Discovery of Novel Dot1L Inhibitors through a Structure-Based Fragmentation Approach.

Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to repression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.

Screening for Inhibitors of Kinase Autophosphorylation.

Autophosphorylation of kinases influences their conformational state and can also regulate enzymatic activity. Recently, this has become an area of interest for drug discovery. Using Alk2 as an example, we present two protocols - one based on phosphate-binding Alphascreen beads, the other on coupled luminescence measurements of ADP formation - that can be used to screen for inhibitors of autophosphorylation.

Non-stoichiometric inhibition in integrated lead finding - a literature review.

INTRODUCTION: Non-stoichiometric inhibition summarizes different mechanisms by which low-molecular weight compounds can reproducibly inhibit high-throughput screening (HTS) and other lead finding assays without binding to a structurally defined site on their molecular target. This disqualifies such molecules from optimization by medicinal chemistry, and therefore their rapid elimination from screening hit lists is essential for productive and effective drug discovery. AREAS COVERED: This review covers recent literature that either investigates the various mechanisms behind non-stoichiometric inhibition or suggests assays and readouts to identify them. In addition, combination of the various methods to distill promising molecules out of raw primary hit lists step-by-step is considered. Emerging technologies to demonstrate target engagement in cells are also discussed. EXPERT OPINION: Over the last few years, awareness of non-stoichiometric inhibitors within screening libraries and HTS hit lists has considerably increased, not only in the pharmaceutical industry but also in the academic drug discovery community. This has resulted in a variety of methods to detect and handle such compounds. These range from in silico approaches to flag suspicious compounds, and counterassays to measure non-stoichiometric inhibition, to biophysical methods that positively demonstrate stoichiometric binding. In addition, novel technologies to verify target engagement within cells are becoming available. While still a time- and resource-consuming nuisance, non-stoichiometric inhibitors therefore do not fundamentally jeopardize the discovery of low molecular weight lead and drug candidates. Rather, they should be viewed as a manageable issue that with appropriate expertise can be overcome through integration of the above-mentioned approaches.

A screening pattern recognition method finds new and divergent targets for drugs and natural products.

Computational target prediction methods using chemical descriptors have been applied exhaustively in drug discovery to elucidate the mechanisms-of-action (MOAs) of small molecules. To predict truly novel and unexpected small molecule-target interactions, compounds must be compared by means other than their chemical structure alone. Here we investigated predictions made by a method, HTS fingerprints (HTSFPs), that matches patterns of activities in experimental screens. Over 1,400 drugs and 1,300 natural products (NPs) were screened in more than 200 diverse assays, creating encodable activity patterns. The comparison of these activity patterns to an MOA-annotated reference panel led to the prediction of 5,281 and 2,798 previously unknown targets for the NP and drug sets, respectively. Intriguingly, there was limited overlap among the targets predicted; the drugs were more biased toward membrane receptors and the NPs toward soluble enzymes, consistent with the idea that they represent unexplored pharmacologies. Importantly, HTSFPs inferred targets that were beyond the prediction capabilities of standard chemical descriptors, especially for NPs but also for the more explored drug set. Of 65 drug-target predictions that we tested in vitro, 48 (73.8%) were confirmed with AC50 values ranging from 38 nM to 29 µM. Among these interactions was the inhibition of cyclooxygenases 1 and 2 by the HIV protease inhibitor Tipranavir. These newly discovered targets that are phylogenetically and phylochemically distant to the primary target provide an explanation for spontaneous bleeding events observed for patients treated with this drug, a physiological effect that was previously difficult to reconcile with the drug's known MOA.

PIKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling.

Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.

A novel class of oral direct renin inhibitors: highly potent 3,5-disubstituted piperidines bearing a tricyclic p3-p1 pharmacophore.

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay.

Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids, and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation; however, most HMT assay methods are either not amenable to a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents following dispensing and after addition to reactions; and the potential for interference from drug-like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well-characterized Transcreener AMP/GMP assay, the authors have developed a robust HTS assay for HMTs that should be broadly applicable to other types of methyltransferases as well.

Light it up: highly efficient multigene delivery in mammalian cells.

Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics, and cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges. Here, we review recent multigene delivery and expression strategies and their exploitation in mammalian cells. We discuss applications in drug discovery assays, interaction studies, and biologics production, which may benefit in the future from our novel approach.

Non-stoichiometric inhibition in biochemical high-throughput screening.

INTRODUCTION: Over the last 2 decades, high-throughput screening (HTS) has become one of the key strategies for the generation of new leads. Non-stoichiometric inhibition is one of the most extensively studied mechanisms responsible for the large percentage of hit compounds from biochemical screens that cannot be developed into leads. Therefore, HTS hit lists need to be sorted rapidly and efficiently into stoichiometrically binding inhibitors and compounds that affect enzyme activity non-stoichiometrically. AREAS COVERED: This article explores the non-stoichiometric inhibition of enzymatic activity in biochemical screens, particularly by compound aggregation, and the authors explain the terminology they use to describe such compound behavior. The paper then provides a short historical overview of both academic and industrial research on compound aggregation specifically. Finally, the article discusses the implications for industrial drug discovery and the measures that can be taken to identify non-stoichiometric and aggregating inhibitors early in this process. EXPERT OPINION: The most pragmatic approach in a lead finding campaign is to focus on the early identification of selective and stoichiometric inhibitors. The combination of multiple approaches (assessing both activity and binding) allows the enrichment of stoichiometric inhibitors at each stage of the flowchart.

Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays.

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. Compared with conventional calorimetry, enthalpy arrays achieve a significant reduction of sample volume and measurement time through the combination of the small size of the detectors and ability to perform measurements in parallel. The current capabilities of the technology for studying enzyme-catalyzed reactions are demonstrated by determining the kinetic parameters for reactions with three model enzymes. In addition, the technology has been used with two classes of enzymes to determine accurate inhibitor constants for competitive inhibitors from measurements at a single inhibitor concentration.

Targeting ceramide metabolism with a potent and specific ceramide kinase inhibitor.

Ceramide kinase (CerK) produces the bioactive lipid ceramide-1-phosphate (C1P) and appears as a key enzyme for controlling ceramide levels. In this study, we discovered and characterized adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231), a potent, specific, and reversible CerK inhibitor that competitively inhibits binding of ceramide to CerK. NVP-231 is active in the low nanomolar range on purified as well as cellular CerK and abrogates phosphorylation of ceramide, resulting in decreased endogenous C1P levels. When combined with another ceramide metabolizing inhibitor, such as tamoxifen, NVP-231 synergistically increased ceramide levels and reduced cell growth. Therefore, NVP-231 represents a novel and promising compound for controlling ceramide metabolism that may provide insight into CerK physiological function.

Trends in drug discovery technologies - meeting highlights from MipTec 2007.

The 10th anniversary of MipTec, which took place in Basel, Switzerland on 7 - 10 May 2007, demonstrated that this meeting has truly evolved into the leading European event for drug discovery technologies. The rich programme included sessions on drug discovery processes, structure-based drug design, pharmacodynamics and biomarkers, maximizing compound value, drug discovery technologies or emerging absorption, distribution, metabolism and excretion/toxicity technologies together with keynote presentations providing the larger picture, an excellent poster session and a well-attended vendor exhibition. In summary, MipTec more than ever provides a much needed hub for scientific exchange and networking within the European drug discovery community.

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format.

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.

Trends in drug discovery technologies - conference report from MipTec 2006.

The 9th annual MipTec conference was held in Basel, Switzerland on 8 - 11 May 2006 and was preceded by a preconference including a variety of well-established speakers highlighting various aspects of the drug discovery process from a more general perspective. The main conference was thoughtfully organised into sessions on structure-based drug design, drug discovery processes, drug discovery technologies, pharmacodynamics and biomarkers, and maximising compound value. Here the authors attempt to 'cherry pick' some personal highlights from the many presentations in the sessions.

Analysis of p53 "latency" and "activation" by fluorescence correlation spectroscopy. Evidence for different modes of high affinity DNA binding.

The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the "activation" phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of "latent" and "activated" p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions.