Yafeng Xue

Discovery of novel potent and highly selective glycogen synthase kinase-3β (GSK3β) inhibitors for Alzheimer's disease: design, synthesis, and characterization of pyrazines.

Glycogen synthase kinase-3β, also called tau phosphorylating kinase, is a proline-directed serine/threonine kinase which was originally identified due to its role in glycogen metabolism. Active forms of GSK3β localize to pretangle pathology including dystrophic neuritis and neurofibrillary tangles in Alzheimers disease (AD) brain. By using a high throughput screening (HTS) approach to search for new chemical series and cocrystallization of key analogues to guide the optimization and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and blood-brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy for Alzheimers disease.

Synthesis and Structure-Activity Relationship of 4-(1,3-Benzothiazol-2-Yl)-Thiophene-2-Sulfonamides as Cyclin-Dependent Kinase 5 (Cdk5)/P25 Inhibitors.

4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a) was found to be a moderately potent inhibitor of cyclin-dependent kinase 5 (cdk5) from a HTS screen. The synthesis and SAR around this hit is described. The X-ray coordinates of ligand 4a with cdk5 are also reported, showing an unusual binding mode to the hinge region via a water molecule.

Benzoxazepines Achieve Potent Suppression of IL-17 Release in Human T-Helper 17 (TH17) Cells through an Induced-Fit Binding Mode to the Nuclear Receptor RORγ

RORγt, an isoform of the retinoic acid‐related orphan receptor gamma (RORc, RORγ), has been identified as the master regulator of T‐helper 17 (TH17) cell function and development, making it an attractive target for the treatment of autoimmune diseases. Validation for this target comes from antibodies targeting interleukin‐17 (IL‐17), the signature cytokine produced by TH17 cells, which have shown impressive results in clinical trials. Through focused screening of our compound collection, we identified a series of N‐sulfonylated benzoxazepines, which displayed micromolar affinity for the RORγ ligand‐binding domain (LBD) in a radioligand binding assay. Optimization of these initial hits resulted in potent binders, which dose‐dependently decreased the ability of the RORγ‐LBD to interact with a peptide derived from steroid receptor coactivator 1, and inhibited the release of IL‐17 secretion from isolated and cultured human TH17 cells with nanomolar potency. A cocrystal structure of inverse agonist 15 (2‐chloro‐6‐fluoro‐N‐(4‐{[3‐(trifluoromethyl)phenyl]sulfonyl}‐2,3,4,5‐tetrahydro‐1,4‐benzoxazepin‐7‐yl)benzamide) bound to the RORγ‐LBD illustrated that both hydrophobic interactions, leading to an induced fit around the substituted benzamide moiety of 15, as well as a hydrogen bond from the amide NH to His479 seemed to be important for the mechanism of action. This structure is compared with the structure of agonist 25 (N‐(2‐fluorophenyl)‐4‐[(4‐fluorophenyl)sulfonyl]‐2,3,4,5‐tetrahydro‐1,4‐benzoxazepin‐6‐amine ) and structures of other known RORγ modulators.

Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein–Protein Interaction

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC50 of 0.44 μM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

From natural products to achiral drug prototypes: Potent thrombin inhibitors based on P2/P3 dihydropyrid-2-one core motifs

A series of dihydropyrid-2-ones was synthesized and tested for inhibitory activity against serine protease enzymes. Moderate to low nanomolar inhibitory activities were obtained against thrombin and excellent selectivity against trypsin was observed.

Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV.

In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.

Fragment Screening of Soluble Epoxide Hydrolase for Lead Generation-Structure-Based Hit Evaluation and Chemistry Exploration.

Soluble epoxide hydrolase (sEH) is involved in the regulation of many biological processes by metabolizing the key bioactive lipid mediator, epoxyeicosatrienoic acids. For the development of sEH inhibitors with improved physicochemical properties, we performed both a fragment screening and a high‐throughput screening aiming at an integrated hit evaluation and lead generation. Followed by a joint dose–response analysis to confirm the hits, the identified actives were then effectively triaged by a structure‐based hit‐classification approach to three prioritized series. Two distinct scaffolds were identified as tractable starting points for potential lead chemistry work. The oxoindoline series bind at the right‐hand side of the active‐site pocket with hydrogen bonds to the protein. The 2‐phenylbenzimidazole‐4‐sulfonamide series bind at the central channel with significant induced fit, which has not been previously reported. On the basis of the encouraging initial results, we envision that a new lead series with improved properties could be generated if a vector is found that could merge the cyclohexyl functionality of the oxoindoline series with the trifluoromethyl moiety of the 2‐phenylbenzimidazole‐4‐sulfonamide series.

Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity.

The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high‐throughput screening (HTS) campaign. The compound bound to MR with pKi=6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X‐ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced‐fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11‐ to 79‐fold over PR and 23‐ to 234‐fold over GR was obtained. Given the U‐shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi=7.3. Two protein–ligand X‐ray structures were determined, confirming the hypothesized binding mode for the designed compounds.

Potent and Orally Bioavailable Inverse Agonists of RORγt Resulting from Structure-Based Design

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a small-molecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorable PK in rodents, with compound 32 representing a promising starting point for future investigations.

Fragment Screening of RORγt Using Cocktail Crystallography: Identification of Simultaneous Binding of Multiple Fragments

The retinoic‐acid‐related orphan receptor γ t (RORγt), as a master regulator of Th17 cell pathology, has become an attractive target for small‐molecule drug discovery for the treatment of Th17‐cell‐related autoimmune diseases. A crystallographic fragment screening was carried out for RORγt using the ligand binding domain. An overall hit rate of 5.5 % was obtained by screening 384 compounds in 96 cocktails. Five distinct hotspots were identified, and four regions of anchoring polar interactions were observed. In addition, significant induced fit was found for the binding of several fragments. Strikingly, a simultaneous binding of three fragments was revealed which presents interesting features including π–π stacking, multiple hydrogen bonds to the protein, and significant induced fit. Overall, the results offer a complete mapping of the ligand binding pocket and provide valuable inspiration in structure‐based design for RORγt lead generation and optimization. The crystallographic screening also resulted in fragment hits that bind at the surface away from the ligand binding pocket. This surface site is near the plausible dimer interface by analogy with other nuclear receptor systems, which can provide initial hints to explore alternative ways to modulate RORγt through protein–protein interactions.