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The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (K D = 3 μM), the NBBA proved to be robust and displayed excellent Z'-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC 50 for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC 50 < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds., Competing Interests: Declaration of competing interest J.D. has acted as a consultant for AstraZeneca, Jubilant, Theras, Roche and Vividion and has funded research agreements with Bristol Myers Squibb, Revolution Medicines and AstraZeneca. G.D., T.M., J.T., V.F., J.K., G.S., M.A., D.S., E.G., S.S., S.A. and G.H. are employed by, or have been previously employed by, AZ and own company shares. G.H. is a previous editorial board member of SLAS Discovery. J.D. has acted as a consultant for AstraZeneca, Jubilant, Theras, Roche and Vividion and has funded research agreements with Bristol Myers Squibb, Revolution Medicines and AstraZeneca. The other authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Identification of high-quality hit chemical matter is of vital importance to the success of drug discovery campaigns. However, this goal is becoming ever harder to achieve as the targets entering the portfolios of pharmaceutical and biotechnology companies are increasingly trending towards novel and traditionally challenging to drug. This demand has fuelled the development and adoption of numerous new screening approaches, whereby the contemporary hit identification toolbox comprises a growing number of orthogonal and complementary technologies including high-throughput screening, fragment-based ligand design, affinity screening (affinity-selection mass spectrometry, differential scanning fluorimetry, DNA-encoded library screening), as well as increasingly sophisticated computational predictive approaches. Herein we describe how an integrated strategy for hit discovery, whereby multiple hit identification techniques are tactically applied, selected in the context of target suitability and resource priority, represents an optimal and often essential approach to maximise the likelihood of identifying quality starting points from which to develop the next generation of medicines., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Targeted protein degradation is an important mechanism carried out by the cellular machinery, one that is gaining momentum as an exploitable strategy for the development of drug-like compounds. Molecules which are able to induce proximity between elusive therapeutic targets of interest and E3 ligases which subsequently leads to proteasomal degradation of the target are beginning to decrease the percentage of the human proteome described as undruggable. Therefore, having the ability to screen for, and understand the mechanism of, such molecules is becoming an increasingly attractive scientific focus. We have established a number of cascade experiments including cell-based assays and orthogonal triage steps to provide annotation to the selectivity and mechanism of action for compounds identified as putative degraders from a primary high throughput screen against a high value oncology target. We will describe our current position, using PROTACs as proof-of-concept, on the analysis of these novel outputs and highlight challenges encountered., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. All authors are or have been previous employees of AstraZeneca. Geoffrey A. Holdgate is on the editorial board of SLAS Discovery, (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC 50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented., Competing Interests: Declaration of competing interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are or have been previous employees of AstraZeneca. Geoffrey A. Holdgate is on the editorial board of SLAS Discovery., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Molecular glues are small molecules, typically smaller than PROTACs, and usually with improved physicochemical properties that aim to stabilise the interaction between two proteins. Most often this approach is used to improve or induce an interaction between the target and an E3 ligase, but other interactions which stabilise interactions to increase activity or to inhibit binding to a natural effector have also been demonstrated. This review will describe the effects of induced proximity, discuss current methods used to identify molecular glues and introduce approaches that could be adapted for molecular glue screening., Competing Interests: Declaration of Competing Interest All authors are employees of AstraZeneca. G. A. Holdgate is on the editorial board for SLAS Discovery., (Copyright © 2023. Published by Elsevier Inc.)

Recently, there has been a change in the types of drug target entering early drug discovery portfolios. A significant increase in the number of challenging targets, or which would have historically been classed as intractable, has been observed. Such targets often have shallow or non-existent ligand-binding sites, can have disordered structures or domains or can be involved in protein-protein or protein-DNA interactions. The nature of the screens required to identify useful hits has, by necessity, also changed. The range of drug modalities explored has also increased and the chemistry required to design and optimise these molecules has adapted. In this review, we discuss this changing landscape and provide insights into the future requirements for small-molecule hit and lead generation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Thermal unfolding methods, applied in both isolated protein and cell-based settings, are increasingly used to identify and characterize hits during early drug discovery. Technical developments over recent years have facilitated their application in high-throughput approaches, and they now are used more frequently for primary screening. Widespread access to instrumentation and automation, the ability to miniaturize, as well as the capability and capacity to generate the appropriate scale and quality of protein and cell reagents have all played a part in these advances. As the nature of drug targets and approaches to their modulation have evolved, these methods have broadened our ability to provide useful chemical start points. Target proteins without catalytic function, or those that may be difficult to express and purify, are amenable to these methods. Here, we provide a review of the applications of thermal unfolding methods applied in hit finding during early drug discovery., Competing Interests: The authors have no conflicts to disclose., (© 2023 Crown.)

The cellular thermal shift assay (CETSA®) has increasingly been used in early drug discovery to provide a measure of cellular target engagement. Traditionally, CETSA has been employed for bespoke questions with small to medium throughput and has predominantly been applied during hit validation rather than in hit identification. Using a CETSA screen versus the kinase CRAF, we assessed 3 key questions: (1) technical feasibility - could the CETSA methodology technically be applied at truly high throughput scale? (2) relevance - could hits suitable for further optimisation be identified? (3) reliability - would the approach identify known chemical equity. Here, we describe the first large scale AlphaLISA SureFire based CETSA (Alpha CETSA) approach allowing us to screen a large library of almost 0.5 million compounds. We discuss the issues overcome in automating and executing the screen and describe the resulting screen output., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Geoffrey A. Holdgate, Hannah Rowlands, Kirsten Tschapalda, Carolyn Blackett, Delyan Ivanov, Darren Plant, Joseph Shaw, Andrew Thomas, Martin Packer reports financial support was provided by AstraZeneca UK Ltd Macclesfield. Laurence Arnold reports financial support was provided by Pelago Bioscience AB. Geoffrey A. Holdgate reports a relationship with Society for Laboratory Automation and Screening that includes: speaking and lecture fees and travel reimbursement. Geoffrey A. Holdgate is an Editorial Board member of SLAS Discovery, (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Enzyme activation remains a largely under-represented and poorly exploited area of drug discovery despite some key literature examples of the successful application of enzyme activators by various mechanisms and their importance in a wide range of therapeutic interventions. Here we describe the background nomenclature, present the current position of this field of drug discovery and discuss the challenges of hit identification for enzyme activation, as well as our perspectives on the approaches needed to overcome these challenges in early drug discovery., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Geoff Holdgate is a member of the Editorial Board for SLAS Discovery, (Copyright © 2022. Published by Elsevier Inc.)

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15 N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.

Rapid triage of compounds acting via undesired mechanisms is a crucial stage in a high-throughput screening (HTS) cascade to ensure time and resource is efficiently assigned to the most propitious hits. Redox cycling compounds (RCCs) produce reactive oxygen species, such as hydrogen peroxide, which can impair protein function and appear as hits against liable targets. Direct measurement of tris(2-carboxyethyl)phosphine (TCEP) oxidation has been demonstrated as a sensitive and accurate measure of redox cycling [1]. However, the current nuclear magnetic resonance (NMR) based detection method is not compatible with the throughput required for triage of a HTS campaign. Here we employ Acoustic Mist Ionisation Mass Spectrometry (AMI-MS) [2] to rapidly measure oxidation of TCEP and accurately identify redox cyclers in a high throughput manner., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Challenged by ageing infrastructure and increasingly demanding screening cascades, AstraZeneca High Throughput Screening department has developed advanced automation systems that can support both current needs and future strategies in drug discovery. Through collaboration with HighRes Biosolutions and other third-party vendors, highly versatile automated modular platforms have been designed. Safety features such as collaborative robots allow enhanced system accessibility, and adaptive scheduling software has improved protocol design and system recovery. These innovations have led to significant improvements in system flexibility while maintaining screening productivity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Large compound libraries utilised for HTS often include metal contaminated compounds which can interfere with assay signal or target biology, and therefore appear as hits. Pursuit of these compounds can divert considerable time and resource away from more propitious hits, yet there is currently no established method of detecting metal impurities in a rapid and effective manner. Here we describe the development and application of a high-throughput method to identify metal contaminants using acoustic mist ionisation mass spectrometry (AMI-MS). Although metals species by themselves are not detectable by AMI-MS, we have identified two compounds that chelate metal ions and enable their detection. 6-(diethylamino)-1,3,5-triazine-2,4(1H,3H)-dithione (DMT) and 1-(3-{[4-(4-cyanophenyl)-1-piperidinyl]carbonyl}-4-methylphenyl)-3-ethylthiourea (TU) can form complexes with a range of metal ions. Using a collection of metal catalysts, we have developed two metal chelator assays that collectively allow for the detection of Ag, Au, Co, Cu, Fe, Pd, Pt and Zn. We employed these assays to profile the hit outputs of a Zn liable target, and a Pd liable target, and identified significant quantities of metal contaminated compounds in the HTS outputs. This work provides a method of rapidly identifying metal impurities in hit compounds and has become part of an established workflow in triaging HTS outputs at AstraZeneca, facilitating faster identification of robust lead series., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Dysregulation of the epigenome is associated with the onset and progression of several diseases, including cancer, autoimmune, cardiovascular, and neurological disorders. Members from the three families of epigenetic proteins (readers, writers, and erasers) have been shown to be druggable using small-molecule inhibitors. Increasing knowledge of the role of epigenetics in disease and the reversibility of these modifications explain why pharmacological intervention is an attractive strategy for tackling epigenetic-based disease. In this review, we provide an overview of epigenetics drug targets, focus on approaches used for initial hit identification, and describe the subsequent role of structure-guided chemistry optimisation of initial hits to clinical candidates. We also highlight current challenges and future potential for epigenetics-based therapies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rationale: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required., Methods: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II., Results: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method., Conclusions: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors., (© 2021 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Targeted protein degradation (TPD) is a recent strategy, utilizing the cell's proteostasis machinery to deplete specific proteins. This represents a paradigm shift in early drug discovery, away from occupancy-driven to event-driven mechanisms.Recent efforts have focused on the development of proteolysis-targeting chimeras (PROTACs). These heterobifunctional molecules combine a target-specific binding moiety linked to an E3 ligase ligand and trigger selective ubiquitination of the target protein, marking it for proteasomal degradation. While these molecules can be highly efficacious, they generally have unfavorable physicochemical properties due to their large size.In contrast, smaller molecules that induce degradation could represent an attractive, simple option to overcoming the limitations of both traditional modulators and PROTACs. These molecules may have a range of mechanisms: recruitment of an E3 ligase (molecular glues), introduction of hydrophobic areas, or inducing local unfolding, each of which triggers degradation.We recently completed a high-throughput screen of 111,000 compounds in a cellular HiBiT assay in an effort to identify such molecules. Preliminary analysis indicates that we have been able to identify alternative small-molecule degraders. We highlight methods for triage, characterization, selectivity, and mode of action. In summary, we believe that these types of small-molecule degraders, which may possibly have more acceptable physicochemical properties than the inherently larger heterobifunctional molecules, are an exciting approach for inducing TPD, and we illustrate that a general screening approach can be successful in identifying useful start points for developing such molecules.

Enzymes represent a significant proportion of the druggable genome and constitute a rich source of drug targets. Delivery of a successful program for developing a modulator of enzyme activity requires an understanding of the enzyme's mechanism and the mode of interaction of compounds. This allows an understanding of how physiological conditions in disease-relevant cells will affect inhibitor potency. As a result, there is increasing interest in evaluating hit compounds from high-throughput screens to determine their mode of interaction with the target. This work revisits the common inhibition modalities and illustrates the impact of substrate concentration relative to K m upon the pattern of changes in IC 50 that are expected for increasing substrate concentration. It proposes a new, high-throughput approach for assessing mode of inhibition, incorporating analyses based on a minimal descriptive model, to deliver a workflow that allows rapid and earlier compound classification immediately after high-throughput screening.

High-throughput assays based on fluorescence polarization (or fluorescence anisotropy) technology have often been employed for primary hit-finding in drug discovery. These binding assays provide a homogeneous format and consistent performance and offer advantages over some other optical methods. Developments in assay design and improvements in fluorescent probes have enabled the application of the technique to even complex biological systems. Here we describe the practical considerations for development of FP assays applied in high-throughput screening, including fluorophore selection, assay design, data analysis, and approaches for detecting compound interference.

It is clear from the analysis of the distribution of approved drug targets that enzymes continue to be a major target class for the pharmaceutical industry. The application of high-throughput screens designed to monitor the activity of these enzyme targets, and the ability of test compounds to modulate this activity, is still the predominant hit finding approach in the industry. The widespread use of enzyme activity-based screens has led to the development of several useful guidelines for the development and validation of robust and reliable assays. Key learnings for the development, validation, and implementation of acoustic mist ionization mass spectrometry for high-throughput enzyme assays are described.

Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA., (© 2018 Macek et al.)

Given the therapeutic and commercial success of small-molecule enzyme inhibitors, as exemplified by kinase inhibitors in oncology, a major focus of current drug-discovery and development efforts is on enzyme targets. Understanding the course of an enzyme-catalysed reaction can help to conceptualize different types of inhibitor and to inform the design of screens to identify desired mechanisms. Exploiting this information allows the thorough evaluation of diverse compounds, providing the knowledge required to efficiently optimize leads towards differentiated candidate drugs. This review highlights the rationale for conducting high-quality mechanistic enzymology studies and considers the added value in combining such studies with orthogonal biophysical methods.

The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.

Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called 'DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

Two structurally distinct series of novel, MAPK-activated kinase-2 prevention of activation inhibitors have been discovered by high throughput screening. Preliminary structure-activity relationship (SAR) studies revealed substructural features that influence the selective inhibition of the activation by p38α of the downstream kinase MK2 in preference to an alternative substrate, MSK1. Enzyme kinetics, surface plasmon resonance (SPR), 2D protein NMR, and X-ray crystallography were used to determine the binding mode and the molecular mechanism of action. The compounds bind competitively to the ATP binding site of p38α but unexpectedly with higher affinity in the p38α-MK2 complex compared with p38α alone. This observation is hypothesized to be the origin of the substrate selectivity. The two lead series identified are suitable for further investigation for their potential to treat chronic inflammatory diseases with improved tolerability over previously studied p38α inhibitors.

The binding of a ligand to its target protein is often accompanied by conformational changes of both the protein and the ligand. This is of particular interest, since structural rearrangements of the macromolecular target and the ligand influence the free energy change upon complex formation. In this study, we use X-ray crystallography, isothermal titration calorimetry, and surface-plasmon resonance biosensor analysis to investigate the binding of pyrazolylaminopyrimidine inhibitors to FGFR1 tyrosine kinase, an important anticancer target. Our results highlight that structurally close analogs of this inhibitor series interact with FGFR1 with different binding modes, which are a consequence of conformational changes in both the protein and the ligand as well as the bound water network. Together with the collected kinetic and thermodynamic data, we use the protein-ligand crystal structure information to rationalize the observed inhibitory potencies on a molecular level.

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work., (© 2012 American Chemical Society)

The kinetics of ligand-target interactions have been recognised as being instrumental in dictating the efficacy of drug action. Increased focus on kinetic signatures in drug discovery has concincided with improvements in label free methodology for measuring kinetic parameters. Simultaneously, focus has also been applied to increasing the quality of compounds, in terms of their physicochemical properties. To facilitate this drive towards higher compound quality, metrics such as ligand efficiency and enthalpic efficiency have been employed. We propose another metric, kinetic efficiency, that may be used pragmatically to help identify those compounds displaying differentiated kinetic behaviour. The combination of these metrics has the potential to improve decision-making in drug discovery leading to higher quality compounds and series., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Affinity-based technologies have become impactful tools to detect, monitor and characterize molecular interactions using recombinant target proteins. This can aid the understanding of biological function by revealing mechanistic details, and even more importantly, enables the identification of new improved ligands that can modulate the biological activity of those targets in a desired fashion. The selection of the appropriate technology is a key step in that process, as each one of the currently available technologies offers a characteristic type of biophysical information about the ligand-binding event. Alongside the indisputable advantages of each of those technologies they naturally display diverse restrictions that are quite frequently related to the target system to be studied but also to the affinity, solubility and molecular size of the ligands. This paper discusses some of the theoretical and experimental aspects of the most common affinity-based methods, what type of information can be gained from each one of those approaches, and what requirements as well as limitations are expected from working with recombinant proteins on those platforms and how those can be optimally addressed.

AZD0530, an orally available Src inhibitor, demonstrated potent antimigratory and anti-invasive effects in vitro, and inhibited metastasis in a murine model of bladder cancer. Antiproliferative activity of AZD0530 in vitro varied between cell lines (IC(50) 0.2 ->10μM). AZD0530 inhibited tumor growth in 4/10 xenograft models tested and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth-inhibition-resistant and -sensitive xenografts. The activity of AZD0530 in NBT-II bladder cancer cells in vitro was consistent with inhibition of cell migration and stabilization of cell-cell adhesion. These data suggest a dominant anti-invasive pharmacology for AZD0530 that may limit tumor progression in a range of cancers. AZD0530 is currently in Phase II clinical trials.

Inhibition of the protein kinase, MEK1, is a potential approach for the treatment of cancer. Inhibitors may act by prevention of activation (PoA), which involves interfering with phosphorylation of nonactivated MEK1 by the upstream kinase, B-RAF. Modulation also may occur by inhibition of catalysis (IoC) during phosphorylation of the downstream substrate, ERK2, by activated MEK1. Here, five MEK inhibitors are characterized in terms of binding affinity, PoA, and IoC. The compounds are a butadiene (U-0126), an N-alkoxy amide (CI-1040), two CI-1040 analogues (an anthranilic acid and an N-alkyl amide), and a cyanoquinoline. Some compounds give different mechanisms of inhibition (ATP-competitive, noncompetitive, or uncompetitive) in PoA compared to IoC or show a change in potency between the assays. The inhibitors also exhibit different shifts in potency when either PoA or IoC is compared with binding to nonactivated MEK. The inhibitor potency ranking, therefore, is dependent upon the assay format. When the ATP concentration equals K m, IoC IC 50 increases in the order CI-1040 approximately cyanoquinoline < anthranilic acid approximately U-0126 < alkyl amide. Conversely, the K d from nonactivated MEK1 for four of the compounds varies between more than 6-fold lower and over 18-fold higher than this IC 50, with U-0126 having the lowest K d and CI-1040 having the highest. In PoA when the ATP concentration equals K m, U-0126 has the lowest IC 50, becoming more potent than CI-1040, the cyanoquinoline, and the anthranilic acid. These observations have implications for understanding structure-activity relationships of MEK inhibitors and illustrate how assays can be designed to favor different compounds.

Modern drug discovery usually involves the rapid screening of large numbers of compounds, either individually or in resolvable mixtures. These compounds may be complex and lead-like or may be small fragments representing optimal scaffolds. Several methods are suitable for detecting binding interactions based on a wide range of different physical platforms. However, the use of thermodynamic measurements has a role to play both in the high-throughput identification of binders and also in the fundamental understanding of molecular interaction, which is central to rational drug design. This review describes the benefits and drawbacks of using thermodynamic characterisation of binding interactions at various stages in the rational drug design process and highlights future opportunities for advances in instrumentation and methodology.

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.

Thermodynamics governs the process of biomolecular recognition. The steps of characterizing, understanding and exploiting binding thermodynamics have the potential to contribute to an improved rational drug design process that is more robust and reliable. It is only relatively recently that instrumentation capable of direct and full thermodynamic characterization has been improved, giving impetus to the application of thermodynamic measurements in drug discovery. This review highlights current instruments and methods that can be employed to measure binding thermodynamics and their use in studies of biomolecular recognition and drug discovery.

The statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (HMG-CoAR), and are utilized to decrease levels of atherogenic lipoproteins in patients with, or who are at high risk of, cardiovascular disease. This study describes the inhibition of a recombinant, catalytic fragment of human HMG-CoAR by a new statin, rosuvastatin (CRESTOR(R)). Binding is reversible and involves an initial complex [inhibition constant involving the enzyme-inhibitor complex (E.I), K (i), approximately 1 nM], which undergoes a slow transition ( t ((1/2)) to reach steady state is 33-360 s) to give tighter association [steady-state inhibition constant involving E.I and the second E.I complex in a two-step mechanism (E.I*), K (i)*, approximately 0.1 nM]. At steady state, rosuvastatin is at least as potent as atorvastatin, cerivastatin and simvastatin. It is more potent than fluvastatin and pravastatin. For rosuvastatin, inhibition kinetics are competitive with respect to HMG-CoA and non-competitive when NADPH is varied. At 37 degrees C, binding is linked to a large favourable enthalpy change [Delta H degrees =-69.0 kJ/mol (-16.5 kcal/mol)] and a small entropic penalty [ T Delta S degrees =-9.6 kJ/mol (-2.3 kcal/mol)]. These characteristics, and the high affinity relative to that of 3 S -HMG-CoA ( K (d) approximately 6.6 microM), are discussed in relation to the crystal structures of complexes with HMG-CoAR.

Characterization of the thermodynamics of binding interactions is important in improving our understanding of bimolecular recognition and forms an essential part of the rational drug design process. Isothermal titration calorimetry (ITC) is rapidly becoming established as the method of choice for undertaking such studies. The power of ITC lies in its unique ability to measure binding reactions by the detection of the heat change during the binding interaction. Since heat changes occur during many physicochemical processes, ITC has a broad application, ranging from chemical and biochemical binding studies to more complex processes involving enthalpy changes, such as enzyme kinetics. Several features of ITC have facilitated its preferential use compared to other techniques that estimate affinity. It is a sensitive, rapid, and direct method with no requirement for chemical modification or immobilization. It is the only technique that directly measures enthalpy of binding and so eliminates the need for van't Hoff analysis, which can be time consuming and prone to uncertainty in parameter values. Although ITC has facilitated the measurement of the thermodynamics governing binding reactions, interpretation of these parameters in structural terms is still a major challenge.

Isothermal titration calorimetry (ITC) follows the heat change when a test compound binds to a target protein. It allows precise measurement of affinity. The method is direct, making interpretation facile, because there is no requirement for competing molecules. Titration in the presence of other ligands rapidly provides information on the mechanism of action of the test compound, identifying the intermolecular complexes that are relevant for structure-based design. Calorimetry allows measurement of stoichiometry and so evaluation of the proportion of the sample that is functional. ITC can characterize protein fragments and catalytically inactive mutant enzymes. It is the only technique which directly measures the enthalpy of binding (delta H degree). Interpretation of delta H degree and its temperature dependence (delta Cp) is usually qualitative, not quantitative. This is because of complicated contributions from linked equilibria and a single change in structure giving modification of several physicochemical properties. Measured delta H degree values allow characterization of proton movement linked to the association of protein and ligand, giving information on the ionization of groups involved in binding. Biochemical systems characteristically exhibit enthalpy-entropy compensation where increased bonding is offset by an entropic penalty, reducing the magnitude of change in affinity. This also causes a lack of correlation between the free energy of binding (delta G degree) and delta H degree. When characterizing structure-activity relationships (SAR), most groups involved in binding can be detected as contributing to delta H degree, but not to affinity. Large enthalpy changes may reflect a modified binding mode, or protein conformation changes. Thus, delta H degree values may highlight a potential discontinuity in SAR, so that experimental structural data are likely to be particularly valuable in molecular design.

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Squalene synthase (SQS) catalyses a step following the final branch in the pathway of cholesterol biosynthesis. Inhibition of this enzyme, therefore, is an approach for the treatment of atherosclerosis with the potential for low side effects. We have characterised the inhibition of rat liver microsomal SQS by 3-(biphenyl-4-yl)quinuclidine (BPQ). BPQ follows slow binding kinetics in that the rate of accumulation of product decreases with time if the inhibitor is added when the assay is started. Preincubation of BPQ and SQS leads to a biphasic dose-response where accumulation of product is linear with time only for the sensitive phase. When the farnesyl pyrophosphate (FPP) substrate is present at 19.6 microM, approximately 77% of the SQS activity is sensitive to the inhibitor (vOs) and the remainder is insensitive (vOi). The apparent inhibition constants (K'i values) are respectively K'is = 4.5 nM and K'ii = 1300 nM. Similar biphasic behaviour is exhibited by other inhibitors and in microsomes prepared from human and marmoset liver. As the concentration of FPP is reduced below 19.6 microM, there is a decrease in the relative contribution from vOi. Conversely, the value of K'is for BPQ remains constant when the FPP concentration is changed, showing noncompetitive kinetics with respect to this substrate. Possible causes of the observed kinetics are discussed. Inhibition by BPQ is said to follow tight binding kinetics because the value of K'is is similar to the concentration of inhibitor binding sites. Thus, to avoid an artefactual variation in potency when the enzyme concentration is varied, it is necessary to allow for the effects of depletion of free inhibitor. Furthermore, estimates of potency that average activity across the two phases are influenced by the relative contributions of each phase. These contributions differ according to the FPP concentration and the species used as the source of microsomes. Thus, it is necessary to separate the phases to compare measurements made in different experiments. Our observations indicate that careful experimental design and data analysis are required to characterise the kinetics of SQS inhibitors.

Squalene synthase (SQS) is a key enzyme in the biosynthetic pathway for cholesterol and is a target for improved agents to lower plasma levels of low-density lipoprotein (LDL). A series of novel 3' substituted quinuclidines have been discovered as inhibitors of the rat liver microsomal enzyme. In this study, we demonstrate the inhibitory effects in vitro and in vivo, of two examples of the series. When microsomes were preincubated with compounds, before addition of substrate, both 3-(biphenyl-4-yl)quinuclidine (BPQ) and 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH) were found to cause biphasic inhibition of the enzyme with apparent inhibition constants (K'i) for the sensitive phases of 12 nM and 15 nM, respectively. The K'i values for the insensitive phases were 1.8 microM and 2.9 microM, respectively. The two examples inhibited equally both steps of the SQS-catalysed reaction, as shown by parallel inhibition of 3H+ release and labelled squalene formation from [1-3H]farnesyl pyrophosphate (FPP). BPQ and BPQ-OH were shown to be inhibitors of hepatic sterol synthesis from mevalonate with ED50 values of 10.6 and 7.1 mg/kg, respectively, after acute oral administration to the rat. BPQ-OH was chosen for further study and, to determine its selectivity of effect on the mevalonate pathway in vivo, the effect of a dose of 70 mg/kg on the pattern of labelled mevalonate incorporation into the various lipid fractions of the rat liver was examined. As expected, the incorporation into squalene and sterol products was inhibited by about 70%. An appearance of label in fractions corresponding to farnesyl and geranylgeranylpyrophosphates, as well as the corresponding alcohols, was observed in treated but not control animals. In addition, the administration of compound resulted in the appearance of peaks of mevalonate-derived radioactivity in an acidic fraction believed to represent metabolites of farnesol. Such results are consistent with inhibition of the mevalonate pathway at, and not before, SQS. In contrast, there was a significant increase in the incorporation of labelled mevalonate into ubiquinone 10, and the synthesis of dolichols was apparently unchanged. The results suggest a specific effect of BPQ-OH on rat liver SQS. The compound is, therefore, an interesting lead for further investigation of this class of compounds.

Inhibition of tyrosine kinases is a possible approach for the treatment of cancer. We have investigated the catalytic mechanism of the epidermal growth factor receptor tyrosine kinase (EGF-RTK) in order to obtain information for use in structure-based searching for inhibitors. Initial rate studies imply that EGF-RTK forms a ternary complex together with ATP and peptide substrate. Investigation of pH and temperature dependence suggests that the kinase reaction requires the ionised form of a carboxylate (pK = 6.3) and the protonated form of another group (pK = 9.1). These characteristics are consistent with a mechanism where the carboxylate of Asp813(pK = 6.3) facilitates deprotonation of the tyrosyl hydroxyl of the peptide substrate, activating it as a nucleophile to attack the gamma-phosphorus of ATP which interacts with a protonated enzyme side-chain (pK = 9.1), possibly the guanidinium group of Arg817. This proposed catalytic mechanism was used to define a query when searching for inhibitors in a database of predicted three-dimensional structures. The procedure involved searching for compounds that mimic the ATP gamma-phosphate, tyrosyl hydroxyl and the tyrosyl aromatic ring, all of which seem to interact strongly with the enzyme during catalysis. This search allowed identification of inhibitors of EGF-RTK which were used to define queries for two-dimensional searching of a larger database, leading to the discovery of 4-(3-chloroanilino)quinazoline (CAQ) which is a potent inhibitor (Ki = 16 nM) of the enzyme. The compound is believed to be the first representative from a new structural class of anilinoquinazoline tyrosine kinase inhibitors. It follows competitive kinetics with respect to ATP and noncompetitive kinetics when the peptide is varied, implying that it functions as an analogue of ATP. CAQ is a novel and potent lead in the search for tyrosine kinase inhibitors as potential agents for the treatment of cancer.