Potent and selective TYK2-JH1 inhibitors highly efficacious in rodent model of psoriasis.

TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.

Anti-leukaemic activity of the TYK2 selective inhibitor NDI-031301 in T-cell acute lymphoblastic leukaemia.

Activation of tyrosine kinase 2 (TYK2) contributes to the aberrant survival of T-cell acute lymphoblastic leukaemia (T-ALL) cells. Here we demonstrate the anti-leukaemic activity of a novel TYK2 inhibitor, NDI-031301. NDI-031301 is a potent and selective inhibitor of TYK2 that induced robust growth inhibition of human T-ALL cell lines. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with the JAK inhibitors tofacitinib and baricitinib. Further investigation revealed that NDI-031301 treatment uniquely leads to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage. Activation of p38 MAPK occurred within 1 h of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacological inhibition of p38 MAPK partially rescued apoptosis induced by TYK2 inhibitor. Finally, daily oral administration of NDI-031301 at 100 mg/kg bid to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumour burden and a significant survival benefit. These results support selective inhibition of TYK2 as a promising potential therapeutic strategy for T-ALL.

Multiconformation, Density Functional Theory-Based pKa Prediction in Application to Large, Flexible Organic Molecules with Diverse Functional Groups.

We consider the conformational flexibility of molecules and its implications for micro- and macro-pKa. The corresponding formulas are derived and discussed against the background of a comprehensive scientific and algorithmic description of the latest version of our computer program Jaguar pKa, a density functional theory-based pKa predictor, which is now capable of acting on multiple conformations explicitly. Jaguar pKa is essentially a complex computational workflow incorporating research and technologies from the fields of cheminformatics, molecular mechanics, quantum mechanics, and implicit solvation models. The workflow also makes use of automatically applied empirical corrections which account for the systematic errors resulting from the neglect of explicit solvent interactions in the algorithm's implicit solvent model. Applications of our program to large, flexible organic molecules representing several classes of functional groups are shown, with a particular emphasis in illustrations laid on drug-like molecules. It is demonstrated that a combination of aggressive conformational search and an explicit consideration of multiple conformations nearly eliminates the dependence of results on the initially chosen conformation. In certain cases this leads to unprecedented accuracy, which is sufficient for distinguishing stereoisomers that have slightly different pKa values. An application of Jaguar pKa to proton sponges, the pKa of which are strongly influenced by steric effects, showcases the advantages that pKa predictors based on quantum mechanical calculations have over similar empirical programs.

WScore: A Flexible and Accurate Treatment of Explicit Water Molecules in Ligand-Receptor Docking.

We have developed a new methodology for protein-ligand docking and scoring, WScore, incorporating a flexible description of explicit water molecules. The locations and thermodynamics of the waters are derived from a WaterMap molecular dynamics simulation. The water structure is employed to provide an atomic level description of ligand and protein desolvation. WScore also contains a detailed model for localized ligand and protein strain energy and integrates an MM-GBSA scoring component with these terms to assess delocalized strain of the complex. Ensemble docking is used to take into account induced fit effects on the receptor conformation, and protein reorganization free energies are assigned via fitting to experimental data. The performance of the method is evaluated for pose prediction, rank ordering of self-docked complexes, and enrichment in virtual screening, using a large data set of PDB complexes and compared with the Glide SP and Glide XP models; significant improvements are obtained.

Docking covalent inhibitors: a parameter free approach to pose prediction and scoring.

Although many popular docking programs include a facility to account for covalent ligands, large-scale systematic docking validation studies of covalent inhibitors have been sparse. In this paper, we present the development and validation of a novel approach for docking and scoring covalent inhibitors, which consists of conventional noncovalent docking, heuristic formation of the covalent attachment point, and structural refinement of the protein-ligand complex. This approach combines the strengths of the docking program Glide and the protein structure modeling program Prime and does not require any parameter fitting for the study of additional covalent reaction types. We first test this method by predicting the native binding geometry of 38 covalently bound complexes. The average RMSD of the predicted poses is 1.52 Å, and 76% of test set inhibitors have an RMSD of less than 2.0 Å. In addition, the apparent affinity score constructed herein is tested on a virtual screening study and the characterization of the SAR properties of two different series of congeneric compounds with satisfactory success.

Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors.

NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.

Docking performance of the glide program as evaluated on the Astex and DUD datasets: a complete set of glide SP results and selected results for a new scoring function integrating WaterMap and glide.

Glide SP mode enrichment results for two preparations of the DUD dataset and native ligand docking RMSDs for two preparations of the Astex dataset are presented. Following a best-practices preparation scheme, an average RMSD of 1.140 Å for native ligand docking with Glide SP is computed. Following the same best-practices preparation scheme for the DUD dataset an average area under the ROC curve (AUC) of 0.80 and average early enrichment via the ROC (0.1 %) metric of 0.12 were observed. 74 and 56 % of the 39 best-practices prepared targets showed AUC over 0.7 and 0.8, respectively. Average AUC was greater than 0.7 for all best-practices protein families demonstrating consistent enrichment performance across a broad range of proteins and ligand chemotypes. In both Astex and DUD datasets, docking performance is significantly improved employing a best-practices preparation scheme over using minimally-prepared structures from the PDB. Enrichment results for WScore, a new scoring function and sampling methodology integrating WaterMap and Glide, are presented for four DUD targets, hivrt, hsp90, cdk2, and fxa. WScore performance in early enrichment is consistently strong and all systems examined show AUC > 0.9 and superior early enrichment to DUD best-practices Glide SP results.

Structure-based identification and neutralization mechanism of tyrosine sulfate mimetics that inhibit HIV-1 entry.

Tyrosine sulfate-mediated interactions play an important role in HIV-1 entry. After engaging the CD4 receptor at the cell surface, the HIV-1 gp120 glycoprotein binds to the CCR5 co-receptor via an interaction that requires two tyrosine sulfates, at positions 10 and 14 in the CCR5-N terminus. Building on previous structure determinations of this interaction, here we report the targeting of these tyrosine sulfate binding sites for drug design through in silico screening of small molecule libraries, identification of lead compounds, and characterization of biological activity. A class of tyrosine sulfate-mimicking small molecules containing a "phenyl sulfonate-linker-aromatic" motif was identified that specifically inhibited binding of gp120 to the CCR5-N terminus as well as to sulfated antibodies that recognize the co-receptor binding region on gp120. The most potent of these compounds bound gp120 with low micromolar affinity and its CD4-induced conformation with K(D)'s as tight as ∼50 nM. Neutralization experiments suggested the targeted site to be conformationally inaccessible prior to CD4 engagement. Primary HIV-1 isolates were weakly neutralized, preincubation with soluble CD4 enhanced neutralization, and engineered isolates with increased dependence on the N terminus of CCR5 or with reduced conformational barriers were neutralized with IC(50) values as low as ∼1 µM. These results reveal the potential of targeting the tyrosine sulfate interactions of HIV-1 and provide insight into how mechanistic barriers, evolved by HIV-1 to evade antibody recognition, also restrict small-molecule-mediated neutralization.

Glutamate receptor agonists: stereochemical aspects.

The neurotransmitter (S)-glutamate [(S)-Glu] is responsible for most of the excitatory neurotransmission in the central nervous system. The effect of (S)-Glu is mediated by both ionotropic and metabotropic receptors. Glutamate receptor agonists are generally α-amino acids with one or more stereogenic centers due to strict requirements in the agonist binding pocket of the activated state of the receptor. By contrast, there are many examples of achiral competitive antagonists. The present review addresses how stereochemistry affects the activity of glutamate receptor ligands. The review focuses mainly on agonists and discusses stereochemical and conformational considerations as well as biostructural knowledge of the agonist binding pockets, which is useful in the design of glutamate receptor agonists. Examples are chosen to demonstrate how stereochemistry not only determines how the agonist binding pocket is filled, but also how it affects the conformational space of the ligand and in this way restricts the recognition of various glutamate receptors, ultimately leading to selectivity.

Biostructural and pharmacological studies of bicyclic analogues of the 3-isoxazolol glutamate receptor agonist ibotenic acid.

We describe an improved synthesis and detailed pharmacological characterization of the conformationally restricted analogue of the naturally occurring nonselective glutamate receptor agonist ibotenic acid (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA, 5) at AMPA receptor subtypes. Compound 5 was shown to be a subtype-discriminating agonist at AMPA receptors with higher binding affinity and functional potency at GluA1/2 compared to GluA3/4, unlike the isomeric analogue (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA, 4) that binds to all AMPA receptor subtypes with comparable potency. Biostructural X-ray crystallographic studies of 4 and 5 reveal different binding modes of (R)-4 and (S)-5 in the GluA2 agonist binding domain. WaterMap analysis of the GluA2 and GluA4 binding pockets with (R)-4 and (S)-5 suggests that the energy of hydration sites is ligand dependent, which may explain the observed selectivity.

Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution.

Generating the appropriate protonation states of drug-like molecules in solution is important for success in both ligand- and structure-based virtual screening. Screening collections of millions of compounds requires a method for determining tautomers and their energies that is sufficiently rapid, accurate, and comprehensive. To maximise enrichment, the lowest energy tautomers must be determined from heterogeneous input, without over-enumerating unfavourable states. While computationally expensive, the density functional theory (DFT) method M06-2X/aug-cc-pVTZ(-f) [PB-SCRF] provides accurate energies for enumerated model tautomeric systems. The empirical Hammett-Taft methodology can very rapidly extrapolate substituent effects from model systems to drug-like molecules via the relationship between pK(T) and pK(a). Combining the two complementary approaches transforms the tautomer problem from a scientific challenge to one of engineering scale-up, and avoids issues that arise due to the very limited number of measured pK(T) values, especially for the complicated heterocycles often favoured by medicinal chemists for their novelty and versatility. Several hundreds of pre-calculated tautomer energies and substituent pK(a) effects are tabulated in databases for use in structural adjustment by the program Epik, which treats tautomers as a subset of the larger problem of the protonation states in aqueous ensembles and their energy penalties. Accuracy and coverage is continually improved and expanded by parameterizing new systems of interest using DFT and experimental data. Recommendations are made for how to best incorporate tautomers in molecular design and virtual screening workflows.

The glutamate receptor GluR5 agonist (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid and the 8-methyl analogue: synthesis, molecular pharmacology, and biostructural characterization.

The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound to the GluR5 agonist binding domain (ABD) are presented, including a watermap analysis, which suggests that water molecules in the agonist binding site are important selectivity determinants.

N-Hydroxypyrazolyl glycine derivatives as selective N-methyl-D-aspartic acid receptor ligands.

A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspartate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major glutamate receptor subgroups show that these 4-substituted N-hydroxypyrazol-5-yl glycine (NHP5G) derivatives are selectively recognized by N-methyl- d-aspartic acid (NMDA) receptors and that the ( R)-enantiomers are preferred. Moreover, several of the compounds are able to discriminate between individual subtypes among the NMDA receptors, providing new pharmacological tools. For example, 4-propyl NHP5G is an antagonist at the NR1/NR2A subtype but an agonist at the NR1/NR2D subtype. Molecular docking studies indicate that the substituent protrudes into a region that may be further exploited to improve subtype selectivity, thereby opening up a design strategy for ligands which can differentiate individual NMDA receptor subtypes.

Improving database enrichment through ensemble docking.

While it may seem intuitive that using an ensemble of multiple conformations of a receptor in structure-based virtual screening experiments would necessarily yield improved enrichment of actives relative to using just a single receptor, it turns out that at least in the p38 MAP kinase model system studied here, a very large majority of all possible ensembles do not yield improved enrichment of actives. However, there are combinations of receptor structures that do lead to improved enrichment results. We present here a method to select the ensembles that produce the best enrichments that does not rely on knowledge of active compounds or sophisticated analyses of the 3D receptor structures. In the system studied here, the small fraction of ensembles of up to 3 receptors that do yield good enrichments of actives were identified by selecting ensembles that have the best mean GlideScore for the top 1% of the docked ligands in a database screen of actives and drug-like "decoy" ligands. Ensembles of two receptors identified using this mean GlideScore metric generally outperform single receptors, while ensembles of three receptors identified using this metric consistently give optimal enrichment factors in which, for example, 40% of the known actives outrank all the other ligands in the database.

Epik: a software program for pK( a ) prediction and protonation state generation for drug-like molecules.

Epik is a computer program for predicting pK(a) values for drug-like molecules. Epik can use this capability in combination with technology for tautomerization to adjust the protonation state of small drug-like molecules to automatically generate one or more of the most probable forms for use in further molecular modeling studies. Many medicinal chemicals can exchange protons with their environment, resulting in various ionization and tautomeric states, collectively known as protonation states. The protonation state of a drug can affect its solubility and membrane permeability. In modeling, the protonation state of a ligand will also affect which conformations are predicted for the molecule, as well as predictions for binding modes and ligand affinities based upon protein-ligand interactions. Despite the importance of the protonation state, many databases of candidate molecules used in drug development do not store reliable information on the most probable protonation states. Epik is sufficiently rapid and accurate to process large databases of drug-like molecules to provide this information. Several new technologies are employed. Extensions to the well-established Hammett and Taft approaches are used for pK(a) prediction, namely, mesomer standardization, charge cancellation, and charge spreading to make the predicted results reflect the nature of the molecule itself rather just for the particular Lewis structure used on input. In addition, a new iterative technology for generating, ranking and culling the generated protonation states is employed.

Functional characterization of Tet-AMPA [tetrazolyl-2-amino-3-(3-hydroxy-5-methyl- 4-isoxazolyl)propionic acid] analogues at ionotropic glutamate receptors GluR1-GluR4. The molecular basis for the functional selectivity profile of 2-Bn-Tet-AMPA.

Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-iPr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible determinants of the GluR4i/GluR1i selectivity. In a mutagenesis study, doubly mutating M686V/I687A in GluR1i in combination with either D399S or E683A increased both the potency and the maximal response of 2-Bn-Tet-AMPA at this receptor to levels similar to those elicited by the agonist at GluR4i. The dependence of the novel selectivity profile of 2-Bn-Tet-AMPA upon residues located outside of the orthosteric site underlines the potential for developing GluR subtype selective ligands by designing compounds with substituents that protrude beyond the (S)-Glu binding pocket.

A tetrazolyl-substituted subtype-selective AMPA receptor agonist.

Replacement of the methyl group of the AMPA receptor agonist 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA) with a benzyl group provided the first AMPA receptor agonist, compound 7, capable of discriminating GluR2-4 from GluR1 by its more than 10-fold preference for the former receptor subtypes. An X-ray crystallographic analysis of this new analogue in complex with the GluR2-S1S2J construct shows that accommodation of the benzyl group creates a previously unobserved pocket in the receptor, which may explain the remarkable pharmacological profile of compound 7.

Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes.

A novel scoring function to estimate protein-ligand binding affinities has been developed and implemented as the Glide 4.0 XP scoring function and docking protocol. In addition to unique water desolvation energy terms, protein-ligand structural motifs leading to enhanced binding affinity are included: (1) hydrophobic enclosure where groups of lipophilic ligand atoms are enclosed on opposite faces by lipophilic protein atoms, (2) neutral-neutral single or correlated hydrogen bonds in a hydrophobically enclosed environment, and (3) five categories of charged-charged hydrogen bonds. The XP scoring function and docking protocol have been developed to reproduce experimental binding affinities for a set of 198 complexes (RMSDs of 2.26 and 1.73 kcal/mol over all and well-docked ligands, respectively) and to yield quality enrichments for a set of fifteen screens of pharmaceutical importance. Enrichment results demonstrate the importance of the novel XP molecular recognition and water scoring in separating active and inactive ligands and avoiding false positives.

Structure-activity relationships of selective GABA uptake inhibitors.

For more than four decades there has been a search for selective inhibitors of GABA transporters. This has led to potent and selective inhibitors of the cloned GABA transporter subtype GAT1, which is responsible for a majority of neuronal GABA transport. The only clinically approved compound with this mechanism of action is Tiagabine. Other GABA transporter subtypes have not been targeted with comparable selectivity and potency. We here review a comprehensive series of competitive inhibitors that provide information about the GABA recognition site and summarise the structure-activity relations in a ligand-based pharmacophore model that suggests how future compounds could be designed. Finally, some of the recent results on subtype-characterised competitive inhibitors and recent lipophilic aromatic GABA uptake inhibitors are reviewed.