Computationally-guided optimization of small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.
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ABSTRACT:
SUBMITTER: Cole DJ
PROVIDER: S-EPMC5591577 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Aurora kinases are a family of cell division regulators that govern the correct assembly of a bipolar mitotic spindle and the fidelity of chromosome segregation. Their overexpression is associated with genomic instability and aneuploidy, and is frequently observed in cancer. Accordingly, competitive inhibitors targeting Aurora kinase activity at the ATP-binding site are being investigated for therapeutic purposes. Despite promising pre-clinical data, these molecules display moderate effects in clinical trials and incomplete selectivity, either against distinct family members, or other kinases. As an alternative approach, protein-protein interaction inhibitors targeting mitotic kinases and their activators can be exploited to achieve increased specificity of action. In this study, a virtual screening of small molecules led to the identification of 25 potential inhibitors of the interaction between Aurora-A and its activator TPX2. In vitro experiments confirmed that 4 hits bind Aurora-A in the low micromolar range and compete for TPX2 binding. Immunofluorescence assays showed that 2 compounds also yield lowered Aurora-A activity and spindle pole defects in cultured osteosarcoma cells. The identified protein-protein interaction inhibitors of the Aurora-A/TPX2 complex might represent lead compounds for further development towards pioneering anti-cancer drugs and provide the proof-of-concept for a new exploitable strategy to target mitotic kinases.
| S-EPMC5458272 | biostudies-literature
Project description:The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.
| S-EPMC4919790 | biostudies-literature
Project description:Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and DNA-damage response (DDR). Inhibition of RPA-DNA interactions represents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to synergize with both common DNA damaging chemotherapeutics and newer targeted anticancer agents. In this letter, a new series of analogues based on our previously reported TDRL-551 (4) compound were designed to improve potency and physicochemical properties. Molecular docking studies guided molecular insights, and further SAR exploration led to the identification of a series of novel compounds with low micromolar RPA inhibitory activity, increased solubility, and excellent cellular up-take. Among a series of analogues, compounds 43, 44, 45, and 46 hold promise for further development of novel anticancer agents.
| S-EPMC7294550 | biostudies-literature
Project description:Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.
| S-EPMC3743924 | biostudies-literature
Project description:The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A.
| S-EPMC3001410 | biostudies-literature
Project description:Structure-based optimization was conducted to improve the potency, selectivity, and cell-based activities of ?-catenin/B-cell lymphoma 9 (BCL9) inhibitors based on the 4'-fluoro- N-phenyl-[1,1'-biphenyl]-3-carboxamide scaffold, which was designed to mimic the side chains of the hydrophobic ?-helical hot spots at positions i, i + 3, and i + 7. Compound 29 was found to disrupt the ?-catenin/BCL9 protein-protein interaction (PPI) with a Ki of 0.47 ?M and >1900-fold selectivity for ?-catenin/BCL9 over ?-catenin/E-cadherin PPIs. The proposed binding mode of new inhibitors was consistent with the results of site-directed mutagenesis and structure-activity relationship studies. Cell-based studies indicated that 29 disrupted the ?-catenin/BCL9 interaction without affecting the ?-catenin/E-cadherin interaction, selectively suppressed transactivation of Wnt/?-catenin signaling, downregulated expression of Wnt target genes, and inhibited viability of Wnt/?-catenin-dependent cancer cells in dose-dependent manners. A comparison of the biochemical and cell-based assay results offered the directions for future inhibitor optimization.
| S-EPMC7787347 | biostudies-literature
Project description:Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity.
| S-SCDT-10_1038-S44318-024-00240-Z | biostudies-other
Project description:We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX2(1-25). While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.DOI: http://dx.doi.org/10.7554/eLife.02667.001.
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Project description:The development of novel therapeutics is urgently required for diseases where existing treatments are failing due to the emergence of resistance. This is particularly pertinent for parasitic infections of the tropics and sub-tropics, referred to collectively as neglected tropical diseases, where the commercial incentives to develop new drugs are weak. One such disease is schistosomiasis, a highly prevalent acute and chronic condition caused by a parasitic helminth infection, with three species of the genus Schistosoma infecting humans. Currently, a single 40-year old drug, praziquantel, is available to treat all infective species, but its use in mass drug administration is leading to signs of drug-resistance emerging. To meet the challenge of developing new therapeutics against this disease, we developed an innovative computational drug repurposing pipeline supported by phenotypic screening. The approach highlighted several protein kinases as interesting new biological targets for schistosomiasis as they play an essential role in many parasite's biological processes. Focusing on this target class, we also report the first elucidation of the kinome of Schistosoma japonicum, as well as updated kinomes of S. mansoni and S. haematobium. In comparison with the human kinome, we explored these kinomes to identify potential targets of existing inhibitors which are unique to Schistosoma species, allowing us to identify novel targets and suggest approved drugs that might inhibit them. These include previously suggested schistosomicidal agents such as bosutinib, dasatinib, and imatinib as well as new inhibitors such as vandetanib, saracatinib, tideglusib, alvocidib, dinaciclib, and 22 newly identified targets such as CHK1, CDC2, WEE, PAKA, MEK1. Additionally, the primary and secondary targets in Schistosoma of those approved drugs are also suggested, allowing for the development of novel therapeutics against this important yet neglected disease.
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Project description:Using high concentration biochemical assays and fragment-based screening assisted by structure-guided design, we discovered a novel class of Rho-kinase inhibitors. Compound 18 was equipotent for ROCK1 (IC(50) = 650 nM) and ROCK2 (IC(50) = 670 nM), whereas compound 24 was more selective for ROCK2 (IC(50) = 100 nM) over ROCK1 (IC(50) = 1690 nM). The crystal structure of the compound 18-ROCK1 complex revealed that 18 is a type 1 inhibitor that binds the hinge region in the ATP binding site. Compounds 18 and 24 inhibited potently the phosphorylation of the ROCK substrate MLC2 in intact human breast cancer cells.
| S-EPMC4516226 | biostudies-literature