The structures of salt-inducible kinase 3 in complex with inhibitors reveal determinants for binding and selectivity.

The salt-inducible kinases (SIKs) 1 to 3, belonging to the AMPK-related kinase family, serve as master regulators orchestrating a diverse set of physiological processes such as metabolism, bone formation, immune response, oncogenesis, and cardiac rhythm. Owing to its key regulatory role, the SIK kinases have emerged as compelling targets for pharmacological intervention across a diverse set of indications. Therefore, there is interest in developing SIK inhibitors with defined selectivity profiles both to further dissect the downstream biology and for treating disease. However, despite a large pharmaceutical interest in the SIKs, experimental structures of SIK kinases are scarce. This is likely due to the challenges associated with the generation of proteins suitable for structural studies. By adopting a rational approach to construct design and protein purification, we successfully crystallized and subsequently solved the structure of SIK3 in complex with HG-9-91-01, a potent SIK inhibitor. To enable further SIK3-inhibitor complex structures we identified an antibody fragment that facilitated crystallization and enabled a robust protocol suitable for structure-based drug design. The structures reveal SIK3 in an active conformation, where the ubiquitin-associated domain is shown to provide further stabilization to this active conformation. We present four pharmacologically relevant and distinct SIK3-inhibitor complexes. These detail the key interaction for each ligand and reveal how different regions of the ATP site are engaged by the different inhibitors to achieve high affinity. Notably, the structure of SIK3 in complex with a SIK3 specific inhibitor offers insights into isoform selectivity.

Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

Design and Evaluation of a Low Hydrogen Bond Donor Count Fragment Screening Set to Aid Hit Generation of PROTACs Intended for Oral Delivery.

The development of orally bioavailable PROTACs presents a significant challenge due to the inflated physicochemical properties of such heterobifunctional molecules. Molecules occupying this "beyond rule of five" space often demonstrate limited oral bioavailability due to the compounding effects of elevated molecular weight and hydrogen bond donor count (among other properties), but it is possible to achieve sufficient oral bioavailability through physicochemical optimization. Herein, we disclose the design and evaluation of a low hydrogen bond donor count (≤1 HBD) fragment screening set to aid hit generation of PROTACs intended for an oral route of delivery. We demonstrate that application of this library can enhance fragment screens against PROTAC proteins of interest and ubiquitin ligases, yielding fragment hits containing ≤1 HBD suitable for optimizing toward orally bioavailable PROTACs.

Fragment screening at AstraZeneca: developing the next generation biophysics fragment set.

Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.

Interbase-FRET binding assay for pre-microRNAs.

The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA-target binding studies.

Fragment-Based Discovery of Novel Allosteric MEK1 Binders.

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-µM affinity for MEK1 with promising physicochemical and ADMET properties.

Hot-Spotting with Thermal Scanning: A Ligand- and Structure-Independent Assessment of Target Ligandability.

Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets. Computational as well as biophysical approaches based on nuclear magnetic resonance (NMR) fragment screening are powerful approaches but suffer from specific constraints that limit their usage. Here, we demonstrate the applicability of high-throughput thermal scanning (HTTS) as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening. By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening (HTS). The few divergences for targets of low ligandability originate from the sensitivity differences of the orthogonal biophysical methods. We thus applied a new strategy making use of modulations in the solvent structure to improve assay sensitivity. This novel approach enables improved ligandability assessments in accordance with NMR-based assessments and more importantly positions the methodology as a valuable option for biophysical fragment screening.

Unified Software Solution for Efficient SPR Data Analysis in Drug Research.

Surface plasmon resonance (SPR) is a powerful method for obtaining detailed molecular interaction parameters. Modern instrumentation with its increased throughput has enabled routine screening by SPR in hit-to-lead and lead optimization programs, and SPR has become a mainstream drug discovery technology. However, the processing and reporting of SPR data in drug discovery are typically performed manually, which is both time-consuming and tedious. Here, we present the workflow concept, design and experiences with a software module relying on a single, browser-based software platform for the processing, analysis, and reporting of SPR data. The efficiency of this concept lies in the immediate availability of end results: data are processed and analyzed upon loading the raw data file, allowing the user to immediately quality control the results. Once completed, the user can automatically report those results to data repositories for corporate access and quickly generate printed reports or documents. The software module has resulted in a very efficient and effective workflow through saved time and improved quality control. We discuss these benefits and show how this process defines a new benchmark in the drug discovery industry for the handling, interpretation, visualization, and sharing of SPR data.

An improved model for fragment-based lead generation at AstraZeneca.

Modest success rates in fragment-based lead generation (FBLG) projects at AstraZeneca (AZ) prompted operational changes to improve performance. In this review, we summarize these changes, emphasizing the construction and composition of the AZ fragment library, screening practices and working model. We describe the profiles of the screening method for specific fragment subsets and statistically assess our ability to follow up on fragment hits through near-neighbor selection. Performance analysis of our second-generation fragment library (FL2) in screening campaigns illustrates the complementary nature of flat and 3D fragments in exploring protein-binding pockets and highlights our ability to deliver fragment hits using multiple screening techniques for various target classes. The new model has had profound impact on the successful delivery of lead series to drug discovery projects.

Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.

Discovery and characterization of MAPK-activated protein kinase-2 prevention of activation inhibitors.

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.

HTS followed by NMR based counterscreening. Discovery and optimization of pyrimidones as reversible and competitive inhibitors of xanthine oxidase.

The identification of novel, non-purine based inhibitors of xanthine oxidase is described. After a high-throughput screening campaign, an NMR based counterscreen was used to distinguish actives, which interact with XO in a reversible manner, from assay artefacts. This approach identified pyrimidone 1 as a reversible and competitive inhibitor with good lead-like properties. A hit to lead campaign gave compound 41, a nanomolar inhibitor of hXO with efficacy in the hyperuricemic rat model after oral dosing.

Non-covalent inhibitors of rhinovirus 3C protease.

The first known non-covalent inhibitors of rhinovirus 3C protease (3CP) have been identified through fragment based screening and hit identification activities.

Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput.

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.