Crystal structure of the EphA4 protein tyrosine kinase domain in the apo- and dasatinib-bound state

AbstractThe Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors

Fragment-to-Lead Medicinal Chemistry Publications in 2018

This Perspective, the fourth in an annual series, summarizes fragment-to-lead (F2L) success stories published during 2018. Topics such as target class, screening methods, physicochemical properties, and ligand efficiency are discussed for the 2018 examples as well as for the combined 111 F2L examples covering 2015-2018. While the overall properties of fragments and leads have remained constant, a number of new trends are noted, for example, broadening of target class coverage and application of FBDD to covalent inhibitors. Moreover, several studies make use of fragment hits that were previously described in the literature, illustrating that fragments are versatile starting points that can be optimized to structurally diverse leads. By focusing on success stories, the hope is that this Perspective will identify and inform best practices in fragment-based drug discovery.</p

Escape from planarity in fragment-based drug discovery : A synthetic strategy analysis of synthetic 3D fragment libraries

In fragment-based drug discovery (FBDD), there is a developing appreciation that 3D fragments could offer opportunities that are not provided by 2D fragments. This review provides an overview of the synthetic strategies that have been used to prepare 3D fragments, as discussed in 25 papers published from 2011 to mid-May 2020. Three distinct strategies are highlighted: (i) diversity-oriented synthesis; (ii) the synthesis and diversification of scaffolds; and (iii) computational design and synthesis (where 3D fragments were computationally enumerated and filtered on the basis of computationally generated 3D shape descriptors and other properties). We conclude that a workflow that combines computational design and one other strategy, together with a consideration of fragment properties, 3D shape and ‘fragment sociability’, could allow 3D fragments to feature more widely in fragment libraries and could facilitate fragment-to-lead optimisation

When fragments link : a bibliometric perspective on the development of fragment-based drug discovery

Fragment-based drug discovery (FBDD) is a highly interdisciplinary field, rich in ideas integrated from pharmaceutical sciences, chemistry, biology, and physics, among others. To enrich our understanding of the development of the field, we used bibliometric techniques to analyze 3642 publications in FBDD, complementing accounts by key practitioners. Mapping its core papers, we found the transfer of knowledge from academia to industry. Co-authorship analysis showed that university–industry collaboration has grown over time. Moreover, we show how ideas from other scientific disciplines have been integrated into the FBDD paradigm. Keyword analysis showed that the field is organized into four interconnected practices: library design, fragment screening, computational methods, and optimization. This study highlights the importance of interactions among various individuals and institutions from diverse disciplines in newly emerging scientific fields. We study the organizational aspects of the development of fragment-based drug discovery (FBDD), using tools from bibliometrics

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity

Screening of a PDE-focused library identifies imidazoles with in vitro and in vivo antischistosomal activity

We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 μM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n = 72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro ‘hit’ NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days post-treatment. Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs

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

Phenyldihydropyrazolones as Novel Lead Compounds Against Trypanosoma cruzi

As over 6 million people are infected with Chagas disease and only limited therapeutic options are available, there is an urgent need for novel drugs. The involvement of cyclic nucleotide phosphodiesterases (PDE) in the lifecycle and biological fitness of a number of protozoan parasites has been described and several of these enzymes are thought to be viable drug targets. Within this context, a PDE-focused library was screened for its ability to affect the viability of Trypanosoma cruzi parasites. 5-(3-(Benzyloxy)-4-methoxyphenyl)-2-isopropyl-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (4), previously reported as a human PDE4 inhibitor, was identified as a hit. Upon optimization on three positions of the phenylpyrazolone scaffold, 2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (34) proved to be the most active compound against intracellular forms of T. cruzi (pIC50 = 6.4) with a 100-fold selectivity with respect to toxicity toward human MRC-5 cells. Evaluation on different life stages and clinically relevant T. cruzi strains revealed that the phenylpyrazolones are not active against the bloodstream form of the Y strain but show submicromolar activity against the intracellular form of the Y- and Tulahuen strains as well as against the nitro-drug-resistant Colombiana strain. In vitro screening of phenylpyrazolones against TcrPDEB1, TcrPDEC, and TcrCYP51 showed that there was a poor correlation between enzyme inhibition and the observed phenotypic effect. Among the most potent compounds, both TcrCYP51 and non-TcrCYP51 inhibitors are identified, which were both equally able to inhibit T. cruzi in vitro

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors (Part 2)

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action

In Silico Veritas: The Pitfalls and Challenges of Predicting

Recently the first community-wide assessments of the prediction of the structures of complexes between proteins and small molecule ligands have been reported in the so-called GPCR Dock 2008 and 2010 assessments. In the current review we discuss the different steps along the protein-ligand modeling workflow by critically analyzing the modeling strategies we used to predict the structures of protein-ligand complexes we submitted to the recent GPCR Dock 2010 challenge. These representative test cases, focusing on the pharmaceutically relevant G Protein-Coupled Receptors, are used to demonstrate the strengths and challenges of the different modeling methods. Our analysis indicates that the proper performance of the sequence alignment, introduction of structural adjustments guided by experimental data, and the usage of experimental data to identify protein-ligand interactions are critical steps in the protein-ligand modeling protocol. © 2011 by the authors; licensee MDPI, Basel, Switzerland