First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Small-molecule inhibition of MAP2K4 is synergistic with RAS inhibitors in KRAS-mutant cancers.

The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.

Scaffold modified Vemurafenib analogues as highly selective mitogen activated protein kinase kinase 4 (MKK4) inhibitors.

The mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified as druggable target for the treatment of acute liver failure in RNAi experiments. In these experiments MKK4 was identified to be a major regulator in hepatocyte regeneration. Inhibitors thereof may serve as medication to promote liver regeneration or reducing hepatocyte death. Just a small number of potent inhibitors with acceptable selectivity towards relevant off-targets are known up to date. Among the known potent inhibitors, selectivity is highly sensitive towards minor modifications of the molecule, which makes it necessary to carefully balance between potency and selectivity. In the herein presented study, a new class of Vemurafenib-derived inhibitors was investigated with α-carbolines as new scaffold. This new scaffold showed a remarkable intrinsic selectivity towards the chosen off-targets, without affecting potency towards MKK4 on a broad range of structural modifications.

Design and synthesis of 1H-pyrazolo[3,4-b]pyridines targeting mitogen-activated protein kinase kinase 4 (MKK4) - A promising target for liver regeneration.

Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a major regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this protein kinase. Starting from the approved BRAFV600E inhibitor vemurafenib (8), that showed a high off-target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4 could be conserved while the selectivity against off-target protein kinases was slightly improved. Further modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7, JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective MKK4 inhibitors in this class.

From off-to on-target: New BRAF-inhibitor-template-derived compounds selectively targeting mitogen activated protein kinase kinase 4 (MKK4).

The mitogen-activated protein kinase (MAP) kinase 4 (MKK4) was found to be a major regulator of liver regeneration and could be a valuable drug target addressing liver related diseases by restoring its intrinsic regenerative capacity. We report on the synthesis and optimization of novel MKK4 inhibitors following a target-hopping strategy from the FDA-approved BRAFV600E inhibitor PLX4032 (8). Applying an iterative multi-parameter optimization process we carved out essential structural features yielding in compounds with a low nanomolar affinity for MKK4 and excellent selectivity profiles against the main off-targets MKK7 and JNK1, which, upon relevant inhibition, would totally abrogate the pro-regenerative effect of MKK4 inhibition, as well as against the off-targets MAP4K5, ZAK and BRAF with selectivity factors ranging from 40 to 430 for our best-balanced compounds 70 and 73.

Design and synthesis of novel fluorescently labeled analogs of vemurafenib targeting MKK4.

The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4.

Fluorescence polarization binding assay to develop inhibitors of inactive p38alpha mitogen-activated protein kinase.

Development of inhibitors that target inactive kinase conformations is becoming a more attractive approach to kinase inhibitor research. The major advantage of this methodology is that targeting the inactive conformation reduces competition with high intracellular adenosine triphosphate (ATP) concentrations. p38alpha Mitogen-activated protein kinase (MAPK) signaling has been identified as the principal mediator of inflammation associated with a spectrum of disorders (e.g., arthritis, Alzheimer's disease, various malignancies). To allow identification and development of p38alpha MAPK inhibitors that preferentially bind to the inactive conformation, a novel fluorescence polarization-based binding assay is presented. The assay is homogeneous, requires low amounts of the kinase and fluoroprobe, and does not rely on radioactivity. It may, therefore, offer an inexpensive alternative to current p38alpha MAPK inhibitor screening methods. The validation of the system with known p38alpha MAPK inhibitors confirmed that the binding assay, rather than the conventional enzyme activity assay, correlates with cellular efficacy. Finally, we show that pyridinyl imidazoles that potently bind to the inactive p38alpha MAPK prevent activation of p38 MAPK in living cells, suggesting that pyridinyl imidazoles other than SB203580 are able to induce the DFG-out conformation that is incompatible with activation (where DFG is a single-letter amino acid code for the aspartate-phenylalanine-glycine sequence at the start of the activation loop).

2-(4-Fluoro-phen-yl)-N-{4-[6-(4-fluoro-phen-yl)-2,3-dihydro-imidazo[2,1-b][1,3]thia-zol-5-yl]pyridin-2-yl}acetamide.

In the crystal structure of the title compound, C(24)H(18)F(2)N(4)OS, the imidazole system makes dihedral angles of 34.3 (1) and 43.9 (1)°, respectively, with the directly attached 4-fluoro-phenyl and pyridine rings. The crystal structure is stabilized by inter-molecular N-H⋯N hydrogen bonding and by an intra-molecular C-H⋯O hydrogen inter-action. The F atom of the 2-(4-fluoro-phen-yl) group is disordered over two positions with site-occupancy factors of 0.75 and 0.25.

4-Chloro-7-methoxy-methyl-2-phenyl-7H-pyrrolo[2,3-b]pyridine.

In the title compound, C(15)H(13)ClN(2)O, the phenyl group makes a dihedral angle of 7.91 (8)° with the pyrrole ring. The crystal structure forms a three-dimensional network stabilized by π-π inter-actions [centroid-centroid distances = 3.807 (1) Å] between the pyridine and phenyl rings and via inter-molecular C-H⋯O hydrogen bonds.

2-(Bicyclo-[2.2.1]hept-5-en-2-yl)-1H-pyrrolo-[2,3-b]pyridine.

The crystal structure of the title compound, C(14)H(14)N(2), displays inter-molecular N-H⋯N hydrogen bonds, forming dimers of enanti-omeric mol-ecules via a crystallographic centre of inversion.

7-[4-(4-Fluoro-phen-yl)-2-methyl-sulfanyl-1H-imidazol-5-yl]tetra-zolo[1,5-a]pyridine.

The crystal structure of the title compound, C(15)H(11)FN(6)S, forms a three-dimensional network stabilized by π-π inter-actions between the imidazole core and the tetra-zole ring of the tetra-zolopyridine-unit; the centroid-centroid distance is 3.627 (1) Å. The crystal structure also displays bifurcated N-H⋯(N,N) hydrogen bonding and C-H⋯F inter-actions. The former involve the NH H atom of the imidazole core and the tetra-zolopyridine N atoms, while the latter involve a methyl H atom, of the methyl-sulfanyl group, and the 4-fluoro-phenyl F atom. In the mol-ecule, the imidazole ring makes dihedral angles of 40.45 (9) and 17.09 (8)°, respectively, with the 4-fluoro-phenyl ring and the tetra-zolopyridine ring mean plane.

4-(4-Fluoro-phen-yl)-1-methoxy-methyl-2-phenyl-1H-imidazole.

In the crystal structure of the title compound, C(17)H(15)FN(2)O, the mol-ecules form a three-dimensional network stabilized by π-π inter-actions between two imidazole rings related by a centre of symmetry. The distance between the centroids is 3.5488 (8) Å. The imidazole ring makes dihedral angles of 14.30 (7) and 33.39 (7)° with the 4-fluoro-phenyl ring and the phenyl ring, respectively.

4-Chloro-1-(4-methyl-phenyl-sulfon-yl)-1H-pyrrolo[2,3-b]pyridine.

The crystal structure of the title compound, C(14)H(11)ClN(2)O(2)S, features a three-dimensional network stabilized by π-π inter-actions between the rings of the 4-methyl-phenyl-sulfonyl protecting group [centroid-centroid distance = 3.623 (1) Å]. The 4-methyl-phenyl-sulfonyl ring makes a dihedral angle of 79.60 (6)° with the 4-chloro-1H-pyrrolo[2,3-b]pyridine unit.

2-(3,4,5-Trimethoxy-phen-yl)-1H-pyrrolo[2,3-b]pyridine.

In the title compound, C(16)H(16)N(2)O(3), the 3,4,5-trimethoxy-phenyl group makes a dihedral angle of 10.04 (7)° toward the 1H-pyrrolo[2,3-b]pyridine system. The crystal structure displays inter-molecular N-H⋯N hydrogen bonds, forming inversion dimers.

(2Z)-2-Fluoro-N-{4-[5-(4-fluoro-phen-yl)-2-methyl-sulfanyl-1H-imidazol-4-yl]-2-pyrid-yl}-3-phenyl-acrylamide.

The asymmetric unit of the title compound, C(24)H(18)F(2)N(4)OS, contains two crystallographically independent mol-ecules, A and B, which are linked into two chains of A and B mol-ecules by inter-molecular N-H⋯O hydrogen bonds. The three-dimensional network is stabilized by π-π inter-actions between the pyridine rings and phenyl rings of different residues, with centroid-centroid distances of 3.793 (1) and 3.666 (2) Å. The mol-ecular conformation is stabilized by intra-molecular N-H⋯F hydrogen bonds (2.15/2.15Å). The imidazole rings make dihedral angles of 39.5 (2)/38.5 (2) and 31.8 (2)/33.2 (2)° with the 4-fluoro-phenyl rings and the pyridine rings, respectively. The methyl group of molecule A is disorderd in a 0.60:0.40 ratio.

A frozen analogue approach to aminopyridinylimidazoles leading to novel and promising p38 MAP kinase inhibitors.

In this study we report the design, synthesis, and biological evaluation of constrained aminopyridinylimidazoles as p38α MAP kinase inhibitors. The frozen analogue approach focused on the pyridinyl unit, using purine bioisosteres as constrained structure analogues. The identification of the most potent bioisostere was followed by a further derivatization to address hydrophobic region II. In combination with C-2 modifications of the imidazole core, we were able to design highly active inhibitors on the p38α MAP kinase. The inhibitor design presented herein represents a promising and highly efficient advancement of recent stages of development in this class of p38 MAP kinase inhibitors. In combination with the highly flexible synthetic strategy, directions toward further investigations of complex C-5 modifications of diarylimidazoles are indicated.

Design, synthesis, and biological evaluation of novel 3-aryl-4-(1H-indole-3yl)-1,5-dihydro-2H-pyrrole-2-ones as vascular endothelial growth factor receptor (VEGF-R) inhibitors.

In this study we report on the design, synthesis, and biological evaluation of pyrrole-2-one 2 to be a highly potent VEGF-R2/3 inhibitor with IC 50 of 31/37 nM. The novel 3,4-diaryl-2 H-pyrrole-2-ones were designed on the basis of the modeled binding mode of the corresponding 1 H-pyrrole-2,5-dione (maleimide) VEGF-R2/3 inhibitor 1 indicating two H-bond ligand-protein interactions in the ATP pocket for the amide 2 but not for the isomer 3. Flexible synthetic routes to 3,4-diaryl-2 H-pyrrole-2-ones and structure-activity relationships for the compounds in a panel of 24 therapeutically relevant protein kinases (IC 50 values) are presented. Accordingly to the in vitro data, compounds 1 and 2 were found to possess highly potent antiangiogenic activities in the cellular HLMEC sprouting assay and also slightly induced apoptosis in HDMECs whereas 3 was determined to be significantly less active. Hence, the pyrrole-2-one moiety was dissected from the corresponding maleimide protein kinase inhibitor as a suitable key pharmacophore.