A small-molecule ARTS mimetic promotes apoptosis through degradation of both XIAP and Bcl-2.

Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is "druggable". A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.

Big Data in Drug Discovery.

Interpretation of Big Data in the drug discovery community should enhance project timelines and reduce clinical attrition through improved early decision making. The issues we encounter start with the sheer volume of data and how we first ingest it before building an infrastructure to house it to make use of the data in an efficient and productive way. There are many problems associated with the data itself including general reproducibility, but often, it is the context surrounding an experiment that is critical to success. Help, in the form of artificial intelligence (AI), is required to understand and translate the context. On the back of natural language processing pipelines, AI is also used to prospectively generate new hypotheses by linking data together. We explain Big Data from the context of biology, chemistry and clinical trials, showcasing some of the impressive public domain sources and initiatives now available for interrogation.

Classifying shape coverage in fragment libraries using a fingerprinting approach.

Fragment screening is one approach to hit identification for early stage drug discovery projects. Like any screening library, diversity is needed in fragment libraries. This includes coverage of shape and electrostatic space, as well as chemotype diversity. A new, easily interpretable shape-based fingerprint is described and its utility in probing fragment library content is demonstrated using a Rule of Three library from Maybridge. This method explicitly considers size as a component of shape. It allows interrogation of shape space on both a per conformer and a per molecule level, and includes a measure of flexibility. This allows for the identification of highly flexible compounds and their exclusion from the analysis. A comparison with two literature methods, the triangle plot approach of Sauer and Schwarz and the plane of best fit method of Firth et al., is also included.

Strategies for small molecule library design.

Compilation of an appropriate set of compounds is essential for the success of a small molecule screen. When very little is known about the target and when no or few ligands have been identified, the screening file is often made as diverse as possible. When structural information on the target or target family is available or ligands of the target are known, it is more efficient to apply a ligand- or target-focused bias, so as to predominantly screen compounds that can be expected to modulate the target. One way to achieve this is to select subsets of existing collections; another is to specifically design and synthesize libraries focused on a particular target, target family or mechanism of action. Despite the number of success stories, designing such libraries is still challenging and requires specialized knowledge, especially in emerging target areas such as protein-protein interactions (PPI), epigenetics and the ubiquitin proteasome pathway. BioFocus has successfully produced so-called SoftFocus(®) libraries for many years, evolving their targets from kinases to GPCRs and ion channels to difficult targets in the epigenetics and PPI fields. This article outlines several of the principles applied to SoftFocus library design, showcasing successes achieved by BioFocus' clients. In addition, screening results for a comprehensive set of BioFocus' kinase libraries against 20 kinase targets are used to demonstrate the power of the SoftFocus approach in delivering both selective and less-selective compounds and libraries against these targets. Trademarks: BioFocus(®), SoftFocus(®), HDRA™, FieldFocus™, Thematic Analysis™, ThemePair™ and ThemePair Fragment™ are trademarks of Galapagos NV and/or its affiliates.

Optimization of 3,5-dimethylisoxazole derivatives as potent bromodomain ligands.

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells, as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown, whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested.

Building in molecular diversity for targeted libraries.

The use of gene-focussed libraries for screening against protein targets can improve timelines for drug discovery projects. This is especially true when the library is based on a novel core scaffold, avoiding the potential need to scaffold hop from early hits. Identification of an appropriate novel scaffold is therefore integral to the success of such a library. In this article we outline a new method to aid scaffold design that combines structure- based virtual screening (VS) with a second phase in which fragmentation of the output is made before the final scaffold design step. Through consideration of a refined set of bound fragments, in the context of the compounds from which they originated, appropriate vectors for appended R-groups can be assigned before validation of the final library.

Discovery of selective and potent inhibitors of gram-positive bacterial thymidylate kinase (TMK).

Thymidylate kinase (TMK) is an essential enzyme in bacterial DNA synthesis. The deoxythymidine monophosphate (dTMP) substrate binding pocket was targeted in a rational-design, structure-supported effort, yielding a unique series of antibacterial agents showing a novel, induced-fit binding mode. Lead optimization, aided by X-ray crystallography, led to picomolar inhibitors of both Streptococcus pneumoniae and Staphylococcus aureus TMK. MICs < 1 µg/mL were achieved against methicillin-resistant S. aureus (MRSA), S. pneumoniae, and vancomycin-resistant Enterococcus (VRE). Log D adjustments yielded single diastereomers 14 (TK-666) and 46, showing a broad antibacterial spectrum against Gram-positive bacteria and excellent selectivity against the human thymidylate kinase ortholog.

The design and application of target-focused compound libraries.

Target-focused compound libraries are collections of compounds which are designed to interact with an individual protein target or, frequently, a family of related targets (such as kinases, voltage-gated ion channels, serine/cysteine proteases). They are used for screening against therapeutic targets in order to find hit compounds that might be further developed into drugs. The design of such libraries generally utilizes structural information about the target or family of interest. In the absence of such structural information, a chemogenomic model that incorporates sequence and mutagenesis data to predict the properties of the binding site can be employed. A third option, usually pursued when no structural data are available, utilizes knowledge of the ligands of the target from which focused libraries can be developed via scaffold hopping. Consequently, the methods used for the design of target-focused libraries vary according to the quantity and quality of structural or ligand data that is available for each target family. This article describes examples of each of these design approaches and illustrates them with case studies, which highlight some of the issues and successes observed when screening target-focused libraries.

Inhibitors of PIM-1 kinase: a computational analysis of the binding free energies of a range of imidazo [1,2-b] pyridazines.

The binding of a selection of competitive imidazo [1,2-b] pyridazine inhibitors of PIM-1 kinase with nanomolar activity has been analyzed using computational methods. Molecular dynamics simulations using umbrella sampling to determine a potential of mean force have been used to accurately predict the relative free energies of binding of these inhibitors, from -4.3 to -9.5 kcal mol(-1), in excellent agreement with the trends observed in previous experimental assays. The relative activity of the inhibitors could not be accounted for by any single effect or interaction within the active site and could only be fully reproduced when the overall free energies were considered, including important contributions from interactions outside the hinge region and using explicit solvent in the active site. The potential of mean force for the displacement of the glycine-rich phosphate binding loop (P-loop) has also been estimated and shown to be an important feature in the binding of these ligands.