Optimization of Naphthyridones into Selective TATA-Binding Protein Associated Factor 1 (TAF1) Bromodomain Inhibitors

Bromodomain containing proteins and the acetyl-lysine binding bromodomains contained therein are increasingly attractive targets for the development of novel epigenetic therapeutics. To help validate this target class and unravel the complex associated biology, there has been a concerted effort to develop selective small molecule bromodomain inhibitors. Herein we describe the structure-based efforts and multiple challenges encountered in optimizing a naphthyridone template into selective TAF1(2) bromodomain inhibitors which, while unsuitable as chemical probes themselves, show promise for the future development of small molecules to interrogate TAF1(2) biology. Key to this work was the introduction and modulation of the basicity of a pendant amine which had a substantial impact on not only bromodomain selectivity but also cellular target engagement.     

1,3-Dimethyl Benzimidazolones Are Potent,Selective Inhibitors of the BRPF1 Bromodomain

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Discovery and characterization of small molecule inhibitors of the BET family bromodomains

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.     

Binding evaluation of fragment-based scaffolds for probing allosteric enzymes

Fragment-based drug discovery has become a powerful method for the generation of drug leads against therapeutic targets. Beyond the identification of novel and effective starting points for drug design, fragments have emerged as reliable tools for assessing protein druggability and identifying protein hot spots. Here, we have examined fragments resulting from the deconstruction of known inhibitors from the glycogen phosphorylase enzyme, a therapeutic target against type 2 diabetes, with two motivations. First, we have analyzed the fragment binding to the multiple binding sites of the glycogen phosphorylase, and then we have investigated the use of fragments to study allosteric enzymes. The work we report illustrates the power of fragmentlike ligands not only for probing the various binding pockets of proteins, but also for uncovering cooperativity between these various binding sites.     

Targeting enzyme inhibitors in drug discovery

Drugs that function as enzyme inhibitors constitute a significant portion of the orally bioavailable therapeutic agents that are in clinical use today. Likewise, much of drug discovery and development efforts at present are focused on identifying and optimizing drug candidates that act through inhibition of specific enzyme targets. The attractiveness of enzymes as targets for drug discovery stems from the high levels of disease association (target validation) and druggability (target tractability) that typically characterize this class of proteins. In this expert opinion the authors describe the existing practices and future directions in drug discovery enzymology, with emphasis on how a detailed understanding of the catalytic mechanism of specific targets can be used to identify and optimize small-molecule compounds that interact with conformationally distinct forms of the enzyme, thus resulting in high potency, high selectivity inhibitors.     

Targeting enzyme inhibitors in drug discovery

Drugs that function as enzyme inhibitors constitute a significant portion of the orally bioavailable therapeutic agents that are in clinical use today. Likewise, much of drug discovery and development efforts at present are focused on identifying and optimizing drug candidates that act through inhibition of specific enzyme targets. The attractiveness of enzymes as targets for drug discovery stems from the high levels of disease association (target validation) and druggability (target tractability) that typically characterize this class of proteins. In this expert opinion the authors describe the existing practices and future directions in drug discovery enzymology, with emphasis on how a detailed understanding of the catalytic mechanism of specific targets can be used to identify and optimize small-molecule compounds that interact with conformationally distinct forms of the enzyme, thus resulting in high potency, high selectivity inhibitors.     

Selective targeting of epigenetic reader domains

Introduction: Epigenetic regulators including writers, erasers, and readers of chromatin marks have been implicated in numerous diseases and are therefore subject of intense academic and pharmaceutical research. While several small-molecule inhibitors targeting writers or erasers are either approved drugs or are currently being evaluated in clinical trials, the targeting of epigenetic readers has lagged behind. Proof-of-principle that epigenetic readers are also relevant drug targets was provided by landmark discoveries of selective inhibitors targeting the BET family of acetyl-lysine readers. More recently, high affinity chemical probes for non-BET acetyl- and methyl-lysine reader domains have also been developed.

Areas covered: This article covers recent advances with the identification and validation of inhibitors and chemical probes targeting epigenetic reader domains. Issues related to epigenetic reader druggability, quality requirements for chemical probes, interpretation of cellular action, unexpected cross-talk, and future challenges are also discussed.

Expert opinion: Chemical probes provide a powerful means to unravel biological functions of epigenetic readers and evaluate their potential as drug targets. To yield meaningful results, potency, selectivity, and cellular target engagement of chemical probes need to be stringently validated. Future chemical probes will probably need to fulfil additional criteria such as strict target specificity or the targeting of readers within protein complexes.     

Anticancer drug discovery by targeting cullin neddylation

Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of neddylation are cullin family proteins, which are the core component of Cullin–RING E3 ubiquitin ligases (CRLs). Given that cullin neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein neddylation from preclinical target validation, to drug screening, then to the clinical trials of neddylation inhibitors. In this review, we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade, followed by a summary of all reported chemical inhibitors of neddylation enzymes. We then discussed the structure-based targeting of protein–protein interaction in neddylation cascade, and finally the available approaches for the discovery of new neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.     

Structure of Class B GPCRs: new horizons for drug discovery

Class B GPCRs of the secretin family are important drug targets in many human diseases including diabetes, neurodegeneration, cardiovascular disease and psychiatric disorders. X-ray crystal structures for the glucagon receptor and corticotropin-releasing factor receptor 1 have now been published. In this review, we analyse the new structures and how they compare with each other and with Class A and F receptors. We also consider the differences in druggability and possible similarity in the activation mechanisms. Finally, we discuss the potential for the design of small-molecule modulators for these important targets in drug discovery. This new structural insight allows, for the first time, structure-based drug design methods to be applied to Class B GPCRs.     

Fragment based drug discovery: practical implementation based on ¹⁹F NMR spectroscopy

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.     

Interaction profiles of protein kinase-inhibitor complexes and their application to virtual screening

A major challenge facing structure-based drug discovery efforts is how to leverage the massive amount of experimental (X-ray and NMR) and virtual structural information generated from drug discovery projects. Many important drug targets have large numbers of protein-inhibitor complexes, necessitating tools to compare and contrast their similarities and differences. This information would be valuable for understanding potency and selectivity of inhibitors and could be used to define target constraints to assist virtual screening. We describe a profile-based approach that enables us to capture the conservation of interactions between a set of protein-ligand receptor complexes. The use of profiles provides a sensitive means to compare multiple inhibitors binding to a drug target. We demonstrate the utility of profile-based analysis of small molecule complexes from the protein-kinase family to identify similarities and differences in binding of ATP, p38, and CDK2 compounds to kinases and how these profiles can be applied to differentiate the selectivity of these inhibitors. Importantly, our virtual screening results demonstrate superior enrichment of kinase inhibitors using profile-based methods relative to traditional scoring functions. Interaction-based analysis should provide a valuable tool for understanding inhibitor binding to other important drug targets.     

Technical Pitfalls and Improvements for High-speed Screening and QSAR Analysis to Predict Inhibitors of the Human Bile Salt Export Pump (ABCB11/BSEP)

Drug-induced hepatotoxicity is one of the major problems encountered in drug discovery and development. Selection of a candidate compound for pre-clinical studies in the drug discovery process is a critical step that can determine the speed and expenditure of clinical development. Because inhibition of human adenosine triphosphate-binding cassette transporter ABCB11 (SPGP/bile salt export pump) has severe consequences, which include intrahepatic cholestasis and hepatotoxicity, resulting from exposure to toxic xenobiotics or drug interactions, in vitro screening methods are necessary for quantifying and characterizing the inhibition of ABCB11. In line with such initiatives, we developed methods for in vitro high-speed screening and quantitative structure-activity relationship (QSAR) analysis to investigate the interaction of ABCB11 with a variety of compounds. We identified one set of chemical fragmentation codes closely linked with inhibition of ABCB11. Furthermore, the high-speed screening method enables us to analyze the kinetics of ABCB11-inhibition by test compounds and to distinguish competitive and non-competitive inhibitors. Troglitazone and novobiocin were found to be competitive inhibitors to taurocholate, whereas porphyrins were non-competitive inhibitors. Kinetics-based classification of inhibitors is considered important to improve the accuracy of our QSAR analysis. The present mini-review addresses technical pitfalls and improvements for high-speed screening and QSAR analysis in the ABCB11 inhibition study.     

Recent advances in designing substrate-competitive protein kinase inhibitors

Protein kinases play central roles in cellular signaling pathways and their abnormal phosphorylation activity is inseparably linked with various human diseases. Therefore, modulation of kinase activity using potent inhibitors is an attractive strategy for the treatment of human disease. While most protein kinase inhibitors in clinical development are mainly targeted to the highly conserved ATP-binding sites and thus likely promiscuously inhibit multiple kinases including kinases unrelated to diseases, protein substrate-competitive inhibitors are more selective and expected to be promising therapeutic agents. Most substrate-competitive inhibitors mimic peptides derived from substrate proteins, or from inhibitory domains within kinases or inhibitor proteins. In addition, bisubstrate inhibitors are generated by conjugating substrate-competitive peptide inhibitors to ATP-competitive inhibitors to improve affinity and selectivity. Although structural information on protein kinases provides invaluable guidance in designing substrate-competitive inhibitors, other strategies including bioinformatics, computational modeling, and high-throughput screening are often employed for developing specific substrate-competitive kinase inhibitors. This review focuses on recent advances in the design and discovery of substrate-competitive inhibitors of protein kinases.     

Chemical genetic approaches to kinase drug discovery

Chemical genetics is an important approach in biological research that utilizes small molecules to study protein function. In the context of kinase drug discovery, chemical genetics has broad applications in identifying and validating targets, demonstrating the druggability of a target and providing potential kinase inhibitor leads for further optimization. The successful application of this approach demands that the small-molecule kinase inhibitors used achieve a desired potency and selectivity. However, given the high number (> 518) and homology of kinases in the human genome, identifying potent and selective kinase inhibitors presents a major challenge. This article reviews recent advances in small-molecule kinase inhibitor design, with an emphasis on selectivity, and also discusses recent progress in the development of analog-sensitive kinase allele (ASKA)-based chemical genetics technology, which creates genetically engineered versions of protein kinases that are fully functional and can be selectively inhibited by a unique reference orthogonal inhibitor. Examples of how ASKA technology can be applied to kinase drug discovery is discussed.     

HTS technologies in biopharmaceutical discovery

The concepts and philosophies of HTS can be productively applied to the discovery of new biopharmaceuticals. It is now possible, comprehensively and systematically, to enumerate, clone, produce and screen all secreted proteins, by building upon knowledge accumulated over the past two decades in HTS, genomics and parallel protein expression technologies. Each of the crucial operational components (comprehensive and high-quality cDNA library construction, proper protein-sequence classification, high-throughput protein production, medically relevant assays, state-of-the-art screening and data management) must be optimized to increase the chances of success. In this review, we draw comparisons between small-molecule and protein screening to illuminate common underlying principles as well as differences between the two operations.     

A novel method for evaluation and screening of caspase inhibitory peptides by the amino acid positional fitness score

Background

Since caspases are key executioners of apoptosis in cases of severe diseases including neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, and viral infection diseases such as AIDS and hepatitis, potent and specific inhibitors of caspases have clinical potential. A series of peptide inhibitors has been designed based on cleavage sites of substrate proteins. However, these peptides are not necessarily the most potent to each caspase. Moreover, so far, it has proved to be difficult to design potent and specific peptide inhibitors of each caspase from sequence data of known cleavage sites in substrate proteins. We have attempted to develop a computational screening system for rapid selection of potent and specific peptide inhibitors from a comprehensive peptide library.

Results

We developed a new method for rapid evaluation and screening of peptide inhibitors based on Amino acid Positional Fitness (APF) score. By using this score, all known peptide inhibitors of each caspases-3,-7,-8, and -9 were rapidly selected in their enriched libraries. In this libraries, there were good correlations between predicted binding affinities of the known peptide inhibitors and their experimental Ki values. Furthermore, a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 was able to be designed by this method. To our knowledge, DNLD is a first reported caspase-3 inhibitory peptide identified by using the computational screening strategy.

Conclusion

Our new method for rapid screening of peptide inhibitors using APF score is an efficient strategy to select potent and specific peptide inhibitors from a comprehensive peptide library. Thus, the APF method has the potential to become a valuable approach for the discovery of the most effective peptide inhibitors. Moreover, it is anticipated that these peptide inhibitors can serve as leads for further drug design and optimization of small molecular inhibitors.