Understanding the Differences of Danusertib's Residence Time in Aurora Kinases A/B: Dissociation Paths and Key Residues Identified using Conventional and Enhanced Molecular Dynamics Simulations.

The accurate experimental estimation of protein-ligand systems' residence time (τ) has become very relevant in drug design projects due to its importance in the last stages of refinement of the drug's pharmacodynamics and pharmacokinetics. It is now well-known that it is not sufficient to estimate the affinity of a protein-drug complex in the thermodynamic equilibrium process in in vitro experiments (closed systems), where the concentrations of the drug and protein remain constant. On the contrary, it is mandatory to consider the conformational dynamics of the system in terms of the binding and unbinding processes between protein and drugs in in vivo experiments (open systems), where their concentrations are in constant flux. This last model has been proven to dictate much of several drugs' pharmacological activities in vivo. At the atomistic level, molecular dynamics simulations can explain why some drugs are more effective than others or unveil the molecular aspects that make some drugs work better in one molecular target. Here, the protein kinases Aurora A/B, complexed with its inhibitor Danusertib, were studied using conventional and enhanced molecular dynamics (MD) simulations to estimate the dissociation paths and, therefore, the computational τ values and their comparison with experimental ones. Using classical molecular dynamics (cMD), three differential residues within the Aurora A/B active site, which seems to play an essential role in the observed experimental Danusertib's residence time against these kinases, were characterized. Then, using WT-MetaD, the relative Danusertib's residence times against Aurora A/B kinases were measured in a nanosecond time scale and were compared to those τ values observed experimentally. In addition, the potential dissociation paths of Danusertib in Aurora A and B were characterized, and differences that might be explained by the differential residues in the enzyme's active sites were found. In perspective, it is expected that this computational protocol can be applied to other protein-ligand complexes to understand, at the molecular level, the differences in residence times and amino acids that may contribute to it.

Derivation of stationary distributions of biochemical reaction networks via structure transformation.

Long-term behaviors of biochemical reaction networks (BRNs) are described by steady states in deterministic models and stationary distributions in stochastic models. Unlike deterministic steady states, stationary distributions capturing inherent fluctuations of reactions are extremely difficult to derive analytically due to the curse of dimensionality. Here, we develop a method to derive analytic stationary distributions from deterministic steady states by transforming BRNs to have a special dynamic property, called complex balancing. Specifically, we merge nodes and edges of BRNs to match in- and out-flows of each node. This allows us to derive the stationary distributions of a large class of BRNs, including autophosphorylation networks of EGFR, PAK1, and Aurora B kinase and a genetic toggle switch. This reveals the unique properties of their stochastic dynamics such as robustness, sensitivity, and multi-modality. Importantly, we provide a user-friendly computational package, CASTANET, that automatically derives symbolic expressions of the stationary distributions of BRNs to understand their long-term stochasticity.

Aurora Kinase B Inhibition: A Potential Therapeutic Strategy for Cancer.

Aurora kinase B (AURKB) is a mitotic serine/threonine protein kinase that belongs to the aurora kinase family along with aurora kinase A (AURKA) and aurora kinase C (AURKC). AURKB is a member of the chromosomal passenger protein complex and plays a role in cell cycle progression. Deregulation of AURKB is observed in several tumors and its overexpression is frequently linked to tumor cell invasion, metastasis and drug resistance. AURKB has emerged as an attractive drug target leading to the development of small molecule inhibitors. This review summarizes recent findings pertaining to the role of AURKB in tumor development, therapy related drug resistance, and its inhibition as a potential therapeutic strategy for cancer. We discuss AURKB inhibitors that are in preclinical and clinical development and combination studies of AURKB inhibition with other therapeutic strategies.

Heteroarene-fused anthraquinone derivatives as potential modulators for human aurora kinase B.

The quest for effective anticancer therapeutics continues to be extensively pursued. Over the past century, several drugs have been developed, however, a majority of these drugs have a poor therapeutic index and increased toxicity profile. Hence, there still exists ample opportunity to discover safe and effective anticancer drugs. Aurora Kinase B (AurB), a member of the Aurora kinase family and a key regulator of mitotic cell division, is found to be frequently overexpressed in a variety of human cancers and has thus emerged as an attractive target for the design of anticancer therapeutics. In the present study, a structure-based scaffold hopping approach was utilized to modify the heterocyclic moiety of (S)-3-(3-aminopyrrolidine-1-carbonyl)-4,11-dihydroxy-2-methylanthra [2,3-b]furan-5,10-dione (anthrafuran 1) to generate a series of heteroarene-fused anthraquinone derivatives, which were then subjected to virtual screening for the identification of potential AurB inhibitors. The obtained hits were subsequently synthesized and evaluated by using a combination of in silico and biophysical techniques for elucidating their in vitro binding and inhibition activity with recombinantly expressed AurB. Four identified hits presented an improved binding profile as compared to their parent analog anthrafuran 1. One derivative, anthrathiophene 2 demonstrated excellent in vitro inhibition of AurB (7.3 µM).

Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle.

The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations. Centromere recruitment of the CPC requires interaction of survivin with histone H3 phosphorylated at threonine 3, and we provide a complete structure of this assembly. Furthermore, we show that the INCENP RRKKRR-motif is required for both centromeric localization of the CPC in metaphase and MKLP2-dependent transport in anaphase. MKLP2 and DNA bind competitively to this motif, and INCENP T59 phosphorylation acts as a switch preventing MKLP2 binding in metaphase. In anaphase, CPC binding promotes the microtubule-dependent ATPase activity of MKLP2. These results explain how centromere targeting of the CPC in mitosis is coupled to its movement to the central spindle in anaphase.

Theoretical Studies Aimed at Finding FLT3 Inhibitors and a Promising Compound and Molecular Pattern with Dual Aurora B/FLT3 Activity.

FLT3 and dual Aurora B/FLT3 inhibitors have shown relevance in the search for promising new anticancer compounds, mainly for acute myeloid leukemia (AML). This study was designed to investigate the interactions between human FLT3 in the kinase domain with several indolin-2-one derivatives, structurally similar to Sunitinib. Molegro Virtual Docker (MVD) software was utilized in docking analyses. The predicted model of the training group, considering nineteen amino acid residues, performed in Chemoface, achieved an R2 of 0.82, suggesting that the binding conformations of the ligands with FLT3 are reasonable, and the data can be used to predict the interaction energy of other FLT3 inhibitors with similar molecular patterns. The MolDock Score for energy for compound 1 showed more stable interaction energy (-233.25 kcal mol-1) than the other inhibitors studied, while Sunitinib presented as one of the least stable (-160.94 kcal mol-1). Compounds IAF70, IAF72, IAF75, IAF80, IAF84, and IAF88 can be highlighted as promising derivatives for synthesis and biological evaluation against FLT3. Furthermore, IAF79 can be considered to be a promising dual Aurora B/FLT3 inhibitor, and its molecular pattern can be exploited synthetically to search for new indolin-2-one derivatives that may become drugs used in the treatment of cancers, including AML.

The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition.

The Aurora B abscission checkpoint delays cytokinesis until resolution of DNA trapped in the cleavage furrow. This process involves PKCε phosphorylation of Aurora B S227. Assessing if this PKCε-Aurora B module provides a more widely exploited genome-protective control for the cell cycle, we show Aurora B phosphorylation at S227 by PKCε also occurs during mitosis. Expression of Aurora B S227A phenocopies inhibition of PKCε in by-passing the delay and resolution at anaphase entry that is associated with non-disjunction and catenation of sister chromatids. Implementation of this anaphase delay is reflected in PKCε activation following cell cycle dependent cleavage by caspase 7; knock-down of caspase 7 phenocopies PKCε loss, in a manner rescued by ectopically expressing/generating a free PKCε catalytic domain. Molecular dynamics indicates that Aurora B S227 phosphorylation induces conformational changes and this manifests in a profound switch in specificity towards S29 TopoIIα phosphorylation, a response necessary for catenation resolution during mitosis.

Structural Characterization of the Aurora Kinase B "DFG-flip" Using Metadynamics.

Aurora kinase B (AKB), a Ser/Thr kinase that plays a crucial role in mitosis, is overexpressed in several cancers. Clinical inhibitors targeting AKB bind to the active DFG "in" conformation of the kinase. It would be beneficial, however, to understand if AKB is susceptible to type II kinase inhibitors that bind to the inactive, DFG "out" conformation, since type II inhibitors achieve higher kinome selectivity and higher potency in vivo. The DFG "out" conformation of AKB is not yet experimentally determined which makes the design of type II inhibitors exceedingly difficult. An alternate approach is to simulate the DFG "out" conformation from the experimentally determined DFG "in" conformation using atomistic molecular dynamics (MD) simulation. In this work, we employed metadynamics (MTD) approach to simulate the DFG "out" conformation of AKB by choosing the appropriate collective variables. We examined structural changes during the DFG-flip and determined the interactions crucial to stabilize the kinase in active and inactive states. Interestingly, the MTD approach also identified a unique transition state (DFG "up"), which can be targeted by small molecule inhibitors. Structural insights about these conformations is essential for structure-guided design of next-generation AKB inhibitors. This work also emphasizes the usefulness of MTD simulations in predicting macromolecular conformational changes at reduced computational costs.

Allosteric modulation of a human protein kinase with monobodies.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

The depolymerase activity of MCAK shows a graded response to Aurora B kinase phosphorylation through allosteric regulation.

Kinesin-13 motors regulate precise microtubule dynamics and limit microtubule length throughout metazoans by depolymerizing microtubule ends. Recently, the kinesin-13 motor family member MCAK (also known Kif2C) has been proposed to undergo large conformational changes during its catalytic cycle, as it switches from being in solution to being bound to microtubules. Here, we reveal that MCAK has a compact conformation in solution through crosslinking and electron microscopy experiments. When MCAK is bound to the microtubule ends, it adopts an extended conformation with the N-terminus and neck region of MCAK interacting with the microtubule. Interestingly, the region of MCAK that interacts with the microtubule is the region phosphorylated by Aurora B and contains an end binding (EB) protein-binding motif. The level of phosphorylation of the N-terminus results in a graded microtubule depolymerase activity. Here, we show that the N-terminus of MCAK forms a platform to integrate Aurora B kinase downstream signals and in response fine-tunes its depolymerase activity during mitosis. We propose that this allosteric control mechanism allows decoupling of the N-terminus from the motor domain of MCAK to allow MCAK depolymerase activity at kinetochores.

KNL1 Binding to PP1 and Microtubules Is Mutually Exclusive.

The kinetochore scaffold 1 (KNL1) protein coordinates the spindle assembly checkpoint (SAC), a signaling pathway that delays chromosome segregation until all sister chromatids are properly attached to spindle microtubules. Recently, microtubules and protein phosphatase 1 (PP1), which both bind the N-terminal domain of KNL1, have emerged as regulators of the SAC; however, how these proteins interact to contribute to SAC signaling is unknown. Here, we use X-ray crystallography, nuclear magnetic resonance spectroscopy, and biochemical assays to show how KNL1 binds both PP1 and microtubules. Unexpectedly, we discovered that PP1 and microtubules bind KNL1 via overlapping binding sites. Further, we showed that Aurora B kinase phosphorylation results in distinct patterns of KNL1 complex disruption. Finally, combining this data with co-sedimentation assays unequivocally demonstrated that microtubules and PP1 binding to KNL1 is mutually exclusive, with preferential formation of the KNL1:PP1 holoenzyme in the presence of PP1.

Phosphomimetic Mutation Destabilizes the Central Core Domain of Human p53.

Transcriptional activity of p53 is modulated by various posttranslational modifications. Earlier studies have reported that Aurora B phosphorylation of p53 leads to loss of its transcriptional activity, subsequently leading to its ubiquitin-mediated proteasomal degradation. To decipher the fate of structural and functional stature of p53 upon phosphorylation by Aurora B, we have generated five phosphomimetic mutants of p53 core domain and characterized their biophysicochemical properties. Our biophysical studies show that the T211E, S215E, and S269E mutants are thermally unstable and show a higher propensity toward aggregation than WT with the loss of DNA binding except for S183E. These results indicate structural and functional destabilization of p53 upon phosphomimetic substitution, which provides a molecular basis toward understanding the process that drives the fate of p53 upon phosphorylation by Aurora B kinase. © 2018 IUBMB Life, 70(10):1023-1031, 2018.

Theoretical Studies on Azaindoles as Human Aurora B Kinase Inhibitors: Docking, Pharmacophore and ADMET Studies.

Aurora kinases are the cell cycle mitotic regulators processing multiple functions during cell division. Altered mechanism of these mitotic kinases may contribute to genomic instability that is most often correlated with tumorigenesis, which has been reported in many human cancers. Selective blockage of the aberrantly expressed Aurora kinases has the potential therapeutic assessment to control the deregulated cell cycle machinery and their associated risks of cancer. Using a combination of docking-, ligand- and structure-based pharmacophore strategies, in the present study, we have tried to predict the anticancer potentiality of our synthesized compounds (A1 to A5 and B1 to B9) against human Aurora B kinase. The results revealed that among all the compounds, compound B7 may act as a best candidate to be an agent of the high binding affinity with a score of 113.464 kcal/mol and good pharmacophoric features with acceptable fit values of both ligand- and structure-based pharmacophore models. Consequently, ADMET properties are also calculated to predict the safer efficacy of the compounds.

PKCɛ switches Aurora B specificity to exit the abscission checkpoint.

The 'NoCut', or Aurora B abscission checkpoint can be activated if DNA is retained in the cleavage furrow after completion of anaphase. Checkpoint failure leads to incomplete abscission and a binucleate outcome. These phenotypes are also observed after loss of PKCɛ in transformed cell models. Here we show that PKCɛ directly modulates the Aurora B-dependent abscission checkpoint by phosphorylating Aurora B at S227. This phosphorylation invokes a switch in Aurora B specificity, with increased phosphorylation of a subset of target substrates, including the CPC subunit Borealin. This switch is essential for abscission checkpoint exit. Preventing the phosphorylation of Borealin leads to abscission failure, as does expression of a non-phosphorylatable Aurora B S227A mutant. Further, depletion of the ESCRT-III component and Aurora B substrate CHMP4C enables abscission, bypassing the PKCɛ-Aurora B exit pathway. Thus, we demonstrate that PKCɛ signals through Aurora B to exit the abscission checkpoint and complete cell division.

Antitumor activity of TY-011 against gastric cancer by inhibiting Aurora A, Aurora B and VEGFR2 kinases.

BACKGROUND: Overexpression of Aurora A and B has been reported in a wide range of tumor types, including gastric cancer. Anti-angiogenesis has been considered as an important therapeutic modality in advanced gastric cancer. Here we identified a novel compound TY-011 with promising antitumor activity by targeting mitotic kinases (Aurora A and B) and angiogenic receptor tyrosine kinase (VEGFR2). METHODS: HTRF® KinEASE™ assay was used to detect the effect of TY-011 against Aurora A, Aurora B and VEGFR2 activities. Docking simulation study was performed to predict the binding mode of TY-011 with Aurora A and B kinases. CCK-8 assay was used to test cell growth. Cell cycle and cell apoptosis was analyzed by flow cytometry. Gastric cancer cell xenograft mouse models were used for in vivo study. TUNEL kit was used to determine the apoptosis of tumor tissues. Immunohistochemistry analysis and HUVEC tube formation assay were performed to determine the anti-angiogenesis ability. Immunofluorescence and western blot were used to test protein expression. RESULTS: TY-011 was identified as a potential Aurora A and B inhibitor by HTRF® KinEASE™ assay. It effectively inhibited cellular Aurora A and B activities in a concentration-dependent manner. TY-011 occupied the ATP-binding site of both Aurora A and B kinases. TY-011 demonstrated prominent inhibitory effects on proliferation of gastric cancer cells. TY-011 treatment induced an obvious accumulation of cells at G2/M phase and a modest increase of cells with >4 N DNA content, which then underwent apoptosis. Meaningfully, orally administration of TY-011 demonstrated superior efficacy against the tumor growth in gastric cancer cell xenograft, with ~90% inhibition rate and 100% tumor regression at 9 mg/kg dose, and TY-011 did not affect the body weight of mice. Interestingly, we observed that TY-011 also antagonized tumor angiogenesis by targeting VEGFR2 kinase. CONCLUSIONS: These results indicate that TY-011 is a well-tolerated, orally active compound that targets mitosis and angiogenesis in tumor growth, and provides strong preclinical support for use as a therapeutic for human gastric cancers.

AIM-1: An Antibiotic-Degrading Metallohydrolase That Displays Mechanistic Flexibility.

Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-ß-lactamases (MBL) have evolved to inactivate most of the commonly used ß-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.

Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase.

Monopolar spindle 1 (Mps1, also known as TTK) is a protein kinase crucial for ensuring that cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules. Incomplete chromosomal attachment leads to abnormal chromosome counts in the daughter cells (aneuploidy), a condition common in many solid cancers. Therefore Mps1 is an established target in cancer therapy. Mps1 kinase inhibitors include reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine), a promiscuous compound first recognized as an inhibitor of the Aurora B mitotic kinase. Here, we present the 3.0-Å resolution crystal structure of the Mps1 kinase domain bound to reversine. Structural comparison of reversine bound to Mps1 and Aurora B, indicates a similar binding pose for the purine moiety of reversine making three conserved hydrogen bonds to the protein main chain, explaining the observed promiscuity of this inhibitor. The cyclohexyl and morpholinoaniline moieties of reversine however, have more extensive contacts with the protein in Mps1 than in Aurora B. This is reflected both in structure-based docking energy calculations, and in new experimental data we present here, that both confirm that the affinity of reversine towards Mps1 is about two orders of magnitude higher than towards Aurora B. Thus, our data provides detailed structural understanding of the existing literature that argues reversine inhibits Mps1 more efficiently than Aurora B based on biochemical and in-cell assays. Proteins 2016; 84:1761-1766. © 2016 Wiley Periodicals, Inc.

Aurora-C Interactions with Survivin and INCENP Reveal Shared and Distinct Features Compared with Aurora-B Chromosome Passenger Protein Complex.

Aurora-C, a member of the Aurora kinase family that can complement Aurora-B function in mitosis is either moderately expressed or repressed in most adult somatic tissues but is active in early embryonic development and expressed at elevated levels in multiple human cancers. Aurora-C overexpression reportedly plays a role in tumorigenic transformation. We performed detailed characterization of Aurora-C interactions with members of the Chromosome Passenger Complex (CPC), Survivin and Inner Centromere Protein (INCENP) in reference to known Aurora-B interactions to understand the functional significance of Aurora-C overexpression in human cancer cells. The results revealed that silencing of Aurora-C or -B individually does not affect localization of the other kinase and the two kinases exist predominantly in independent complexes in vivo. Presence of Aurora-C and -B in molecular complexes of varying as well as overlapping sizes and co-existence in INCENP overexpressing cells indicated oligomerization of ternary complexes under different physiological conditions in vivo. Furthermore, Aurora-C and -B stabilized INCENP through interaction with and phosphorylation of the IN box domain while Aurora-C was activated following Survivin phosphorylation on Serine 20. Phosphorylation of Survivin residue Serine 20 by Aurora-C and -B appears important for proper chromosome segregation. Taken together, our study suggests that Aurora-C, expressed at low levels in somatic cells, functions as a catalytic component of the CPC together with Aurora-B through mitosis. Elevated expression of Aurora-C in cancer cells alters the structural and functional characteristics of the Aurora-B-CPC leading to chromosomal instability.

A Dual Non-ATP Analogue Inhibitor of Aurora Kinases A and B, Derived from Resorcinol with a Mixed Mode of Inhibition.

Aurora kinases are the most commonly targeted mitotic kinases in the intervention of cancer progression. Here, we report a resorcinol derivative, 5-methyl-4-(2-thiazolylazo) resorcinol (PTK66), a dual inhibitor of Aurora A and Aurora B kinases. PTK66 is a surface binding non-ATP analogue inhibitor that shows a mixed pattern of inhibition against both of Aurora A and B kinases. The in vitro IC50 is approximately 47 and 40 µm for Aurora A and Aurora B kinases, respectively. In cellular systems, PTK66 exhibits a substantially low cytotoxicity at micromolar concentrations but it can induce aneuploidy under similar dosages as a consequence of Aurora kinase inhibition. This result was corroborated by a drop in the histone H3 (S10) phosphorylation level detected via Western blot analysis using three different cell types. Altogether, our findings indicate that the ligand containing resorcinol backbone is one of the novel scaffolds targeting the Aurora family of kinases, which could be a target for antineoplastic drug development.

Secondary ubiquitin-RING docking enhances Arkadia and Ark2C E3 ligase activity.

RING-domain E3 ligases enhance transfer of ubiquitin to substrate proteins by stabilizing the RING-bound thioester-linked E2∼ubiquitin conjugate in a defined conformation that primes the active site for nucleophilic attack. Here we report that the monomeric RING domains from the human E3 ligases Arkadia and Ark2C bind directly to free ubiquitin with an affinity comparable to that of other dedicated ubiquitin-binding domains. Further work showed that the Ark-like RING domain and the noncovalently bound ubiquitin molecule coordinately stabilize the E2-conjugated ubiquitin (donor ubiquitin) in the 'closed' conformation. Our studies identify the RING domain of Arkadia as a ubiquitin-binding domain and provide insight into a new ubiquitin-dependent mechanism used by monomeric RING domains to activate ubiquitin transfer. This study also suggests how substrates that have been monoubiquitinated could be favored for further ubiquitination.