Hinge Interaction Research Articles

Design and synthesis of novel Thiazolo[5,4-b]pyridine derivatives as potent and selective EGFR-TK inhibitors targeting resistance Mutations in non-small cell lung cancer

A novel series of substituted thiazolo[5,4-b]pyridine analogues were rationally designed and synthesized via a multi-step synthetic pathway, including Suzuki cross-coupling reaction. The anticancer activity of all forty-five synthesized derivatives was evaluated against HCC827, H1975, and A549 cancer cell lines utilizing the standard MTT assay. A significant number of the thiazolo[5,4-b]pyridine derivatives exhibited potent anticancer activity. Notably, compounds 10b, 10c, 10h, 10i, and 10k emerged as the most promising anticancer agents. The lead compound, N-(3-(6-(2-aminopyrimidin-5-yl)thiazolo[5,4-b]pyridin-2-yl)-2-methylphenyl)-2,5-difluorobenzenesulfonamide (10k), displayed remarkable potency with IC50 values of 0.010 μM, 0.08 μM, and 0.82 μM against the HCC827, NCI–H1975 and A-549 cancer cell lines, respectively, which were comparable to the clinically approved drug Osimertinib. Importantly, the potent derivatives 10b, 10c, 10h, 10i, and 10k exhibited selective cytotoxicity towards cancer cells and showing no toxicity against the normal BEAS-2B cell line at concentrations exceeding 35 μM. Mechanistic studies revealed that the active compound 10k acts as an EGFR-TK autophosphorylation inhibitor in HCC827 cells. Furthermore, apoptosis assays demonstrated that compound 10k induced substantial early apoptosis (31.9 %) and late apoptosis (8.8 %) in cancer cells, in contrast to the control condition exhibiting only 2.0 % early and 1.6 % late apoptosis. Molecular docking simulations of the synthesized compounds revealed that they formed essential hinge interactions and established hydrogen bonding with Cys797, indicating potential target engagement. These findings highlight the potential of the synthesized thiazolo [(Woodburn, 1999; Zigrossi et al., 2022) 5,45,4-b]pyridine derivatives as promising anticancer agents, warranting further investigation for the development of novel targeted therapies against non-small cell lung cancer.

Suppressor screening reveals common kleisin–hinge interaction in condensin and cohesin, but different modes of regulation

Cohesin and condensin play fundamental roles in sister chromatid cohesion and chromosome segregation, respectively. Both consist of heterodimeric structural maintenance of chromosomes (SMC) subunits, which possess a head (containing ATPase) and a hinge, intervened by long coiled coils. Non-SMC subunits (Cnd1, Cnd2, and Cnd3 for condensin; Rad21, Psc3, and Mis4 for cohesin) bind to the SMC heads. Here, we report a large number of spontaneous extragenic suppressors for fission yeast condensin and cohesin mutants, and their sites were determined by whole-genome sequencing. Mutants of condensin's non-SMC subunits were rescued by impairing the SUMOylation pathway. Indeed, SUMOylation of Cnd2, Cnd3, and Cut3 occurs in midmitosis, and Cnd3 K870 SUMOylation functionally opposes Cnd subunits. In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1-mediated chromatin remodeling. In addition, distinct regulations were discovered for condensin and cohesin hinge mutants. Mutations in the N-terminal helix bundle [containing a helix-turn-helix (HTH) motif] of kleisin subunits (Cnd2 and Rad21) rescue virtually identical hinge interface mutations in cohesin and condensin, respectively. These mutations may regulate kleisin's interaction with the coiled coil at the SMC head, thereby revealing a common, but previously unknown, suppression mechanism between the hinge and the kleisin N domain, which is required for successful chromosome segregation. We propose that in both condensin and cohesin, the head (or kleisin) and hinge may interact and collaboratively regulate the resulting coiled coils to hold and release chromosomal DNAs.

Towards discovery of novel scaffold with potent antiangiogenic activity; design, synthesis of pyridazine based compounds, impact of hinge interaction, and accessibility of their bioactive conformation on VEGFR-2 activities

Pyridazine scaffolds are considered privileged structures pertaining to its novelty, chemical stability, and synthetic feasibility. In our quest towards the development of novel scaffolds for effective vascular endothelial growth 2 (VEGFR-2) inhibition with antiangiogenic activity, four novel series of pyridazines were designed and synthesised. Five of the synthesised compounds; namely (8c, 8f, 15, 18b, and 18c) exhibited potent VEGFR-2 inhibitory potency (>80%); with IC50 values ranging from low micromolar to nanomolar range; namely compounds 8c, 8f, 15, 18c with (1.8 µM, 1.3 µM, 1.4 µM, 107 nM), respectively. Moreover, 3-[4-{(6-oxo-1,6-dihydropyridazin-3-yl)oxy}phenyl]urea derivative (18b) exhibited nanomolar potency towards VEGFR-2 (60.7 nM). In cellular assay, the above compounds showed excellent inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells at 10 μM concentration. Finally, an extensive molecular simulation study was performed to investigate the probable interaction with VEGFR-2.

Novel Mycobacterium tuberculosis PknG inhibitors. A computational-experimental study

Background: Tuberculosis is a chronic disease caused by the bacillus Mycobacterium tuberculosis (Mtb) that remains a leading cause of mortality worldwide. The search for new protein targets and their inhibitory compounds became a priority due to the emergence of multidrug and extremely drug resistant strains. Pkns are the main kinase family in Mtb, consisting of 11 members including pknG, a protein that plays a central role in energy metabolism and the infection process making it an excellent target for drug design. Methods & Materials: In the present work, we have used docking and molecular dynamics simulations to search for a competitive inhibitor of the ATP binding site of pknG. Three different libraries of small compounds: 1) a standard purchasable compound library of 2.7 million compounds; 2) a kinase focused set from GSK consisting of 360 compounds, and 3) a small fragment based library of 600 compounds. The docking was performed with the rDock program using a pharmacophoric restraint on the kinase hinge interactions. Docking top rank compounds were subject to a protocol of dynamic undocking for further evaluation. Finally, the best 12 compounds from the first set, all the fragments, and the best 23 GSK compounds were tested for inhibition of in vitro kinase activity. Results: Until now we found actives in the fragment, including one fragment with IC50 near 100 μM; which is promising due to the small size of the compound. GSK set reported one compound with IC50 near 40 μM. Conclusion: The fragment based library was the one with the higher number of actives. We look forward to use the GSK compound core to future development of new inhibitors and also to further evolve the hit fragments into full-size drug like compounds.

Lactose repressor hinge domain independently binds DNA.

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ∼4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.

Monoclonal antibody fragment removal mediated by mixed mode resins

Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required.

Discovery and the structural basis of a novel p21-activated kinase 4 inhibitor

Functional versatility and elevated expression in cancers have endowed p21-activated kinase 4 (PAK4) as one of the first-in-class anti-cancer drug target. In this study, a novel PAK4 inhibitor, KY-04031 (N2-(2-(1H-indol-3-yl)ethyl)-N4-(1H-indazol-5-yl)-6-methoxy-1,3,5-triazine-2,4-diamine), was discovered using a high-throughput screening. Analysis of the complex crystal structure illustrated that both indole and indazole of KY-04031 are responsible for PAK4 hinge interaction. Moreover, the molecule’s triazine core was found to mimic the ribose of the natural ATP substrate. The cell-based anti-cancer potency of KY-04031 was less effective than the pyrroloaminopyrazoles; however, the unique molecular feature of KY-04031 can be exploited in designing new PAK4 inhibitors.

7,8-dichloro-1-oxo-β-carbolines as a versatile scaffold for the development of potent and selective kinase inhibitors with unusual binding modes.

Development of both potent and selective kinase inhibitors is a challenging task in modern drug discovery. The innate promiscuity of kinase inhibitors largely results from ATP-mimetic binding to the kinase hinge region. We present a novel class of substituted 7,8-dichloro-1-oxo-β-carbolines based on the distinct structural features of the alkaloid bauerine C whose kinase inhibitory activity does not rely on canonical ATP-mimetic hinge interactions. Intriguingly, cocrystal structures revealed an unexpected inverted binding mode and the presence of halogen bonds with kinase backbone residues. The compounds exhibit excellent selectivity over a comprehensive panel of human protein kinases while inhibiting selected kinases such as the oncogenic PIM1 at low nanomolar concentrations. Together, our biochemical and structural data suggest that this scaffold may serve as a valuable template for the design and development of specific inhibitors of various kinases including the PIM family of kinases, CLKs, DAPK3 (ZIPK), BMP2K (BIKE), and others.

Discovery of a novel class of non-ATP site DFG-out state p38 inhibitors utilizing computationally assisted virtual fragment-based drug design (vFBDD)

Discovery of a new class of DFG-out p38α kinase inhibitors with no hinge interaction is described. A computationally assisted, virtual fragment-based drug design (vFBDD) platform was utilized to identify novel non-aromatic fragments which make productive hydrogen bond interactions with Arg 70 on the αC-helix. Molecules incorporating these fragments were found to be potent inhibitors of p38 kinase. X-ray co-crystal structures confirmed the predicted binding modes. A lead compound was identified as a potent (p38α IC(50)=22 nM) and highly selective (≥ 150-fold against 150 kinase panel) DFG-out p38 kinase inhibitor.

Identification of Novel TSH Interaction Sites by Systematic Binding Analysis of the TSHR Hinge Region

In which ways the binding of the thyroid stimulating hormone to the extracellular domain of its receptor leads to activation of the thyroid-stimulating hormone receptor (TSHR) is currently only incompletely understood. It is known that TSH binding to the TSHR depends on the interaction with the leucine-rich repeat and sulfation at Y385 of the hinge region. Recently it was also shown that electrostatic interactions between positive charges of bovine (b) TSH and the residues E297, E303, and D382 of the hinge region contribute to hormone-TSHR binding. After the identification of these first TSH binding sites in the hinge region, it was apparent that multiple positions in this region remained to be characterized for their roles in hormone binding. The goal of this study was therefore to clarify whether further contact points of TSH exist in the structurally undefined hinge region. Therefore, we systematically analyzed 41 uncharacterized residues of the TSHR hinge region as single mutants regarding differences between cell surface expression and bTSH binding. Indeed, we identified further amino acids of the hinge region with influence on bTSH binding. Some of these contribute to a new binding domain from human TSHR position F381 to D386. These hinge mutants with influence on bTSH binding were also analyzed for binding of the superagonistic human TSH analog TR1401 demonstrating that these positions also have an impact on TR1401 binding. Moreover, side chain variations revealed that different amino acid properties like the negative charge, aromatic as well as hydrophilic characteristics, contribute to maintain the hormone-TSHR hinge interaction.

Both Interaction Surfaces within Cohesin's Hinge Domain Are Essential for Its Stable Chromosomal Association

SummaryBackgroundThe cohesin complex that mediates sister chromatid cohesion contains three core subunits: Smc1, Smc3, and Scc1. Heterotypic interactions between Smc1 and Smc3 dimerization domains create stable V-shaped Smc1/Smc3 heterodimers with a hinge at the center and nucleotide-binding domains (NBDs) at the ends of each arm. Interconnection of each NBD through their association with the N- and C-terminal domains of Scc1 creates a tripartite ring, within which sister DNAs are thought to be entrapped (the ring model). Crystal structures show that the Smc1/Smc3 hinge has a toroidal shape, with independent “north” and “south” interaction surfaces on an axis of pseudosymmetry. The ring model predicts that sister chromatid cohesion would be lost by transient hinge opening.ResultsWe find that mutations within either interface weaken heterodimerization of isolated half hinges in vitro but do not greatly compromise formation of cohesin rings in vivo. They do, however, reduce the residence time of cohesin on chromosomes and cause lethal defects in sister chromatid cohesion. This demonstrates that mere formation of rings is insufficient for cohesin function. Stable cohesion requires cohesin rings that cannot easily open.ConclusionsEither the north or south hinge interaction surface is sufficient for the assembly of V-shaped Smc1/Smc3 heterodimers in vivo. Any tendency of Smc proteins with weakened hinges to dissociate will be suppressed by interconnection of their NBDs by Scc1. We suggest that transient hinge dissociation caused by the mutations described here is incompatible with stable sister chromatid cohesion because it permits chromatin fibers to escape from cohesin rings.

Pyrimidine-based inhibitors of CaMKIIδ

Non-ATP competitive pyrimidine-based inhibitors of CaMKIIdelta were identified. Computational studies were enlisted to predict the probable mode of binding. The results of the computational studies led to the design of ATP competitive inhibitors with optimized hinge interactions. Inhibitors of this class possessed improved enzyme and cellular activity compared to early leads.

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC–hinge interactions were observed in constructs including at least one unpaired guanine at the 5′-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3′-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5′-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3′-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3′–hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3′-hinge was more important than the left. Increasing the length of the U3 hinge–ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5′- and 3′-hinge interactions with the ETS or by shifting the base pairing of the U3 3′-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3′-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing.