Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

The molecular interaction pattern of lenvatinib enables inhibition of wild-type or kinase-mutated FGFR2-driven cholangiocarcinoma.

Fibroblast growth factor receptor (FGFR)-2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.

Development of Selective Pyrido[2,3-d]pyrimidin-7(8H)-one-Based Mammalian STE20-Like (MST3/4) Kinase Inhibitors.

Mammalian STE20-like (MST) kinases 1-4 play key roles in regulating the Hippo and autophagy pathways, and their dysregulation has been implicated in cancer development. In contrast to the well-studied MST1/2, the roles of MST3/4 are less clear, in part due to the lack of potent and selective inhibitors. Here, we re-evaluated literature compounds, and used structure-guided design to optimize the p21-activated kinase (PAK) inhibitor G-5555 (8) to selectively target MST3/4. These efforts resulted in the development of MR24 (24) and MR30 (27) with good kinome-wide selectivity and high cellular potency. The distinct cellular functions of closely related MST kinases can now be elucidated with subfamily-selective chemical tool compounds using a combination of the MST1/2 inhibitor PF-06447475 (2) and the two MST3/4 inhibitors developed. We found that MST3/4-selective inhibition caused a cell-cycle arrest in the G1 phase, whereas MST1/2 inhibition resulted in accumulation of cells in the G2/M phase.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The reengineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

Shifting the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one inhibitors towards the salt-inducible kinase (SIK) subfamily.

Salt-inducible kinases 1-3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.

Do Go Chasing Waterfalls: Enoyl Reductase (FabI) in Complex with Inhibitors Stabilizes the Tetrameric Structure and Opens Water Channels.

The enzyme enoyl-ACP reductase (FabI) is the limiting step of the membrane's fatty acid biosynthesis in bacteria and a druggable target for novel antibacterial agents. The FabI active form is a homotetramer, which displays the highest affinity to inhibitors. Herein, molecular dynamics studies were carried out using the structure of FabI in complex with known inhibitors to investigate their effects on tetramerization. Our results suggest that multimerization is essential for the integrity of the catalytic site and that inhibitor binding enables the multimerization by stabilizing the substrate binding loop (SBL, L:195-200) coupled with changes in the H4/5 (QR interface). We also observed that AFN-1252 (naphtpyridinone derivative) promotes unique conformational changes affecting monomer-monomer interfaces. These changes are induced by AFN-1252 interaction with key residues in the binding sites (Ala95, Tyr146, and Tyr156). In addition, the analysis of water trajectories indicated that AFN-1252 complexes allow more water molecules to enter the binding site than triclosan and MUT056399 complexes. FabI-AFN-1252 simulations show accumulation of water molecules near the Tyr146/147 pocket, which can become a hotspot to the design of novel FabI inhibitors.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a ß-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 µM) and C11 (EC50 = 11.4 µM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 µM) and 27 (LN6023, EC50 = 3.5 µM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Regulatory spine RS3 residue of protein kinases: a lipophilic bystander or a decisive element in the small-molecule kinase inhibitor binding?

In recent years, protein kinases have been one of the most pursued drug targets. These determined efforts have resulted in ever increasing numbers of small-molecule kinase inhibitors reaching to the market, offering novel treatment options for patients with distinct diseases. One essential component related to the activation and normal functionality of a protein kinase is the regulatory spine (R-spine). The R-spine is formed of four conserved residues named as RS1-RS4. One of these residues, RS3, located in the C-terminal part of αC-helix, is usually accessible for the inhibitors from the ATP-binding cavity as its side chain is lining the hydrophobic back pocket in many protein kinases. Although the role of RS3 has been well acknowledged in protein kinase function, this residue has not been actively considered in inhibitor design, even though many small-molecule kinase inhibitors display interactions to this residue. In this minireview, we will cover the current knowledge of RS3, its relationship with the gatekeeper, and the role of RS3 in kinase inhibitor interactions. Finally, we comment on the future perspectives how this residue could be utilized in the kinase inhibitor design.

The autoinhibited state of MKK4: Phosphorylation, putative dimerization and R134W mutant studied by molecular dynamics simulations.

Protein kinases are crucial components of the cell-signalling machinery that orchestrate and convey messages to their downstream targets. Most often, kinases are activated upon a phosphorylation to their activation loop, which will shift the kinase into the active conformation. The Dual specificity mitogen-activated protein kinase kinase 4 (MKK4) exists in a unique conformation in its inactive unphosphorylated state, where its activation segment appears in a stable α-helical conformation. However, the precise role of this unique conformational state of MKK4 is unknown. Here, by all-atom molecular dynamics simulations (MD simulations), we show that this inactive state is unstable as monomer even when unphosphorylated and that the phosphorylation of the activation segment further destabilizes the autoinhibited α-helix. The specific phosphorylation pattern of the activation segment has also a unique influence on MKK4 dynamics. Furthermore, we observed that this specific inactive state is stable as a dimer, which becomes destabilized upon phosphorylation. Finally, we noticed that the most frequent MKK4 mutation observed in cancer, R134W, which role has not been disclosed to date, contributes to the dimer stability. Based on these data we postulate that MKK4 occurs as a dimer in its inactive autoinhibited state, providing an additional layer for its activity regulation.