we have proposed a working model to depict the regulatory role of the Usp25 UBR in the functional display of the enzyme.

The N-terminal ubiquitin-binding region of ubiquitin-specific protease 28 modulates its deubiquitination function: NMR structural and mechanistic insights.

The deubiquitinase ubiquitin-specific protease 28 (Usp28) contains a ubiquitin-binding region (UBR) composed of one ubiquitin-associated domain (UBA) and one ubiquitin-interacting motif (UIM) at its N-terminus. It is of interest that an additional small ubiquitin-like modifier (SUMO)-interacting motif (SIM) is located next to its UIM. To date, the functional role of the Usp28 UBR is still not understood. To elucidate the regulatory mechanism of the UBR on the full functional display of Usp28, in the present study, NMR and biochemical approaches were applied. The solution structure of Usp28 UBR was obtained, and the key residues responsible for ubiquitin and SUMO1/2 recognition were identified. In addition, we find that the ubiquitin-binding ability of Usp28 UBR was required for full enzymatic activity of Usp28, whereas binding of SUMO1/2 impaired the catalytic activity of the enzyme by competitively blocking its interactions with ubiquitin substrates. Our findings provide a first insight into understanding how the enzymatic activity of Usp28 is regulated by its non-catalytic UBR and endogenous ligands.