Fragment-based Drug Discovery Research Articles

HT-SuMD: making molecular dynamics simulations suitable for fragment-based screening. A comparative study with NMR

Fragment-based lead discovery (FBLD) is one of the most efficient methods to develop new drugs. We present here a new computational protocol called High-Throughput Supervised Molecular Dynamics (HT-SuMD), which makes it possible to automatically screen up to thousands of fragments, representing therefore a new valuable resource to prioritise fragments in FBLD campaigns. The protocol was applied to Bcl-XL, an oncological protein target involved in the regulation of apoptosis through protein–protein interactions. Initially, HT-SuMD performances were validated against a robust NMR-based screening, using the same set of 100 fragments. These independent results showed a remarkable agreement between the two methods. Then, a virtual screening on a larger library of additional 300 fragments was carried out and the best hits were validated by NMR. Remarkably, all the in silico selected fragments were confirmed as Bcl-XL binders. This represents, to date, the largest computational fragments screening entirely based on MD.

Comparing Fragment Binding Poses Prediction Using HSP90 as a Key Study: When Bound Water Makes the Difference

Fragment-Based Drug Discovery (FBDD) approaches have gained popularitynot only in industry but also in academic research institutes. However, the computational prediction of the binding mode adopted by fragment-like molecules within a protein binding site is still a very challenging task. One of the most crucial aspects of fragment binding is related to the large amounts of bound waters in the targeted binding pocket. The binding affinity of fragmentsmay not be sufficientto displace the bound water molecules. In the present work, we confirmed the importance of the bound water molecules in the correct prediction of the fragment binding mode. Moreover, we investigate whether the use of methods based on explicit solvent molecular dynamics simulations can improve the accuracy of fragment posing. The protein chosen for this study is HSP-90.

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

Fragment-based lead discovery of a novel class of small molecule antagonists of neuropeptide B/W receptor subtype 1 (GPR7)

Here, we report the discovery of a new class of NPBWR1 antagonists identified from a fragment-based screen. Compound 1 (cAMP IC50 = 250 µM; LE = 0.29) emerged as an initial hit. Further optimization of 1 by SAR-by-catalogue and chemical modification produced 21a (cAMP IC50 = 30 nM; LE = 0.39) with a 6700-fold increase in potency from fragment 1. Somewhat surprisingly, Schild analysis of compound 21a suggested that in vitro inhibition of NPW-mediated effects on upon cAMP accumulation were saturable, and that compound 21a dose-dependently increased [125I]-hNPW23 dissociation rate constants from NPBWR1 in kinetic binding studies. Collectively, these data are inconsistent with a classic surmountable, orthosteric mechanism of inhibition. The benzimidazole inhibitors reported herein may therefore represent a mechanistically differentiated class of compounds with which to form a better appreciation of the pharmacology and physiological roles of this central neuropeptide system.

Targeting the Initiator Protease of the Classical Pathway of Complement Using Fragment-Based Drug Discovery.

The initiating protease of the complement classical pathway, C1r, represents an upstream and pathway-specific intervention point for complement-related autoimmune and inflammatory diseases. Yet, C1r-targeted therapeutic development is currently underrepresented relative to other complement targets. In this study, we developed a fragment-based drug discovery approach using surface plasmon resonance (SPR) and molecular modeling to identify and characterize novel C1r-binding small-molecule fragments. SPR was used to screen a 2000-compound fragment library for binding to human C1r. This led to the identification of 24 compounds that bound C1r with equilibrium dissociation constants ranging between 160–1700 µM. Two fragments, termed CMP-1611 and CMP-1696, directly inhibited classical pathway-specific complement activation in a dose-dependent manner. CMP-1611 was selective for classical pathway inhibition, while CMP-1696 also blocked the lectin pathway but not the alternative pathway. Direct binding experiments mapped the CMP-1696 binding site to the serine protease domain of C1r and molecular docking and molecular dynamics studies, combined with C1r autoactivation assays, suggest that CMP-1696 binds within the C1r active site. The group of structurally distinct fragments identified here, along with the structure–activity relationship profiling of two lead fragments, form the basis for future development of novel high-affinity C1r-binding, classical pathway-specific, small-molecule complement inhibitors.

The QSAR Paradigm in Fragment-Based Drug Discovery: From the Virtual Generation of Target Inhibitors to Multi-Scale Modeling.

Fragment-Based Drug Design (FBDD) has established itself as a promising approach in modern drug discovery, accelerating and improving lead optimization, while playing a crucial role in diminishing the high attrition rates at all stages in the drug development process. On the other hand, FBDD has benefited from the application of computational methodologies, where the models derived from the Quantitative Structure-Activity Relationships (QSAR) have become consolidated tools. This mini-review focuses on the evolution and main applications of the QSAR paradigm in the context of FBDD in the last five years. This report places particular emphasis on the QSAR models derived from fragment-based topological approaches to extract physicochemical and/or structural information, allowing to design potentially novel mono- or multi-target inhibitors from relatively large and heterogeneous databases. Here, we also discuss the need to apply multi-scale modeling, to exemplify how different datasets based on target inhibition can be simultaneously integrated and predicted together with other relevant endpoints such as the biological activity against non-biomolecular targets, as well as in vitro and in vivo toxicity and pharmacokinetic properties. In this context, seminal papers are briefly analyzed. As huge amounts of data continue to accumulate in the domains of the chemical, biological and biomedical sciences, it has become clear that drug discovery must be viewed as a multi-scale optimization process. An ideal multi-scale approach should integrate diverse chemical and biological data and also serve as a knowledge generator, enabling the design of potentially optimal chemicals that may become therapeutic agents.

Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains.

As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed 19F (PrOF) NMR and 1H CPMG NMR are two fragment screening assays that have complementary advantages. Here, we sought to combine these two NMR-based assays into a new screening workflow. This combination of protein- and ligand-observed experiments allows for a time- and resource-efficient multiplexed screen of mixtures of fragments and proteins. PrOF NMR is first used to screen mixtures against two proteins. Hit mixtures for each protein are identified then deconvoluted using 1H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and Plasmodium falciparum (Pf) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, PfGCN5, and fragments binding both proteins, respectively. Select hits were characterized, revealing a broad range of affinities from low µM to mM dissociation constants. Follow-up experiments supported a low-affinity second binding site on PfGCN5. This approach can be used to bias fragment screens towards more selective hits at the onset of inhibitor development in a resource- and time-efficient manner.

Abstract 3030: Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity

Abstract The MAPK pathway is frequently dysregulated in cancer, resulting in constitutive phosphorylation and activation of its downstream signaling node ERK1/2 (ERK). The direct targeting of activated ERK is being explored in several phase I clinical trials. A good understanding of the relationship between inhibition of pathway activity and inhibition of tumor growth allows dose scheduling in the clinic to maximise response to an inhibitor. We have recently described the development of a novel, potent and selective small molecule inhibitor of both ERK activity and phosphorylation using fragment-based drug discovery. Using this inhibitor as a tool compound, we explored the relationship between duration of ERK inhibition and inhibition of tumor growth. We examined the in vivo activity of our ERK inhibitor in a Colo205 xenograft model. Inhibition of the ERK pathway was monitored by measuring phosphorylation of a direct substrate of ERK, RSK90 (pRSK), and phosphorylated ERK itself (pERK) to determine pharmacodynamic profiles. We observed that pathway inhibition was consistent with the pharmacokinetic profile of the ERK inhibitor and that the duration of pathway inhibition was primarily determined by dosing frequency rather than the total dose of ERK inhibitor. High, single doses (50 or 40 mg/kg) of ERK inhibitor produce strong maximal pathway inhibition at 2 h post dose (pRSK levels 8% of control levels for a single dose of 40 mg/kg) but pRSK levels have returned to levels close to control values by 12 h post dose. In contrast, 3x equally spaced doses of 20 mg/kg have a lower maximal pathway inhibition at 2 h post dose (pRSK levels 29% of control) but extend pathway inhibition beyond 12 h (pRSK levels 21% of control at 14 h). Despite these two different pharmacodynamic profiles, we observed comparable tumor growth inhibition following daily dosing either at 1 × 50 mg/kg (maximal tumor growth inhibition of 21%) or 3 × 20 mg/kg (maximal tumor growth inhibition of 15%). These data suggest that a short duration of ERK inhibition rather than constant suppression can be sufficient to provide anti-tumor activity. This may allow flexibility in clinical dosing schedules to improve tolerability without a loss in efficacy. Citation Format: Christopher Hindley, Luke Bevan, Hannah Braithwaite, Aurélie Courtin, Lynsey Fazal, Roberta Ferraldeschi, Vanessa Martins, Sandra Muench, Nicola Wallis, Nicola Wilsher, John Lyons, Joanne Munck. Different pharmacodynamic profiles of ERK1/2 inhibition can elicit comparable anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3030.

Abstract 1039: Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2

Abstract The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Recent advances have shown that genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signalling and inhibits proliferation of RTK-driven cancer cell lines. SHP2 is now understood to act upstream of RAS and plays a role in KRAS-driven cancers, an area of research which is rapidly growing. Considering that RTK deregulation often leads to a wide range of cancers and the newly appreciated role of SHP2 in KRAS-driven cancers, SHP2 inhibitors are therefore a promising therapeutic approach. SHP2 contains two N-terminal tandem SH2 domains (N-SH2, C-SH2), a catalytic phosphatase domain and a C-terminal tail. SHP2 switches between “open” active and “closed” inactive forms due to autoinhibitory interactions between the N-SH2 domain and the phosphatase domain. Historically, phosphatases were deemed undruggable as there had been no advancements with active site inhibitors. We hypothesised that fragment screening would be highly applicable and amenable to this target to enable alternative means of inhibition through identification of allosteric binding sites. Here we describe the first reported fragment screen against SHP2. Using our fragment-based PyramidTM approach, screening was carried out on two constructs of SHP2; a closed autoinhibited C-terminal truncated form (phosphatase and both SH2 domains), as well as the phosphatase-only domain. A combination of screening methods such as X-ray crystallography and NMR were employed to identify fragment hits at multiple sites on SHP2, including the tunnel-like allosteric site reported by Chen et al, 2016. Initial fragment hits had affinities for SHP2 in the range of 1mM as measured by ITC. Binding of these hits was improved using structure-guided design to generate compounds which inhibit SHP2 phosphatase activity and are promising starting points for further optimization. Citation Format: Philip J. Day, Valerio Berdini, Juan Castro, Gianni Chessari, Thomas G. Davies, James E. Day, Satoshi Fukaya, Chris Hamlett, Keisha Hearn, Steve Hiscock, Rhian Holvey, Satoru Ito, Yasuo Kodama, Kenichi Matsuo, Yoko Nakatsuru, Nick Palmer, Amanda Price, Tadashi Shimamura, Jeffrey D.St. Denis, Nicola G. Wallis, Glyn Williams, Christopher N. Johnson. Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1039.

Application of Fragment-Based Drug Discovery to Versatile Targets.

Fragment-based drug discovery (FBDD) is a powerful method to develop potent small-molecule compounds starting from fragments binding weakly to targets. As FBDD exhibits several advantages over high-throughput screening campaigns, it becomes an attractive strategy in target-based drug discovery. Many potent compounds/inhibitors of diverse targets have been developed using this approach. Methods used in fragment screening and understanding fragment-binding modes are critical in FBDD. This review elucidates fragment libraries, methods utilized in fragment identification/confirmation, strategies applied in growing the identified fragments into drug-like lead compounds, and applications of FBDD to different targets. As FBDD can be readily carried out through different biophysical and computer-based methods, it will play more important roles in drug discovery.

Astex's journey of fragment-based drug discovery using cryo-EM

Astex's journey of fragment-based drug discovery using cryo-EM

Prospecting for new catechol-O-methyltransferase (COMT) inhibitors as a potential treatment for Parkinson’s disease: a study by molecular dynamics and structure-based virtual screening

Parkinson’s disease (PD) is a neurodegenerative, chronic, and progressive disease, common in the elderly. The catechol-O-methyltransferase (COMT) is a monomeric enzyme involved in dopamine (DA) degradation, the neurotransmitter in deficit in patients with PD. The reference treatment of PD consists of levodopa (L-dopa) administration, which is the precursor of DA. The inhibition of COMT is an adjuvant treatment in PD since it keeps DA levels constant. The goal of this study was to identify drug candidates capable of inhibiting COMT for the treatment of PD and identify important fragments of these molecules. Initially, we analyzed the flexibility of COMT and defined its main conformations in solution regarding the absence (system I) and presence of the S-adenosyl-L-methionine (SAM) cofactor (system II) through molecular dynamics (MD) simulations. Two regions in these structures were selected for molecular docking, firstly the entire cavity where the cofactor and substrates are bound and secondly the specific biding region of the enzyme substrates. Based on the conformations of the MD, the virtual screening (VS) was performed against FDA Approved and Zinc Natural Products databases aiming at the selection of the best compounds. Subsequently, the absorption, distribution, metabolization, excretion, and toxicity (ADMET) properties, as well as drug-score and drug-likeness indexes of the most promising compounds were analyzed. After a detailed analysis of the compounds selected by structure-based VS, it was possible to highlight the fragments most frequently involved in their stability: 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, 9H-Benz(c)indole(3,2,1-ij)(1,5)naphthyridin-9-one and (10R,13S)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17dodecahydrocyclopenta[a]phenanthren-3-one. The identification of these potential fragments is essential for the prospection of more specific inhibitors against COMT using the technique of Fragment-based lead discovery (FBLD). Besides, this study allowed us to identify the potential COMT inhibitors through a complete understanding of molecular-level interactions based on the flexibility of this protein. Communicated by Ramaswamy H. Sarma

Library Design Strategies To Accelerate Fragment-Based Drug Discovery.

Fragment-based drug discovery (FBDD) has become an established approach for the generation of early lead candidates. However, despite its success and inherent advantages, hit-to-candidate progression for FBDD is not necessarily faster than that of traditional high-throughput screening. Thus, new technology-driven library design strategies have emerged as a means to facilitate more efficient fragment screening and/or subsequent fragment-to-hit chemistry. This minireview discusses such strategies, which cover the use of labeled fragments for NMR spectroscopy, X-ray crystallographic screening of specialized fragments, covalent linkage for mass spectrometry, dynamic combinatorial chemistry, and fragments optimized for easy elaboration.

CcpNmr AnalysisScreen, a new software programme with dedicated automated analysis tools for fragment-based drug discovery by NMR

Fragment-based drug discovery or FBDD is one of the main methods used by industry and academia for identifying drug-like candidates in early stages of drug discovery. NMR has a significant impact at any stage of the drug discovery process, from primary identification of small molecules to the elucidation of binding modes for guiding optimisations. The essence of NMR as an analytical tool, however, requires the processing and analysis of relatively large amounts of single data items, e.g. spectra, which can be daunting when managed manually. One bottleneck in FBDD by NMR is a lack of adequate and well-integrated resources for NMR data analysis that are freely available to the community. Thus, scientists typically resort to manually inspecting large datasets and relying predominantly on subjective interpretations. In this manuscript, we present CcpNmr AnalysisScreen, a software package that provides computational tools for automated analysis of FBDD data by NMR. We outline how the quality of collected spectra can be evaluated quickly, and how robust workflows can be optimised for reliable and rapid hit identification. With an intuitive graphical user interface and powerful algorithms, AnalysisScreen enables easy analysis of the large datasets needed in the early process of drug discovery by NMR.

FBLD2012（Fragment-based Lead Discovery Conference 2012）参加報告

FBLD2012（Fragment-based Lead Discovery Conference 2012）参加報告

Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M.tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.

Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband 19 F NMR-Based Screening.

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

Fragment Linking Strategies for Structure-Based Drug Design.

Fragment-based drug discovery is a strategy widely used in both academia and pharmaceutical companies to generate small-molecule protein inhibitors and drug candidates. Among the approaches reported in the literature (growing, linking, and merging), the linking approach theoretically offers the opportunity to rapidly gain in binding energy. Nevertheless, this approach is poorly represented when considering the compounds currently in clinical trials. Here, we report an exhaustive view of the cases published so far in the literature, together with the methods used to identify the two initial fragments either simultaneously or successively. We review the different types of linkers published and discuss how these linkers are designed to obtain the lead compound. Mixing merging and linking methods, where the linker is duplicated from a known inhibitor, appears as an interesting strategy. To reach superadditivity, we propose to grow one of the fragments in order to minimize the distance between the two binders and then link the resulting compounds using flexible alkyl-derived linkers.

Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.