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This Perspective is the seventh in an annual series that summarizes successful Fragment-to-Lead (F2L) case studies published in a given year. A tabulated summary of relevant articles published in 2021 is provided, and features such as target class, screening methods, and ligand efficiency are discussed, both for the 2021 examples and for the combined examples over the years 2015-2021. In addition, trends and new developments in the field are summarized. In particular, the use of structural information in fragment-based drug discovery is discussed.

Fragment-based drug discovery (FBDD) continues to evolve and make an impact in the pharmaceutical sciences. We summarize successful fragment-to-lead studies that were published in 2020. Having systematically analyzed annual scientific outputs since 2015, we discuss trends and best practices in terms of fragment libraries, target proteins, screening technologies, hit-optimization strategies, and the properties of hit fragments and the leads resulting from them. As well as the tabulated Fragment-to-Lead (F2L) programs, our 2020 literature review identifies several trends and innovations that promise to further increase the success of FBDD. These include developing structurally novel screening fragments, improving fragment-screening technologies, using new computer-aided design and virtual screening approaches, and combining FBDD with other innovative drug-discovery technologies.

Soellner published on the interplay between allosteric and adenosine triphosphate (ATP)-competitive inhibitors of ABL kinase, showing that the latter preferably binds to different conformational states of ABL compared to allosteric agents that specifically target the ABL myristate pocket (STAMP) and deducing that asciminib cannot bind to ABL simultaneously with ATP-competitive drugs. These results are to some extent in line with ours, although our analyses of dose-response matrices from combinations of asciminib with imatinib, nilotinib or dasatinib, show neither synergy nor antagonism, but suggest additive antiproliferative effects on BCR-ABL-dependent KCL22 cells. Furthermore, our X-ray crystallographic, solution nuclear magnetic resonance (NMR), and isothermal titration calorimetry studies show that asciminib can bind ABL concomitantly with type-1 or -2 ATP-competitive inhibitors to form ternary complexes. Concomitant binding of asciminib with imatinib, nilotinib, or dasatinib might translate to benefit some chronic myeloid leukaemia patients.

The second biannual Alpine Winter Conference on Medicinal and Synthetic Chemistry (short: Alpine Winter Conference) took place January 19-23, 2020, in St. Anton in western Austria. There were roughly 180 attendees from around the globe, making this mid-sized conference particularly conducive to networking and exchanging ideas over the course of four and a half days. This report summarizes the key events and presentations given by researchers working in both industry and academia.

The pharmaceutical industry is highly reliant on researchers who not only possess the technical knowledge but also the professional skills to collaborate in drug development. To prepare future practitioners to thrive in this interdisciplinary environment, Innovative Training Networks (ITNs) have become increasingly important in doctoral training. In this piece, we explore the benefits of these ITNs in training future practitioners in drug discovery. Through a bibliometric review, we find that the top researchers in fragment-based drug discovery have a high degree of collaboration and mobility across institutes. We then investigate which aspects of the ITN training program enable PhD students to gain these skills. We find that secondments, the short-term stays that students have in partner research institutes, are useful in preparing students to have both broad knowledge of drug discovery and specialization in their field of interest. Aside from imparting technical skills, we find that the collaborative environment in ITNs enables students to communicate better and to work effectively in teams. Doctoral students benefit by being exposed to relevant experiences that they can later apply as they navigate through the complex web of relationships and competencies in the industry. We conclude by recommending best practices to further improve ITNs in the training of future practitioners.

FGF19 signaling through the FGFR4/β-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.

Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their poor pharmacokinetic properties, they are not well suited for antiparasitic therapy. Recently, an allosteric binding pocket was discovered on human FPPS, but its existence on trypanosomal FPPS was unclear. We applied NMR and X-ray fragment screening to T. brucei FPPS and report herein on four fragments bound to this previously unknown allosteric site. Surprisingly, non-bisphosphonate active-site binders were also identified. Moreover, fragment screening revealed a number of additional binding sites. In an early structure-activity relationship (SAR) study, an analogue of an active-site binder was unexpectedly shown to bind to the allosteric site. Overlaying identified fragment binders of a parallel T. cruzi FPPS fragment screen with the T. brucei FPPS structure, and medicinal chemistry optimisation based on two binders revealed another example of fragment "pocket hopping". The discovery of binders with new chemotypes sets the framework for developing advanced compounds with pharmacokinetic properties suitable for the treatment of parasitic infections by inhibition of FPPS in T. brucei parasites.

The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain.

The second biannual Alpine Winter Conference on Medicinal and Synthetic Chemistry (short: Alpine Winter Conference) took place January 19-23, 2020, in St. Anton in western Austria. There were roughly 180 attendees from around the globe, making this mid-sized conference particularly conducive to networking and exchanging ideas over the course of four and a half days. This report summarizes the key events and presentations given by researchers working in both industry and academia.

This Miniperspective is the third in a series reviewing fragment-to-lead publications from a given year. Following our reviews for 2015 and 2016, this Miniperspective provides tabulated summaries of relevant articles published in 2017 along with some general observations. In addition, we discuss insights obtained from analysis of the combined data set of 85 examples from all three years of publications.

Fragment-based drug discovery (FBDD) has grown and matured to a point where it is valuable to keep track of its extent and details of application. This Perspective summarizes successful fragment-to-lead stories published in 2019. It is the fifth in a series that started with literature published in 2015. The analysis of screening methods, optimization strategies, and molecular properties of hits and leads are presented in the hope of informing best practices for FBDD. Moreover, FBDD is constantly evolving, and the latest technologies and emerging trends are summarized. These include covalent FBDD, FBDD for the stabilization of proteins or protein-protein interactions, FBDD for enzyme activators, new screening technologies, and advances in library design and chemical synthesis.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

The popularity of fragment-based drug discovery (FBDD) is demonstrated by the number of recent successful fragment-to-lead (F2L) publications. This Miniperspective provides a tabulated summary of the F2L literature published in the year 2016, along with discussion of general trends. It uses the same format as our summary of the 2015 literature and is intended to be a resource for both FBDD practitioners and medicinal chemists in general.

Tyrosine kinase inhibitors (TKIs) that inhibit the transphosphorylation activity of the BCR-ABL1 oncoprotein by binding the ATP-binding site of the catalytic domain of protein kinases are well established as being effective drugs for the treatments of chronic myeloid leukemia (CML). However, the off-target kinase activities of these non-specific TKIs are associated with adverse events that can limit their suitability for the treatment of patients and can negatively impact quality of life. Therefore, a new drug combining high efficacy with minimal side-effects could provide substantial therapeutic advantages. Asciminib is a new investigational agent that at concentrations However, in isothermal calorimetry studies asciminib showed strong binding affinity (KD 0.5 nM) to a much larger construct ofwild-typeABL1 that contains the catalytic, SH2 and SH3 domains (residues 46-534, ABL46-534), with thermodynamic parameters (ΔH = -72.8 kJ/mol, ΔS = -65.3 J/mol/K, resulting in ΔG = -53.2 kJ/mol) indicating strong enthalpy-driven, entropically unfavorable binding. This binding translates to inhibition of tyrosine phosphorylation catalysed by the ABL64-515 construct with a mean IC50 value of 2.6 ± 0.8 nM (radiometric filter binding assay) and 0.5 ± 0.1 nM (fluorescence resonance energy transfer assay). This data shows that asciminib inhibits the kinase activity of ABL1 by an allosteric mechanism which does not involve direct interaction with the ATP-binding pocket. X-ray crystallographic studies of a ternary complex between asciminib, nilotinib and the ABL146-534 protein possessing Thr315Ile and Asp382Asn substitutions show that asciminib binds to ABL1 in a deep-pocket on the C-lobe of the kinase domain, referred to as the myristate (or myristoyl) pocket. Nuclear magnetic resonance studies confirm that asciminib can also form asciminib-ABL1-TKI ternary complexes with thewild-typeABL83-534 protein in solution. Native ABL1 kinase is post-translationally myristoylated at the N-terminal glycine residue and this myristate group plays an important role in autoregulating the kinase by binding to a pocket (myristate pocket) on the catalytic domain of the protein to induce the formation of an inactive conformation of the enzyme. This regulatory mechanism is lost in BCR-ABL1 since the N-terminal region is replaced in the fusion protein with a fragment of the BCR protein, thus rendering the ABL1 kinase constitutively active. The binding of asciminib in this pocket therefore mimics that of myristate, thus stabilizing the inactive state of the ABL1 kinase. Consistent with this binding mode to the ABL1 kinase, asciminib potently inhibits BCR-ABL1 driven proliferation of leukemia-derived cell lines, while having little effect on cells that do not express BCR-ABL1 (Figure 1). Thein vitrodata translates into anti-tumor activity in mouse models of CML where asciminib dose-dependently inhibited the growth of subcutaneous KCL22 cell xenografts, with 30 mg/kg administered twice-daily by oral gavage affording 92% tumor regression (Figure 2). Asciminib, the first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, has several important features as a potential treatment of CML. At physiologically achievable concentrations asciminib can overcome mutations on the ATP-binding site of BCR-ABL1 that impede the binding of TKIs which lead to drug resistance in patients with CML. Secondly, by not inhibiting kinases such as EGFR, KIT, CSF1R, PDGFR or the sSRC-family kinases that are associated with off-target activities of TKIs such as bosutinib, dasatinib, imatinib, nilotinib and ponatinib, asciminib is not expected to be associated with cross-intolerance. Thirdly, asciminib can bind to the ABL1 kinase domain together with ATP-competitive TKIs to form ternary complexes, such that appropriate drug combinations should greatly impede the emergence of drug resistant kinase mutations. Disclosures Schuld: Novartis Pharma AG:Current Employment, Current equity holder in publicly-traded company.Grzesiek:Novartis Pharma:Research Funding.Jahnke:Novartis:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.Barys:Novartis Pharma AG:Current Employment.Cowan-Jacob:Novartis Pharma AG:Current Employment.Loo:Novartis Pharma AG:Current Employment.Wiget:Novartis Pharma AG:Current Employment.Manley:Novartis Pharma AG:Current Employment.

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation. OA hybrid

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment. Refereed/Peer-reviewed

Modulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.

This Miniperspective is the third in a series reviewing fragment-to-lead publications from a given year. Following our reviews for 2015 and 2016, this Miniperspective provides tabulated summaries of relevant articles published in 2017 along with some general observations. In addition, we discuss insights obtained from analysis of the combined data set of 85 examples from all three years of publications.

The article "Boeszoermenyi A, Ogorek B, Jain A, Arthanari H, Wagner G (2020) The precious fluorine on the ring: fluorine NMR for biological systems. J Biomol NMR. https ://doi.org/10.1007/s10858-020-00331-z" was written for the "Special Issue: NMR in Pharmaceutical Discovery and Development". However, unfortunately, it was published in an earlier issue of this journal owing to a publisher error. Further, the ORCID ID of author Wolfgang Jahnke is updated in the article. The original article has been corrected.

Die Wirksamkeit und Vertraglichkeit eines Medikaments kann verbessert werden, indem dieses gezielt an den gewunschten Wirkungsort gebracht wird. Bisphosphonate sind Prototypen derartiger Medikamente; allerdings ist ihre Affinitat zum Knochen eher zu stark und lasst sich nicht modulieren, ohne die Aktivitat auf das Enzym Farnesylpyrophosphat-Synthase (FPPS) zu verlieren. Eine Konsequenz der hohen Knochenaffinitat ist auserdem eine niedrige und stark variable orale Bioverfugbarkeit. Wir beschreiben hier Inhibitoren von FPPS mit einem Monophosphonat als Knochenanker, der eine moderate Affinitat zum Knochen verleiht, die uberdies noch moduliert werden kann. Uber einen NMR-basierten Knochenbindungstest wurde ein Zusammenhang von Struktur und Knochenaffinitat etabliert. Das Konzept, Wirkstoffe durch Anhangen einer Monophosphonatgruppe und ohne Verlust der oralen Bioverfugbarkeit gezielt zum Knochen zu bringen, kann weitreichende Anwendungsmoglichkeiten auch fur andere Wirkstoffe haben.

Imatinib (Glivec or Gleevec) potently inhibits the tyrosine kinase activity of BCR-ABL, a constitutively activated kinase, which causes chronic myelogenous leukemia (CML). Here we report the first almost complete backbone assignment of c-ABL kinase domain in complex with imatinib.

Help in determining biomolecular structure by NMR spectroscopy is found in a new method recently proposed by Reif, Hennig, and Griesinger, which enables the direct measurement of angles between bond vectors (see picture; X, Y=

The popularity of fragment-based drug discovery (FBDD) is demonstrated by the number of recent successful fragment-to-lead (F2L) publications. This Miniperspective provides a tabulated summary of the F2L literature published in the year 2016, along with discussion of general trends. It uses the same format as our summary of the 2015 literature and is intended to be a resource for both FBDD practitioners and medicinal chemists in general.

Binding of tyrosine kinase inhibitors such as imatinib was shown to induce a novel open-inhibited conformation of BCR-ABL, in which Tyr245 is exposed and prone to phosphorylation. To evaluate whether this leads to priming of the kinase in cellular systems, we probed activation of downstream signaling as a result of Tyr245 phosphorylation in a series of cellular washout experiments. While a spike in Tyr245 phosphorylation was observed both in overexpression and endogenous settings, no induction of downstream signaling was detected, showing that the priming hypothesis is not relevant for the therapeutic situation.

The objective of this work was to determine the maximum gauge pressure in the vapour phase above the liquid in different design types of dangerous goods packagings under normal conditions of carriage. The design types investigated were steel and plastic packagings with a volume of approximately 6 l. Two different methods were applied. In method 1, the pressure inside the packaging filled with a certain filling substance (dichloromethane) was directly measured under simulated conditions of carriage (degree of filling: 90%; filling temperature: 15°C; temperature during storage: 31°C). The maximum measured gauge pressures were between 89 mbar for a light plastic jerrican and 336 mbar for a steel drum. In method 2, the gauge pressure was calculated. The consideration of a rigid packaging combined with the assumption of a vapour pressure of zero during filling and sealing can serve as a worst case scenario. The calculated gauge pressure is approximately 1061 mbar. This procedure leads to the highest safety factor and does not require any experimental investigations. For a more realistic approximation of the gauge pressure of a non-rigid packaging, a packaging-specific function of relative expansion can be used, which is determined by a hydraulic pressure test. The calculated values ranged from 105 to 347 mbar. Method 2 provides conservative results. No hazardous filling substance is needed, and it allows a prediction of gauge pressure for other temperatures, substances and filling degrees. Therefore, this method could serve as alternative to UN Model Regulations 6.1.5.5.4 (a). Copyright © 2014 John Wiley & Sons, Ltd.

Summary KRAS is frequently mutated in several of the most lethal types of cancer; however, the KRAS protein has proven a challenging drug target. K-RAS4B must be localized to the plasma membrane by prenylation to activate oncogenic signaling, thus we endeavored to target the protein-membrane interface with small-molecule compounds. While all reported lead compounds have low affinity for KRAS in solution, the potency of Cmpd2 was strongly enhanced when prenylated K-RAS4B is associated with a lipid bilayer. We have elucidated a unique mechanism of action of Cmpd2, which simultaneously engages a shallow pocket on KRAS and associates with the lipid bilayer, thereby stabilizing KRAS in an orientation in which the membrane occludes its effector-binding site, reducing RAF binding and impairing activation of RAF. Furthermore, enrichment of Cmpd2 on the bilayer enhances potency by promoting interaction with KRAS. This insight reveals a novel approach to developing inhibitors of membrane-associated proteins.

In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using 19F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery., PLoS ONE, 12 (4), ISSN:1932-6203

The objective of this experiment was to verify that in regards to the leakproofness bubble test for packagings of dangerous goods, a reduction of the air overpressure from 0.2 to 0.1 bar can be compensated for by reducing the water surface tension to a value of approximately 33.2 mN/m by adding a wetting agent. It was experimentally proven that this method will yield the same leak diameters. This is important to avoid irreversible deformations during the leaktesting of intermediate bulk containers (IBCs) while using a test overpressure of 0.2 bar. Bubble test experiments were carried out on artificial borehole-shaped leaks manufactured of two different materials high density polyethylene (HDPE) and stainless steel by ultrashort pulse laser technology and with two different immersion test liquids (deionized water and a 0.1% Lutensol FSA fabric softener active 10 solution). The characteristic diameters of the boreholes investigated were from 11.5 to 30.3 µm in length. Copyright © 2013 John Wiley & Sons, Ltd.

Protein kinases are involved in many essential cellular processes and their deregulation can lead to a variety of diseases, including cancer. The pharmaceutical industry has invested heavily in the identification of kinase inhibitors to modulate these disease-promoting pathways, resulting in several successful drugs. However, the field is challenging as it is difficult to identify novel selective inhibitors with good pharmacokinetic/pharmacodynamic properties. In addition, resistance to kinase inhibitor treatment frequently arises. The identification of non-ATP site targeting (‘allosteric’) inhibitors, the identification of kinase activators and the expansion of kinase target space to include the less studied members of the family, including atypical- and pseudo-kinases, are potential avenues to overcome these challenges. In this perspective, the opportunities and challenges of following these approaches and others will be discussed.

After 20 years of sometimes quiet growth, fragment-based drug discovery (FBDD) has become mainstream. More than 30 drug candidates derived from fragments have entered the clinic, with two approved and several more in advanced trials. FBDD has been widely applied in both academia and industry, as evidenced by the large number of papers from universities, non-profit research institutions, biotechnology companies and pharmaceutical companies. Moreover, FBDD draws on a diverse range of disciplines, from biochemistry and biophysics to computational and medicinal chemistry. As the promise of FBDD strategies becomes increasingly realized, now is an opportune time to draw lessons and point the way to the future. This Review briefly discusses how to design fragment libraries, how to select screening techniques and how to make the most of information gleaned from them. It also shows how concepts from FBDD have permeated and enhanced drug discovery efforts.

Protein-ligand interactions are at the heart of drug discovery research. NMR spectroscopy is an excellent technology to identify and validate protein-ligand interactions. A plethora of NMR methods are available which are powerful, robust and information-rich, but also have pitfalls and limitations. In this review, we will focus on how to choose between different experiments, and assess their strengths and liabilities. We introduce the concept of the validation cross, which helps to categorize experiments according to their information content and to simplify the choice of the right experiment in order to address a specific question. Additionally, we will provide the framework for drawing correct conclusions from experimental results in order to accurately evaluate such interactions. Out of scope for this review are methods for subsequent characterization of the interaction such as quantitative KD determination, binding mode analysis, or structure determination.

Safety risks may ensue when the chemicals contained in polyethylene packagingsa age and damage them. To prevent subsequent accidents, the European Dangerous Goods Regulations have laid down requirements for testing the chemical compatibility of liquid dangerous goods transported in polyethylene packagings. The test procedures include 6 months in which the chemical is prestored in the packaging. After this time, the respective design-type tests are performed. Alternative methods with so-called standard liquids, simulating the different types of damaging effects, are also possible. If a packaging has successfully passed the design type tests with a standard liquid, other dangerous goods may also be transported in this packaging, as long as it is demonstrated that they have a less damaging effect than the standard liquid. However, in this area there is only little information and research available. A new potentially effective and time-saving method for comparing the stress crack damaging influence of liquids with standard liquids was proposed by a major German chemical company. The validation of this method, which was carried out on two polyethylene materials, showed the general applicability of the method. Two kinds of wetting solutions were applied to simulate cracking under stress. The influence of prestorage, test temperatures, wetting agents and material was examined. FEM calculations were carried out to ascertain the influence of the sample shape. Copyright © 2012 John Wiley & Sons, Ltd.

The key point within the scope of this research project was to find out whether there was a risk of creating an explosive atmosphere by permeation of flammable liquid compounds during transport of dangerous goods in freight containers under normal conditions of carriage. Therefore, all aspects that had an influence on the formation of such an atmosphere had to be considered. The most important influencing factors were permeation, air change in the freight container and ambient temperature. The first step was to investigate the permeation with different packaging materials, charge and temperatures. Furthermore, the air change rates of different freight containers were measured. A few climate tests with containers on ships, e.g. to Singapore, were performed to assess normal conditions of carriage. Another important point was measuring the solvent (toluene) concentration in the gas phase in a freight container loaded with plastic intermediate bulk containers (IBCs) filled with toluene. To confirm that the measured values were in the right range, the toluene concentration in the gas phase in a container was calculated with different packaging materials, air change rates and temperatures. The results of the measurements and calculations have shown that safety layers in the packaging wall, e.g. the copolymer of ethylene and vinyl alcohol (EVOH) and polyamide, can reduce the rate of permeation by more than a decimal power, but the lower explosive limit of toluene is easily reached within a few hours at 40°C charge temperature if there is no barrier. Copyright © 2012 John Wiley & Sons, Ltd.

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity. We present an NMR-based conformational assay to monitor the conformation of this crucial helix_I and show that myristate ligands that bend helix_I are functional antagonists, whereas ligands that bind to the myristate pocket but do not induce this conformational change are kinase agonists. Activation of c-Abl by allosteric agonists has been confirmed in a biochemical assay.

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr-Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Together with the NS5 polymerase, the NS3 helicase has a pivotal function in flavivirus RNA replication and constitutes an important drug target. We captured the dengue virus NS3 helicase at several stages along the catalytic pathway including bound to single-stranded (ss) RNA, to an ATP analogue, to a transition-state analogue and to ATP hydrolysis products. RNA recognition appears largely sequence independent in a way remarkably similar to eukaryotic DEAD box proteins Vasa and eIF4AIII. On ssRNA binding, the NS3 enzyme switches to a catalytic-competent state imparted by an inward movement of the P-loop, interdomain closure and a change in the divalent metal coordination shell, providing a structural basis for RNA-stimulated ATP hydrolysis. These structures demonstrate for the first time large quaternary changes in the flaviviridae helicase, identify the catalytic water molecule and point to a beta-hairpin that protrudes from subdomain 2, as a critical element for dsRNA unwinding. They also suggest how NS3 could exert an effect as an RNA-anchoring device and thus participate both in flavivirus RNA replication and assembly.

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

Current structural understanding of kinases is largely based on x-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. ABL kinase is an important drug target in the treatment of chronic myelogenous leukemia. Here, we present the first characterization of ABL kinase in complex with three clinical inhibitors (imatinib, nilotinib, and dasatinib) by modern solution NMR techniques. Structural and dynamical results were derived from complete backbone resonance assignments, experimental residual dipolar couplings, and (15)N relaxation data. Residual dipolar coupling data on the imatinib and nilotinib complexes show that the activation loop adopts the inactive conformation, whereas the dasatinib complex preserves the active conformation, which does not support contrary predictions based upon molecular modeling. Nanosecond as well as microsecond dynamics can be detected for certain residues in the activation loop in the inactive and active conformation complexes.

The versatility of NMR and its broad applicability to several stages in the drug discovery process is well known and generally considered one of the major strengths of NMR (Pellecchia et al., Nature Rev Drug Discov 1:211-219, 2002; Stockman and Dalvit, Prog Nucl Magn Reson Spectrosc 41:187-231, 2002; Lepre et al., Comb Chem High throughput screen 5:583-590, 2002; Wyss et al., Curr Opin Drug Discov Devel 5:630-647, 2002; Jahnke and Widmer, Cell Mol Life Sci 61:580-599, 2004; Huth et al., Methods Enzymol 394:549-571, 2005b; Klages et al., Mol Biosyst 2:318-332, 2006; Takeuchi and Wagner, Curr Opin Struct Biol 16:109-117, 2006; Zartler and Shapiro, Curr Pharm Des 12:3963-3972, 2006). Indeed, NMR is the only biophysical technique which can detect and quantify molecular interactions, and at the same time provide detailed structural information with atomic level resolution. NMR should therefore be ideally suited and widely requested as a tool for drug discovery research, and numerous examples of drug discovery projects which have substantially benefited from NMR contributions or were even driven by NMR have been described in the literature. However, not all pharmaceutical companies have rigorously implemented NMR as integral tool of their research processes. Some companies invest with limited resources, and others do not use biomolecular NMR at all. This discrepancy in assessing the value of a technology is striking, and calls for clarification--under which circumstances can NMR provide added value to the drug discovery process? What kind of contributions can NMR make, and how is it implemented and integrated for maximum impact? This perspectives article suggests key areas of impact for NMR, and a model of integrating NMR with other technologies to realize synergies and maximize their value for drug discovery.

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed “trigger sequence,” that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.