Structure-Guided Discovery of Selective USP7 Inhibitors with In Vivo Activity.

Inhibition of ubiquitin-specific protease 7, USP7, has been proposed as a mechanism to affect many disease processes, primarily those implicated in oncology. The bound crystal structure of a published high-throughput screening hit with low-micromolar affinity for USP7 identified three regions of the compound for structure-guided optimization. Replacing one side of the compound with different aromatic moieties gave little improvement in affinity, and the central piperidine could not be improved.

Identification of a Novel Subtype-Selective α-Adrenoceptor Antagonist.

α-, α-, and α-adrenoceptors (α-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease.

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the for ATP.

Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors.

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery.  However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits.

Fragment Merging Using a Graph Database Samples Different Catalogue Space than Similarity Search.

Fragment merging is a promising approach to progressing fragments directly to on-scale potency: each designed compound incorporates the structural motifs of overlapping fragments in a way that ensures compounds recapitulate multiple high-quality interactions. Searching commercial catalogues provides one useful way to quickly and cheaply identify such merges and circumvents the challenge of synthetic accessibility, provided they can be readily identified. Here, we demonstrate that the Fragment Network, a graph database that provides a novel way to explore the chemical space surrounding fragment hits, is well-suited to this challenge.

Exploration of piperidine 3D fragment chemical space: synthesis and 3D shape analysis of fragments derived from 20 regio- and diastereoisomers of methyl substituted pipecolinates.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we report the synthesis of piperidine-based 3D fragment building blocks - 20 regio- and diastereoisomers of methyl substituted pipecolinates using simple and general synthetic methods.

PAC-FragmentDEL - photoactivated covalent capture of DNA-encoded fragments for hit discovery.

We describe a novel approach for screening fragments against a protein that combines the sensitivity of DNA-encoded library technology with the ability of fragments to explore what will bind. Each of the members of the library consists of a fragment which is linked to a photoactivatable diazirine moiety. Split and pool synthesis combines each fragment with a set of linkers with the version of the library reported here containing some 70k different compounds, each with an individual DNA code.

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons.

The Effect of Core Replacement on S64315, a Selective MCL-1 Inhibitor, and Its Analogues.

Following the identification of thieno[2,3-]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned.

Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit.

Structure-Guided Discovery of Potent and Selective DYRK1A Inhibitors.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors.

Fragment-Based Discovery of Novel Non-Hydroxamate LpxC Inhibitors with Antibacterial Activity.

UDP-3--acyl--acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation.

Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1.

Fragment-derived modulators of an industrial β-glucosidase.

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity.

Rapid optimisation of fragments and hits to lead compounds from screening of crude reaction mixtures.

Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product.

Exploring IDP-Ligand Interactions: tau K18 as A Test Case.

Over the past decade intrinsically disordered proteins (IDPs) have emerged as a biologically important class of proteins, many of which are of therapeutic relevance. Here, we investigated the interactions between a model IDP system, tau K18, and nine literature compounds that have been reported as having an effect on tau in order to identify a robust IDP-ligand system for the optimization of a range of biophysical methods. We used NMR, surface plasmon resonance (SPR) and microscale thermophoresis (MST) methods to investigate the binding of these compounds to tau K18; only one showed unambiguous interaction with tau K18.

Correction to: H, C, N backbone and IVL methyl group resonance assignment of the fungal β-glucosidase from Trichoderma reesei.

In the original publication of the article, the name of one of the authors is incorrect. The author's name is Eiso AB, but was modified to A. B.

H, C, N backbone and IVL methyl group resonance assignment of the fungal β-glucosidase from Trichoderma reesei.

β-glucosidases have received considerable attention due to their essential role in bioethanol production from lignocellulosic biomass. β-glucosidase can hydrolyse cellobiose in cellulose degradation and its low activity has been considered as one of the main limiting steps in the process. Large-scale conversions of cellulose therefore require high enzyme concentration which increases the cost.

Design and Synthesis of 56 Shape-Diverse 3D Fragments.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we describe a workflow for the design and synthesis of 56 3D disubstituted pyrrolidine and piperidine fragments that occupy under-represented areas of fragment space (as demonstrated by a principal moments of inertia (PMI) analysis).

Water Networks Can Determine the Affinity of Ligand Binding to Proteins.

Solvent organization is a key but underexploited contributor to the thermodynamics of protein-ligand recognition, with implications for ligand discovery, drug resistance, and protein engineering. Here, we explore the contribution of solvent to ligand binding in the virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity.

Establishing Drug Discovery and Identification of Hit Series for the Anti-apoptotic Proteins, Bcl-2 and Mcl-1.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods.

Structure-Guided Discovery of a Selective Mcl-1 Inhibitor with Cellular Activity.

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when observed in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small molecule inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using nuclear magnetic resonance and X-ray-derived structural information.

1D NMR WaterLOGSY as an efficient method for fragment-based lead discovery.

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-x, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available.

Predicting how drug molecules bind to their protein targets.

There have been substantial advances in the application of molecular modelling and simulation to drug discovery in recent years, as massive increases in computer power are coupled with continued development in the underlying methods and understanding of how to apply them. Here, we survey recent advances in one particular area-predicting how a known ligand binds to a particular protein. We focus on the four contributing classes of calculation: predicting where a binding site is on a protein; characterizing where chemical functional groups will bind to that site; molecular docking to generate a binding mode for a ligand and dynamics simulations to refine that pose and allow for protein conformation change.

Using Fragment-Based Approaches to Discover New Antibiotics.

Fragment-based lead discovery has emerged over the past two decades as a successful approach to generate novel lead candidates in drug discovery programs. The two main advantages over conventional high-throughput screening (HTS) are more efficient sampling of chemical space and tighter control over the physicochemical properties of the lead candidates. Antibiotics are a class of drugs with particularly strict property requirements for efficacy and safety.