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. We use an iteration of the database containing >120 million catalogue compounds to find fragment merges for four crystallographic screening campaigns and contrast the results with a traditional fingerprint-based similarity search. The two approaches identify complementary sets of merges that recapitulate the observed fragment-protein interactions but lie in different regions of chemical space. We further show our methodology is an effective route to achieving on-scale potency by retrospective analyses for two different targets; in analyses of public COVID Moonshot and Mycobacterium tuberculosis EthR inhibitors, potential inhibitors with micromolar IC50 values were identified. This work demonstrates the use of the Fragment Network to increase the yield of fragment merges beyond that of a classical catalogue search.

Fragment-derived modulators of an industrial ß-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. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.

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). A key, and unique, underpinning design feature of this fragment collection is that assessment of fragment shape and conformational diversity (by considering conformations up to 1.5 kcal mol-1 above the energy of the global minimum energy conformer) is carried out prior to synthesis and is also used to select targets for synthesis. The 3D fragments were designed to contain suitable synthetic handles for future fragment elaboration. Finally, by comparing our 3D fragments with six commercial libraries, it is clear that our collection has high three-dimensionality and shape diversity.

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. Several near neighbors of this compound were synthesized and their interactions with tau K18 characterized using additional NMR methods, including 1D ligand-observed NMR, diffusion-ordered spectroscopy (DOSY) and 19F NMR. This study demonstrates that it is possible to detect and characterize IDP-ligand interactions using biophysical methods. However, care must be taken to account for possible artefacts, particularly the impact of compound solubility and where the protein has to be immobilized.

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 Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavorable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP's enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand, or both. This suggests that solvent structure is an evolutionary constraint on protein sequence that contributes to ligand affinity and selectivity.

Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.

rDock: a fast, versatile and open source program for docking ligands to proteins and nucleic acids.

Identification of chemical compounds with specific biological activities is an important step in both chemical biology and drug discovery. When the structure of the intended target is available, one approach is to use molecular docking programs to assess the chemical complementarity of small molecules with the target; such calculations provide a qualitative measure of affinity that can be used in virtual screening (VS) to rank order a list of compounds according to their potential to be active. rDock is a molecular docking program developed at Vernalis for high-throughput VS (HTVS) applications. Evolved from RiboDock, the program can be used against proteins and nucleic acids, is designed to be computationally very efficient and allows the user to incorporate additional constraints and information as a bias to guide docking. This article provides an overview of the program structure and features and compares rDock to two reference programs, AutoDock Vina (open source) and Schrödinger's Glide (commercial). In terms of computational speed for VS, rDock is faster than Vina and comparable to Glide. For binding mode prediction, rDock and Vina are superior to Glide. The VS performance of rDock is significantly better than Vina, but inferior to Glide for most systems unless pharmacophore constraints are used; in that case rDock and Glide are of equal performance. The program is released under the Lesser General Public License and is freely available for download, together with the manuals, example files and the complete test sets, at http://rdock.sourceforge.net/

Lead-oriented synthesis: Investigation of organolithium-mediated routes to 3-D scaffolds and 3-D shape analysis of a virtual lead-like library.

Synthetic routes to six 3-D scaffolds containing piperazine, pyrrolidine and piperidine cores have been developed. The synthetic methodology focused on the use of N-Boc α-lithiation-trapping chemistry. Notably, suitably protected and/or functionalised medicinal chemistry building blocks were synthesised via concise, connective methodology. This represents a rare example of lead-oriented synthesis. A virtual library of 190 compounds was then enumerated from the six scaffolds. Of these, 92 compounds (48%) fit the lead-like criteria of: (i) -1⩽AlogP⩽3; (ii) 14⩽number of heavy atoms⩽26; (iii) total polar surface area⩾50Å(2). The 3-D shapes of the 190 compounds were analysed using a triangular plot of normalised principal moments of inertia (PMI). From this, 46 compounds were identified which had lead-like properties and possessed 3-D shapes in under-represented areas of pharmaceutical space. Thus, the PMI analysis of the 190 member virtual library showed that whilst scaffolds which may appear on paper to be 3-D in shape, only 24% of the compounds actually had 3-D structures in the more interesting areas of 3-D drug space.

Probing linker design in citric acid-ciprofloxacin conjugates.

A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.

Discovery of selective small-molecule activators of a bacterial glycoside hydrolase.

Fragment-based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small-molecule enzyme activators. Herein, we describe the discovery and characterization of small-molecule activators of a glycoside hydrolase (a bacterial O-GlcNAc hydrolase). A ligand-observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in kcat /KM  values (kcat =catalytic rate constant; KM =Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both KM  and Vmax  values (Vmax =maximum rate of reaction). The compounds appear to stabilize an active "closed" form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.

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. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B  (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau  (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-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 α1-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. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

Fragment screening by weak affinity chromatography: comparison with established techniques for screening against HSP90.

The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening. The study presented here compares screening of 111 fragments against the ATPase domain of HSP90 by all of these methods, with isothermal titration calorimetry (ITC) used to confirm the most potent hits. The study demonstrates that WAC is comparable to the established methods of ligand-based NMR and SPR as a hit-id method, with hit correlations of 88% and 83%, respectively. The stability of HSP90 WAC columns was also evaluated and found to give 90% reproducibility even after 207 days of storage. A good correlation was obtained between the various technologies, validating WAC as an effective technology for fragment screening.

MTSA--a Matlab program to fit thermal shift data.

Thermal shift analysis is becoming widely used as a method to identify initial hit ligands for inhibitor discovery or to identify ligands that may aid crystallization. The data analysis software provided by the equipment manufacturers or in the public domain is cumbersome to use. We have assessed a number of different approaches to generate a value for the melting temperature (T(m)) and implemented these methods in the program MTSA within the commercial software Matlab to provide an easy-to-use and rapid way to process experimental thermal shift data. The program outputs the T(m), the quality of the fit, and the deviation from a standard value, the thermal shift ΔT(m). Our analysis of these results includes a discussion of some issues with previous publications in this area. We conclude that the most suitable value for T(m) should be taken from the midpoint determined for a curve fitted to the experimental data with a five-parameter equation. In addition, we found that different ranking of ligand binding can be obtained using the different techniques when screening for binding of weak ligands such as fragments. Therefore, the technique should be used with caution for such screening.

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 Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

Hsp90 inhibitors and drugs from fragment and virtual screening.

We have previously reported the structure-based optimisation of a number of series of potent compounds progressed as clinical candidates for oncology through inhibition of the ATPase activity of the molecular chaperone, Hsp90. The starting point for these candidates was compounds discovered using a combination of structure-based hit identification methods. This chapter summarises the overall story of how these methods were applied. Virtual screening of commercially available compounds identified a number of classes of compounds. At the same time, an initial fragment screen identified 17 fragments of various classes that bound to the N-terminal domain of Hsp90 with weak (0.5-10 mM) affinity. A subsequent screen identified a total of 60 compounds. This collection of fragments and virtual screening hits were progressed in a number of ways. Although two fragments could be observed binding together in the active site, the synthetic effort required to link these fragments was judged too high. For the resorcinol class of fragments, limited library synthesis generated compounds in the 1-10 µM range. In addition, the resorcinol substructure was used to select commercially available compounds that were filtered using focussed docking in the Hsp90 active site to select further sets of compounds for assay. This identified structural motifs that were exploited during lead optimisation to generate AUY922, currently in Phase II clinical trials. In a separate campaign, features identified in the structures of fragments, evolved fragments and virtual screening hits bound to Hsp90 were combined to generate an oral series of compounds, progressed to preclinical candidates. The crystal structures were determined of many of the fragments bound to Hsp90 and provide examples of both maintenance and change of protein conformation on fragment binding. Finally, we analyse the extent to which our initial set of fragments recapitulates the key structural features of the Hsp90 inhibitors published to date.

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. cis-Piperidines, accessed through a pyridine hydrogenation were transformed into their trans-diastereoisomers using conformational control and unified reaction conditions. Additionally, diastereoselective lithiation/trapping was utilised to access trans-piperidines. Analysis of a virtual library of fragments derived from the 20 cis- and trans-disubstituted piperidines showed that it consisted of 3D molecules with suitable molecular properties to be used in fragment-based drug discovery programs.

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. Incubation of the library with a protein sample is followed by photoactivation, washing and subsequent PCR and sequencing which allows the individual fragment hits to be identified. We illustrate how the approach allows successful hit fragment identification using only microgram quantities of material for two targets. PAK4 is a kinase for which conventional fragment screening has generated many advance leads. The as yet undrugged target, 2-epimerase, presents a more challenging active site for identification of hit compounds. In both cases, PAC-FragmentDEL identified fragments validated as hits by ligand-observed NMR measurements and crystal structure determination of off-DNA sample binding to the proteins.

Comparative assessment of different histidine-tags for immobilization of protein onto surface plasmon resonance sensorchips.

Surface plasmon resonance (SPR) is widely used to assess the kinetics and thermodynamics of binding of two molecules. The major challenge is immobilization of one molecule onto the sensorchip for robust detection of binding of the other molecule. We have compared a number of immobilization strategies for noncovalent attachment of an example protein (the substrate binding protein SiaP) by hexa-histidine (His), deca-His, and double-His tags to a nickel-nitrilotriacetic acid (NTA) surface. The stability of immobilization was assessed, and the binding of two low molecular weight ligands, Neu5Ac and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), at different temperatures studied. The hexa-His tagged SiaP washed off from the surface too rapidly for ligand binding to be measured reliably. Systematic variation of chip loading identified conditions under which the deca-His tagged SiaP could generate reliable results. The double-His tagged protein performed as well as covalently attached deca-His tagged protein at 15, 25, and 35 °C. The observed ligand binding kinetics were comparable for all immobilization strategies, and thermodynamic values calculated from SPR are in agreement with solution-based isothermal titration calorimetry measurements. Extended trials suggest that covalent attachment is preferable for screening campaigns, whereas the double-His-tag strategy allows rapid regeneration of the chip, for example, when tight binding compounds are assessed.

Predicting fragment binding poses using a combined MCSS MM-GBSA approach.

Improved methods are required to predict the position and orientation (pose) of binding to the target protein of low molecular weight compounds identified in fragment screening campaigns. This is particularly important to guide initial chemistry to generate structure-activity relationships for the cases where a high resolution structure cannot be obtained. We have assessed the benefit of an implicit solvent method for assessment of fragment binding poses generated by the Multiple Copy Simultaneous Search (MCSS) method in CHARMm. Additionally, the effect of using multiple receptor structures for a flexible receptor is investigated. The original MCSS performance -50% of fragment positions accurately predicted and scored - was increased up to 67% by scoring MCSS energy minima with a Molecular Mechanics Generalized Born approach with molecular volume integration and Surface Area model (MM-GBSA). The same increase in performance (but occasionally for different targets) was observed when using the docking program GOLD followed by MM-GBSA rescoring. The combined results from both methods resulted in a higher success rate emphasizing that a comparison of different docking methods can increase the correct identification of binding poses. For a receptor where multiple structures are available, Hsp90, the average performance on randomly adding receptor structures was also investigated. The results suggest that predictions using these docking methods can be used with some confidence to guide chemical optimization, if the structure of the target either remains relatively fixed on ligand binding, or if a number of crystal structures are available with diverse ligands bound and there is information on the positions of key water molecules in the binding site.