Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

Insights on JAK2 Modulation by Potent, Selective, and Cell-Permeable Pseudokinase-Domain Ligands.

JAK2 is a non-receptor tyrosine kinase that regulates hematopoiesis through the JAK-STAT pathway. The pseudokinase domain (JH2) is an important regulator of the activity of the kinase domain (JH1). V617F mutation in JH2 has been associated with the pathogenesis of various myeloproliferative neoplasms, but JAK2 JH2 has been poorly explored as a pharmacological target. In light of this, we aimed to develop JAK2 JH2 binders that could selectively target JH2 over JH1 and test their capacity to modulate JAK2 activity in cells. Toward this goal, we optimized a diaminotriazole lead compound into potent, selective, and cell-permeable JH2 binders leveraging computational design, synthesis, binding affinity measurements for the JH1, JH2 WT, and JH2 V617F domains, permeability measurements, crystallography, and cell assays. Optimized diaminotriazoles are capable of inhibiting STAT5 phosphorylation in both WT and V617F JAK2 in cells.

Conversion of a False Virtual Screen Hit into Selective JAK2 JH2 Domain Binders Using Convergent Design Strategies.

The Janus kinase 2 (JAK2) pseudokinase domain (JH2) is an ATP-binding domain that regulates the activity of the catalytic tyrosine kinase domain (JH1). Dysregulation of JAK2 JH1 signaling caused by the V617F mutation in JH2 is implicated in various myeloproliferative neoplasms. To explore if JAK2 activity can be modulated by a small molecule binding to the ATP site in JH2, we have developed several ligand series aimed at selectively targeting the JAK2 JH2 domain. We report here the evolution of a false virtual screen hit into a new JAK2 JH2 series. Optimization guided by computational modeling has yielded analogues with nanomolar affinity for the JAK2 JH2 domain and >100-fold selectivity for the JH2 domain over the JH1 domain. A crystal structure for one of the potent compounds bound to JAK2 JH2 clarifies the origins of the strong binding and selectivity. The compounds expand the platform for seeking molecules to regulate JAK2 signaling, including V617F JAK2 hyperactivation.

Structural basis for ligand reception by anaplastic lymphoma kinase.

The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with learning and memory1 and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity2. Aberrant ALK signalling causes numerous cancers3. In particular, full-length ALK is an important driver in paediatric neuroblastoma4,5, in which it is either mutated6 or activated by ligand7. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides8,9. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma.

Indoloxytriazines as binding molecules for the JAK2 JH2 pseudokinase domain and its V617F variant.

Small molecules that selectively bind to the pseudokinase JH2 domain over the JH1 kinase domain of JAK2 kinase are sought. Virtual screening led to the purchase of 17 compounds among which 9 were found to bind to V617F JAK2 JH2 with affinities of 40 - 300 µM in a fluorogenic assay. Ten analogues were then purchased yielding 9 additional active compounds. Aminoanilinyltriazine 22 was particularly notable as it shows no detectable binding to JAK2 JH1, and it has a 65-µM dissociation constant K d with V617F JAK2 JH2. A crystal structure for 22 in complex with wild-type JAK2 JH2 was obtained to elucidate the binding mode. Additional de novo design led to the synthesis of 19 analogues of 22 with the most potent being 33n with K d values of 2-3 µM for WT and V617F JAK2 JH2, and with 16-fold selectivity relative to binding with WT JAK2 JH1.

Metadynamics as a Postprocessing Method for Virtual Screening with Application to the Pseudokinase Domain of JAK2.

With standard scoring methods, top-ranked compounds from virtual screening by docking often turn out to be inactive. For this reason, metadynamics, a method used to sample rare events, was studied to further evaluate docking poses with the aim of reducing false positives. Specifically, virtual screening was performed with Glide SP to seek potential molecules to bind to the ATP site in the pseudokinase domain of JAK2 kinase, and promising compounds were selected from the top-ranked 1000 based on visualization. Rescoring with Glide XP, GOLD, and MM/GBSA was unable to differentiate well between active and inactive compounds. Metadynamics was then used to gauge the relative binding affinity from the required time or the potential of mean force needed to dissociate the ligand from the bound complex. With consideration of previously known binders of varying affinities, metadynamics was able to differentiate between the most active compounds and inactive or weakly active ones, and it could identify correctly most of the selected virtual screening compounds as false positives. Thus, metadynamics has the potential to be a viable postprocessing method for virtual screening, minimizing the expense of buying or synthesizing inactive compounds.

Selective Janus Kinase 2 (JAK2) Pseudokinase Ligands with a Diaminotriazole Core.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

Bayesian analysis of isothermal titration calorimetry for binding thermodynamics.

Isothermal titration calorimetry (ITC) is the only technique able to determine both the enthalpy and entropy of noncovalent association in a single experiment. The standard data analysis method based on nonlinear regression, however, provides unrealistically small uncertainty estimates due to its neglect of dominant sources of error. Here, we present a Bayesian framework for sampling from the posterior distribution of all thermodynamic parameters and other quantities of interest from one or more ITC experiments, allowing uncertainties and correlations to be quantitatively assessed. For a series of ITC measurements on metal:chelator and protein:ligand systems, the Bayesian approach yields uncertainties which represent the variability from experiment to experiment more accurately than the standard data analysis. In some datasets, the median enthalpy of binding is shifted by as much as 1.5 kcal/mol. A Python implementation suitable for analysis of data generated by MicroCal instruments (and adaptable to other calorimeters) is freely available online.

Optimization of Pyrazoles as Phenol Surrogates to Yield Potent Inhibitors of Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is implicated in the regulation of inflammation, cell proliferation, and neurological disorders. MIF is also an enzyme that functions as a keto-enol tautomerase. Most potent MIF tautomerase inhibitors incorporate a phenol, which hydrogen bonds to Asn97 in the active site. Starting from a 113-µm docking hit, we report results of structure-based and computer-aided design that have provided substituted pyrazoles as phenol alternatives with potencies of 60-70 nm. Crystal structures of complexes of MIF with the pyrazoles highlight the contributions of hydrogen bonding with Lys32 and Asn97, and aryl-aryl interactions with Tyr36, Tyr95, and Phe113 to the binding.

Adding a Hydrogen Bond May Not Help: Naphthyridinone vs Quinoline Inhibitors of Macrophage Migration Inhibitory Factor.

Coordination of the ammonium group of Lys32 in the active site of human macrophage migration inhibitory factor (MIF) using a 1,7-naphthyridin-8-one instead of a quinoline is investigated. Both gas- and aqueous-phase DFT calculations for model systems indicate potential benefits for the added hydrogen bond with the lactam carbonyl group, while FEP results are neutral. Three crystal structures are reported for complexes of MIF with 3a, 4a, and 4b, which show that the desired hydrogen bond is formed with O-N distances of 2.8-3.0 Å. Compound 4b is the most potent new MIF inhibitor with Ki and Kd values of 90 and 94 nM; it also has excellent aqueous solubility, 288 µg/mL. Consistent with the FEP results, the naphthyridinones are found to have similar potency as related quinolines in spite of the additional protein-ligand hydrogen bond.

How Nothing Boosts Affinity: Hydrophobic Ligand Binding to the Virtually Vacated S1' Pocket of Thermolysin.

We investigated the hydration state of the deep, well-accessible hydrophobic S1' specificity pocket of the metalloprotease thermolysin with purposefully designed ligands using high-resolution crystallography and isothermal titration calorimetry. The S1' pocket is known to recognize selectively a very stringent set of aliphatic side chains such as valine, leucine, and isoleucine of putative substrates. We engineered a weak-binding ligand covering the active site of the protease without addressing the S1' pocket, thus transforming it into an enclosed cavity. Its sustained accessibility could be proved by accommodating noble gas atoms into the pocket in the crystalline state. The topology and electron content of the enclosed pocket with a volume of 141 Å3 were analyzed using an experimental MAD-phased electron density map that was calibrated to an absolute electron number scale, enabling access to the total electron content within the cavity. Our analysis indicates that the S1' pocket is virtually vacated, thus free of any water molecules. The thermodynamic signature of the reduction of the void within the pocket by growing aliphatic P1' substituents (H, Me, iPr, iBu) reveals a dramatic, enthalpy-dominated gain in free energy of binding resulting in a factor of 41 000 in Kd for the H-to-iBu transformation. Substituents placing polar decoy groups into the pocket to capture putatively present water molecules could not collect any evidence for a bound solvent molecule.

Paying the Price of Desolvation in Solvent-Exposed Protein Pockets: Impact of Distal Solubilizing Groups on Affinity and Binding Thermodynamics in a Series of Thermolysin Inhibitors.

In lead optimization, open, solvent-exposed protein pockets are often disregarded as prospective binding sites. Because of bulk-solvent proximity, researchers are instead enticed to attach charged polar groups at inhibitor scaffolds to improve solubility and pharmacokinetic properties. It is rarely considered that solvent effects from water reorganization in the first hydration shell of protein-ligand complexes can have a significant impact on binding. We investigate the thermodynamic fingerprint of thermolysin inhibitors featuring terminal charged ammonium groups that are gradually pulled from a distal, solvent-exposed position into the flat, bowl-shaped S2' pocket. Even for the most remote attachment, costs for partial desolvation of the polar group next to the protein-solvent interface are difficult to compensate by interactions with the protein or surrounding water molecules. Through direct comparison with hydrophobic analogues, a significant 180-fold affinity loss was recorded, which questions popular strategies to attach polar ligand-solubilizing groups at the exposed terminus of substituents accommodated in flat open pockets.

Elucidating the Origin of Long Residence Time Binding for Inhibitors of the Metalloprotease Thermolysin.

Kinetic parameters of protein-ligand interactions are progressively acknowledged as valuable information for rational drug discovery. However, a targeted optimization of binding kinetics is not easy to achieve, and further systematic studies are necessary to increase the understanding about molecular mechanisms involved. We determined association and dissociation rate constants for 17 inhibitors of the metalloprotease thermolysin by surface plasmon resonance spectroscopy and correlated kinetic data with high-resolution crystal structures in complex with the protein. From the structure-kinetics relationship, we conclude that the strength of interaction with Asn112 correlates with the rate-limiting step of dissociation. This residue is located at the beginning of a ß-strand motif that lines the binding cleft and is commonly believed to align a substrate for catalysis. A reduced mobility of the Asn112 side chain owing to an enhanced engagement in charge-assisted hydrogen bonds prevents the conformational adjustment associated with ligand release and transformation of the enzyme to its open state. This hypothesis is supported by kinetic data of ZFPLA, a known pseudopeptidic inhibitor of thermolysin, which blocks the conformational transition of Asn112. Interference with this retrograde induced-fit mechanism results in variation of the residence time of thermolysin inhibitors by a factor of 74 000. The high conservation of this structural motif within the M4 and M13 metalloprotease families underpins the importance of this feature and has significant implications for drug discovery.

Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein-Bound Ligands.

A previously studied congeneric series of thermolysin inhibitors addressing the solvent-accessible S2' pocket with different hydrophobic substituents showed modulations of the surface water layers coating the protein-bound inhibitors. Increasing stabilization of water molecules resulted in an enthalpically more favorable binding signature, overall enhancing affinity. Based on this observation, we optimized the series by designing tailored P2' substituents to improve and further stabilize the surface water network. MD simulations were applied to predict the putative water pattern around the bound ligands. Subsequently, the inhibitors were synthesized and characterized by high-resolution crystallography, microcalorimetry, and surface plasmon resonance. One of the designed inhibitors established the most pronounced water network of all inhibitors tested so far, composed of several fused water polygons, and showed 50-fold affinity enhancement with respect to the original methylated parent ligand. Notably, the inhibitor forming the most perfect water network also showed significantly prolonged residence time compared to the other tested inhibitors.

Active Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads To Address a Catalytic Dyad.

Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1' pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1' are promising starting points for fragment-growing strategies.

High-Throughput Crystallography: Reliable and Efficient Identification of Fragment Hits.

Today the identification of lead structures for drug development often starts from small fragment-like molecules raising the chances to find compounds that successfully pass clinical trials. At the heart of the screening for fragments binding to a specific target, crystallography delivers structural information essential for subsequent drug design. While it is common to search for bound ligands in electron densities calculated directly after an initial refinement cycle, we raise the important question whether this strategy is viable for fragments characterized by low affinities. Here, we describe and provide a collection of high-quality diffraction data obtained from 364 protein crystals treated with diverse fragments. Subsequent data analysis showed that ∼25% of all hits would have been missed without further refining the resulting structures. To enable fast and reliable hit identification, we have designed an automated refinement pipeline that will inspire the development of optimized tools facilitating the successful application of fragment-based methods.

Experimental Active-Site Mapping by Fragments: Hot Spots Remote from the Catalytic Center of Endothiapepsin.

Successful optimization of a given lead scaffold requires thorough binding-site mapping of the target protein particular in regions remote from the catalytic center where high conservation across protein families is given. We screened a 361-entry fragment library for binding to the aspartic protease endothiapepsin by crystallography. This enzyme is frequently used as a surrogate for the design of renin and ß-secretase inhibitors. A hit rate of 20% was achieved, providing 71 crystal structures. Here, we discuss 45 binding poses of fragments accommodated in pockets remote from the catalytic dyad. Three major hot spots are discovered in remote binding areas: Asp81, Asp119, and Phe291. Compared to the dyad binders, bulkier fragments occupy these regions. Many of the discovered fragments suggest an optimization concept on how to grow them into larger ligands occupying adjacent binding pockets that will possibly endow them with the desired selectivity for one given member of a protein family.

Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits: A Case Study.

Fragment-based lead discovery (FBLD) has become a pillar in drug development. Typical applications of this method comprise at least two biophysical screens as prefilter and a follow-up crystallographic experiment on a subset of fragments. Clearly, structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment-bound structures. We therefore set out to screen 361 fragments for binding to endothiapepsin, a representative of the challenging group of aspartic proteases, employing six screening techniques and crystallography in parallel. Crystallography resulted in the very high number of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening cascade, building on the results from two or more screening methods, would have failed to predict at least 73% of these hits. We thus conclude that, at least in the present case, the frequently applied biophysical prescreening filters deteriorate the number of possible X-ray hits while only the immediate use of crystallography enables exhaustive retrieval of a maximum of fragment structures, which represent a rich source guiding hit-to-lead-to-drug evolution.

Impact of Surface Water Layers on Protein--Ligand Binding: How Well Are Experimental Data Reproduced by Molecular Dynamics Simulations in a Thermolysin Test Case?

Drug binding involves changes of the local water structure around proteins including water rearrangements across surface-solvation layers around protein and ligand portions exposed to the newly formed complex surface. For a series of thermolysin-binding phosphonamidates, we discovered that variations of the partly exposed P2'-substituents modulate binding affinity up to 10 kJ mol(-1) with even larger enthalpy/entropy partitioning of the binding signature. The observed profiles cannot be completely explained by desolvation effects. Instead, the quality and completeness of the surface water network wrapping around the formed complexes provide an explanation for the observed structure-activity relationship. We used molecular dynamics to compute surface water networks and predict solvation sites around the complexes. A fairly good correspondence with experimental difference electron densities in high-resolution crystal structures is achieved; in detail some problems with the potentials were discovered. Charge-assisted contacts to waters appeared as exaggerated by AMBER, and stabilizing contributions of water-to-methyl contacts were underestimated.