Discovery of a Highly Selective BET BD2 Inhibitor from a DNA-Encoded Library Technology Screening Hit.

Second-generation bromodomain and extra terminal (BET) inhibitors, which selectively target one of the two bromodomains in the BET proteins, have begun to emerge in the literature. These inhibitors aim to help determine the roles and functions of each domain and assess whether they can demonstrate an improved safety profile in clinical settings compared to pan-BET inhibitors. Herein, we describe the discovery of a novel BET BD2-selective chemotype using a structure-based drug design from a hit identified by DNA-encoded library technologies, showing a structural differentiation from key previously reported greater than 100-fold BD2-selective chemotypes GSK620, GSK046, and ABBV-744. Following a structure-based hypothesis for the selectivity and optimization of the physicochemical properties of the series, we identified 60 (GSK040), an in vitro ready and in vivo capable BET BD2-inhibitor of unprecedented selectivity (5000-fold) against BET BD1, excellent selectivity against other bromodomains, and good physicochemical properties. This novel chemical probe can be added to the toolbox used in the advancement of epigenetics research.

Recent advances in identifying protein targets in drug discovery.

Phenotype-based screening has emerged as an alternative route for discovering new chemical entities toward first-in-class therapeutics. However, clarifying their mode of action has been a significant bottleneck for drug discovery. For target protein identification, conventionally bioactive small molecules are conjugated onto solid supports and then applied to isolate target proteins from whole proteome. This approach requires a high binding affinity between bioactive small molecules and their target proteins. Besides, the binding affinity can be significantly hampered after structural modifications of bioactive molecules with linkers. To overcome these limitations, two major strategies have recently been pursued: (1) the covalent conjugation between small molecules and target proteins using photoactivatable moieties or electrophiles, and (2) label-free target identification through monitoring target engagement by tracking the thermal, proteolytic, or chemical stability of target proteins. This review focuses on recent advancements in target identification from covalent capturing to label-free strategies.

Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening.

The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.

A Pharmacological Chaperone Therapy for Acute Intermittent Porphyria.

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

In Silico Investigation of the Anti-Tumor Mechanisms of Epigallocatechin-3-Gallate.

The EGCG, an important component of polyphenol in green tea, is well known due to its numerous health benefits. We employed the reverse docking method for the identification of the putative targets of EGCG in the anti-tumor target protein database and these targets were further uploaded to public databases in order to understand the underlying pharmacological mechanisms and search for novel EGCG-associated targets. Similarly, the pharmacological linkage between tumor-related proteins and EGCG was manually constructed in order to provide greater insight into the molecular mechanisms through a systematic integration with applicable bioinformatics. The results indicated that the anti-tumor mechanisms of EGCG may involve 12 signaling transduction pathways and 33 vital target proteins. Moreover, we also discovered four novel putative target proteins of EGCG, including IKBKB, KRAS, WEE1 and NTRK1, which are significantly related to tumorigenesis. In conclusion, this work may provide a useful perspective that will improve our understanding of the pharmacological mechanism of EGCG and identify novel potential therapeutic targets.

Vitamin C supplementation expands the therapeutic window of BETi for triple negative breast cancer.

BACKGROUND: Bromodomain and extra-terminal inhibitors (BETi) have shown efficacy for the treatment of aggressive triple negative breast cancer (TNBC). However, BETi are plagued by a narrow therapeutic window as manifested by severe toxicities at effective doses. Therefore, it is a limitation to their clinical implementation in patient care. METHODS: The impact of vitamin C on the efficacy of small compounds including BETi was assessed by high-throughput screening. Co-treatment of TNBC by BETi especially JQ1 and vitamin C was evaluated in vitro and in vivo. FINDINGS: High-throughput screening revealed that vitamin C improves the efficacy of a number of structurally-unrelated BETi including JQ1, I-BET762, I-BET151, and CPI-203 in treating TNBC cells. The synergy between BETi and vitamin C is due to suppressed histone acetylation (H3ac and H4ac), which is in turn caused by upregulated histone deacetylase 1 (HDAC1) expression upon vitamin C addition. Treatment with JQ1 at lower doses together with vitamin C induces apoptosis and inhibits the clonogenic ability of cultured TNBC cells. Oral vitamin C supplementation renders a sub-therapeutic dose of JQ1 able to inhibit human TNBC xenograft growth and metastasis in mice. INTERPRETATION: Vitamin C expands the therapeutic window of BETi by sensitizing TNBC to BETi. Using vitamin C as a co-treatment, lower doses of BETi could be used to achieve an increased therapeutic index in patients, which will translate to a reduced side effect profile. FUND: University of Miami Sylvester Comprehensive Cancer Center, Bankhead Coley Cancer Research program (7BC10), Flight Attendant Medical Research Institute, and NIH R21CA191668 (to GW) and 1R56AG061911 (to CW and CHV).

Identifying Protein Features Responsible for Improved Drug Repurposing Accuracies Using the CANDO Platform: Implications for Drug Design.

Drug repurposing is a valuable tool for combating the slowing rates of novel therapeutic discovery. The Computational Analysis of Novel Drug Opportunities (CANDO) platform performs shotgun repurposing of 2030 indications/diseases using 3733 drugs/compounds to predict interactions with 46,784 proteins and relating them via proteomic interaction signatures. The accuracy is calculated by comparing interaction similarities of drugs approved for the same indications. We performed a unique subset analysis by breaking down the full protein library into smaller subsets and then recombining the best performing subsets into larger supersets. Up to 14% improvement in accuracy is seen upon benchmarking the supersets, representing a 100⁻1000-fold reduction in the number of proteins considered relative to the full library. Further analysis revealed that libraries comprised of proteins with more equitably diverse ligand interactions are important for describing compound behavior. Using one of these libraries to generate putative drug candidates against malaria, tuberculosis, and large cell carcinoma results in more drugs that could be validated in the biomedical literature compared to using those suggested by the full protein library. Our work elucidates the role of particular protein subsets and corresponding ligand interactions that play a role in drug repurposing, with implications for drug design and machine learning approaches to improve the CANDO platform.

Nanoscale synthesis and affinity ranking.

Most drugs are developed through iterative rounds of chemical synthesis and biochemical testing to optimize the affinity of a particular compound for a protein target of therapeutic interest. This process is challenging because candidate molecules must be selected from a chemical space of more than 1060 drug-like possibilities 1 , and a single reaction used to synthesize each molecule has more than 107 plausible permutations of catalysts, ligands, additives and other parameters 2 . The merger of a method for high-throughput chemical synthesis with a biochemical assay would facilitate the exploration of this enormous search space and streamline the hunt for new drugs and chemical probes. Miniaturized high-throughput chemical synthesis3-7 has enabled rapid evaluation of reaction space, but so far the merger of such syntheses with bioassays has been achieved with only low-density reaction arrays, which analyse only a handful of analogues prepared under a single reaction condition8-13. High-density chemical synthesis approaches that have been coupled to bioassays, including on-bead 14 , on-surface 15 , on-DNA 16 and mass-encoding technologies 17 , greatly reduce material requirements, but they require the covalent linkage of substrates to a potentially reactive support, must be performed under high dilution and must operate in a mixture format. These reaction attributes limit the application of transition-metal catalysts, which are easily poisoned by the many functional groups present in a complex mixture, and of transformations for which the kinetics require a high concentration of reactant. Here we couple high-throughput nanomole-scale synthesis with a label-free affinity-selection mass spectrometry bioassay. Each reaction is performed at a 0.1-molar concentration in a discrete well to enable transition-metal catalysis while consuming less than 0.05 milligrams of substrate per reaction. The affinity-selection mass spectrometry bioassay is then used to rank the affinity of the reaction products to target proteins, removing the need for time-intensive reaction purification. This method enables the primary synthesis and testing steps that are critical to the invention of protein inhibitors to be performed rapidly and with minimal consumption of starting materials.

Vitamin C Sensitizes Melanoma to BET Inhibitors.

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo-/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR.

BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT.

A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-ß. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6 → BALB/c) was performed using in vivo expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial strategy can suppress pre-clinical GVHD and posit, in vivo EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses.

Dietary Compound Resveratrol Is a Pan-BET Bromodomain Inhibitor.

The chemopreventive and anticancer effects of resveratrol (RSV) are widely reported in the literature. Specifically, mechanisms involving epigenetic regulation are promising targets to regulate tumor development. Bromodomains act as epigenetic readers by recognizing lysine acetylation on histone tails and boosting gene expression in order to regulate tissue-specific transcription. In this work, we showed that RSV is a pan-BET inhibitor. Using Differential Scanning Fluorimetry (DSF), we showed that RSV at 100 µM increased the melting temperature (∆Tm) of BET bromodomains by around 2.0 °C. The micromolar dissociation constant (Kd) range was characterized using Isothermal Titration Calorimetry (ITC). The RSV Kd value accounted to 6.6 µM in case of BRD4(1). Molecular docking proposed the binding mode of RSV against BRD4(1) mimicking the acetyl-lysine interactions. All these results suggest that RSV can also recognize epigenetic readers domains by interacting with BET bromodomains.

Natural formulas and the nature of formulas: Exploring potential therapeutic targets based on traditional Chinese herbal formulas.

By comparing the target proteins (TPs) of classic traditional Chinese medicine (TCM) herbal formulas and modern drugs used for treating coronary artery disease (CAD), this study aimed to identify potential therapeutic TPs for treating CAD. Based on the theory of TCM, the Xuefu-Zhuyu decoction (XZD) and Gualou-Xiebai-Banxia decoction (GXBD), both of which are classic herbal formulas, were selected for treating CAD. Data on the chemical ingredients and corresponding TPs of the herbs in these two formulas and data on modern drugs approved for treating CAD and related TPs were retrieved from professional TCM and bioinformatics databases. Based on the associations between the drugs or ingredients and their TPs, the TP networks of XZD, GXBD, and modern drugs approved for treating CAD were constructed separately and then integrated to create a complex master network in which the vertices represent the TPs and the edges, the ingredients or drugs that are linked to the TPs. The reliability of this master network was validated through statistical tests. The common TPs of the two herbal formulas have a higher possibility of being targeted by modern drugs in comparison with the formula-specific TPs. A total of 114 common XZD and GXBD TPs that are not yet the target of modern drugs used for treating CAD should be experimentally investigated as potential therapeutic targets for treating CAD. Among these TPs, the top 10 are NOS3, PTPN1, GABRA1, PRKACA, CDK2, MAOB, ESR1, ADH1C, ADH1B, and AKR1B1. The results of this study provide a valuable reference for further experimental investigations of therapeutic targets for CAD. The established method shows promise for searching for potential therapeutic TPs based on herbal formulas. It is crucial for this work to select beneficial therapeutic targets of TCM, typical TCM syndromes, and corresponding classic formulas.

An NMR-Guided Screening Method for Selective Fragment Docking and Synthesis of a Warhead Inhibitor.

Selective hits for the glutaredoxin ortholog of Brucella melitensis are determined using STD NMR and verified by trNOE and (15)N-HSQC titration. The most promising hit, RK207, was docked into the target molecule using a scoring function to compare simulated poses to experimental data. After elucidating possible poses, the hit was further optimized into the lead compound by extension with an electrophilic acrylamide warhead. We believe that focusing on selectivity in this early stage of drug discovery will limit cross-reactivity that might occur with the human ortholog as the lead compound is optimized. Kinetics studies revealed that lead compound 5 modified with an ester group results in higher reactivity than an acrylamide control; however, after modification this compound shows little selectivity for bacterial protein versus the human ortholog. In contrast, hydrolysis of compound 5 to the acid form results in a decrease in the activity of the compound. Together these results suggest that more optimization is warranted for this simple chemical scaffold, and opens the door for discovery of drugs targeted against glutaredoxin proteins-a heretofore untapped reservoir for antibiotic agents.

From the RNA world to the clinic.

The study of RNA has continually emphasized the structural and functional versatility of RNA molecules. This versatility has inspired translational and clinical researchers to explore the utility of RNA-based therapeutic agents for a wide variety of medical applications. Several RNA therapeutics, with diverse modes of action, are being evaluated in large late-stage clinical trials, and many more are in early clinical development. Hundreds of patients are enrolled in large trials testing messenger RNAs to combat cancer, small interfering RNAs to treat renal and hepatic disorders, and aptamers to combat ocular and cardiovascular disease. Results from these studies are generating considerable interest among the biomedical community and the public and will be important for the future development of this emerging class of therapeutic agents.

Drug target identification using network analysis: Taking active components in Sini decoction as an example.

Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound.

[Progress of methodology for identifying target protein of natural active small molecules].

Drug targets are special molecules that can interact with drugs and exert pharmacological functions in human body. The natural active small molecules are the bioactive basis of traditional Chinese medicine, and the mechanism study is a hot topic now, especially for the identification of their target proteins. However, little progress has been made in this field until now. Here, we summarized the recent technologies and methods for the identification of target proteins of natural bioactive small molecules, and introduced the main research methods, principles and successful cases in this field. We also explored the applicability and discussed the advantages and disadvantages among different methods. We hope this review can be used as a reference for the researchers who engaged in natural pharmaceutical chemistry, pharmacology and chemical biology.

[Application of drug-target prediction technology in network pharmacology of traditional Chinese medicine].

In recent years, network pharmacology has been developed rapidly, and especially, the concept of ″network target″ has brought a new era in the field of traditional Chinese medicine (TCM). The integrity and systematicness emphasized in network pharmacology comply with the characteristics of holistic view and treatment in Chinese medicine. It can provide deeper insights into the underlying mechanisms of TCM theories, including the illustration on action mechanism of Chinese medicine, selection of pharmacodynamic materials and the combination principles of various Chinese herbs, etc. Therefore, this theory is more suitable for TCM academic characteristics and practical conditions. The key problem in network pharmacology is how to efficiently and quickly identify the interactions between large amounts of drugs and target proteins. As an efficient and high throughput way, drug-target prediction technology can reduce costs, quickly predict the component targets, and provide foundation for the application of TCM network pharmacology. In view of the large amount of compounds and target databases, different prediction methods and technologies have been developed, and used to predict the drug-target interactions. Many virtual screening technologies have been successfully applied to network pharmacology. Based on different prediction principles, drug-target prediction technology can be generally divided into four types： ligand-based prediction, receptor-based prediction, machine learning and combined prediction. In this paper, we are going to review the prediction methods of drug-target interactions and give acomprehensive elaboration of their application in network pharmacology of TCM, hoping to provide beneficial references for various Chinese medicine researchers.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

Cell-based peptide screening to access the undruggable target space.

Only 20-30% of drug target proteins can be accessed by common drug classes, like small molecules or therapeutic antibodies. The vast majority of the remaining proteins are considered "undruggable" and include drug target proteins, like transcription factors, scaffold or adapter proteins, which play important roles in disease. However over the last years innovative compound classes including nucleotide derived drugs (e.g. siRNA, antisense), macrocyclic compounds and cell-permeable peptides matured significantly and hold now the potential to modulate these hard to access target proteins for therapeutic use. This article will focus on the discovery of cell-permeable peptides and discuss intracellular screening systems for peptides, which yield highly relevant peptides, because peptide selection takes place in eukaryotic cells, under conditions, which are very similar to the later therapeutic use.