Structural basis for the design of allosteric inhibitors of the Aurora kinase A enzyme in the cancer chemotherapy

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Structural basis for the design of allosteric inhibitors of the Aurora kinase A enzyme in the cancer chemotherapy Valéria Barbosa de Souzaa, Daniel Fábio Kawanob,c,

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a

Department of Pharmacology, Faculty of Medical Sciences, University of Campinas - Unicamp, Campinas, SP, Brazil Faculty of Pharmaceutical Sciences, University of Campinas - Unicamp, Campinas, SP, Brazil c Institute of Chemistry, University of Campinas - Unicamp, Campinas, SP, Brazil b

ARTICLE INFO

BACKGROUND

Keywords: Aurora kinase A TPX2 Cancer Cell cycle Allosteric inhibitors

Aurora kinases are essential enzymes for the control of cell cycle. The specific role of aurora kinase A (AURKA) is the regulation of spindle assembly and stability by promoting centrosome maturation and separation. Because AURKA is an essential protein for the normal occurrence of the cycle, mutations and deregulations in the activities of this protein are associated with several cancers. The kinase activity of AURKA is controlled by autocatalytic phosphorylation, which is facilitated after binding to a regulator protein, the Target Protein for Xenopuskinesin-like protein 2 (TPX2). Scope of review: This review highlights the physiological and pathophysiological properties of AURKA, the structure of the AURKA/TPX2 complex and the main structural features that can be explored for the design of selective AURKA inhibitors. Major conclusions: The design of selective AURKA inhibitors remains as a challenge as most of the currently available inhibitors target only the ATP binding cleft and are nonselective among kinases. However, by exploring the inactive form of the kinase, researchers get access to an adjacent hydrophobic pocket, allowing the design of more selective inhibitors. Additionally, the possibility of designing potent allosteric AURKA inhibitors look very promising from the clinical perspective, since it tends to yield the most selective class of compounds. General significance: Herein we detailed the binding modes of the most selective AURKA inhibitors currently reported. We believe this will aid researchers in defining the structural patterns necessary for selective AURKA inhibition, guiding the design of more potent compounds to be therapeutically explored in cancer patients.

1. Introduction Aurora kinases are a family of highly conserved serine/threonine kinases involved in the control of the cell cycle and that are related to the regulation of some essential processes necessary for cell division, such as: (1) mitotic spindle assembly checkpoint, (2) centromere separation, (3) transition between cell cycle phases G2 and M, (4) chromatin modification (5) chromatid separation and (6) cytokinesis. The human genome contains genes that encode three different aurora kinases: aurora kinase A (AURKA), B (AURKB) and C (AURKC), which share a high degree of sequence identity in the kinase domain (70% amino acid identity between AURKA and AURKB) but with distinct subcellular localizations [1–3]. All the three kinases are observed at the centrosome in different phases of mitosis but the role of AURKA is in the regulation of spindle assembly and stability by promoting centrosome maturation and

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separation. On the other hand, AURKB is a chromosomal passenger protein (CPC), i.e., it regulates chromosome segregation during cytokinesis by forming a complex with three other CPCs, borealin, survivin and the inner centromere protein (INCENP), which control the targeting, enzymatic activity and stability of AURKB. In adults, AURKC is predominantly expressed in the testes, with a subcellular localization similar to AURKB. However, AURKC is highly expressed during the early cell divisions and it seems to be responsible for the essential CPC functions in the embryonic development [4,5]. AURKA and AURKB exhibit oncogenic properties and are overexpressed in many types of cancers, especially in polyploid cells containing multiple centrosomes, while the role of AURKC in cancer remains controversial [6]. However, AURKA has been the kinase of major interest for the development of new drugs because it fits the criteria of a reliable oncogene. This kinase maps to the chromosomal region 20q13.2, which is amplified in several types of primary tumors and cancer cell lines.

Corresponding author at: Faculty of Pharmaceutical Sciences, University of Campinas - Unicamp. Rua Cândido Portinari 200, 13083-871 Campinas, SP, Brazil. E-mail addresses: [email protected], [email protected] (D.F. Kawano).

https://doi.org/10.1016/j.bbagen.2019.129448 Received 4 May 2019; Received in revised form 18 October 2019; Accepted 22 October 2019 0304-4165/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Valéria Barbosa de Souza and Daniel Fábio Kawano, BBA - General Subjects, https://doi.org/10.1016/j.bbagen.2019.129448

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Aberrant expression of AURKA results in the interruption of mitotic progression, apparently due to the loss of the ability of the chromosomes to be properly oriented in the spindle [5]. Despite the impairment in the chromosomal alignment, these cells exit mitosis since the overexpression of AURKA also inactivates the spindle assembly checkpoint, then promoting the failure of cytokinesis and tetraploid progeny. In cells that do not express p53, they will continue through the subsequent stages of the cell cycle, eventually becoming polyploid and aneuploid, with amplified centrosomes [5,7].

Additionally, there is a strong evidence linking therapeutic resistance to a subpopulation of cells with the stem cell phenotype and the overexpression of AURKA is associated with cancer stem cells (CSCs), then contributing to the maintenance of their properties such as self-renewal, heterogeneity and differentiation [19]. AURKA can activate the Wnt signaling pathways by interacting with the AXIN protein and stabilizing the β-catenin. The β-catenin/TCF4 (T-cell factor) complex also may activate AURKA by the transcription of its gene targets and the inhibition of the Glycogen synthase kinase 3β (GSK3β). Therefore, the AURKA/Wnt signal pathway forms a positive regulation loop that strengthens the expression of CSCs [7]. Overexpression of AURKA and its association with genetic instability and aneuploidy in tumors suggests that a wide range of neoplasms would respond therapeutically to their inhibitors. Several pharmaceutical companies and research institutions reported the development of AURKA and AURKB inhibitors and, although cell death was observed due to the inhibition of both targets, such inhibitors would induce apoptosis by distinct mechanisms [20]. Some researchers have also focused on the ability of AURKA inhibitors in potentiating the effect of traditional chemotherapies, and several specific or pan-aurora inhibitors have been developed and submitted to clinical evaluation [2]. Nevertheless, AURKA has not yet been extensively explored, both in terms of drug development and in terms of the general understanding of its biological role in cellular pathophysiology.

2. Physiological and pathophysiological roles of Aurora kinase A The Aurora kinase A (AURKA) is also known as Aurora-2, serine/ threonine kinase 15 (STK15), serine/threonine kinase 6 (STK6), breast tumor amplified kinase (BTAK), aurora-related kinase 1 (ARK1), Homo sapiens Aurora/IPL1-related kinase (HsAirk1), Eg2, IpI and Aurora kinase 1 (IAK1). All these nomenclatures indicate that it is a member of the Aurora/IPL1-related kinase family of the serine/threonine kinases [2]. The first denomination of Aurora was Ipl (increase-inploidy 1), identified in 1993 in Saccharomyces cerevisiae [8]. AURKA plays multiple roles in cell division in the transition from the phases G2 to M, including the separation of the centromere, maturation and assembly of the spindles [9]. During mitosis, AURKA is positioned at the poles of the mitotic spindle and along the spindle microtubules, where it promotes the duplication and separation of the centrosomes, the main microtubule organizing centers of the cell. In this stage, AURKA assure the proper formation of the spindle by recruiting and activating through phosphorylation several target proteins such as TACC, a microtubule-associated protein that stabilizes the centrosomal microtubules, and Kinesin 5, a motor protein involved in the formation of the bipolar mitotic spindle [10,11]. The kinase activity of AURKA is regulated by autocatalytic phosphorylation, which is facilitated after binding of the kinase to TPX2 (Target Protein for Xenopuskinesin-like protein 2), a microtubule-associated protein that targets AURKA to mitotic spindle microtubules in a process regulated by the guanosine triphosphatase, Ran [12]. Therefore, abnormal AURKA activities may conduce to errorprone cell division and consequent cell death [13], while the kinase overexpression is usually associated with the presence of excessive centrosomes with multipolar spindles and defective cell cycle checkpoint functions that consistently arise because of the failures during cytokinesis [2]. Because AURKA is an essential protein for the normal occurrence of the cell cycle, mutations and deregulations in the activities of this protein are associated with several types of cancer. Researchers as Nakamura et al. [14] and Xia et al. [15] reported the ability of AURKA in promoting the initiation of some tumors, being overexpressed in several cell lines, such as neuroblastoma, breast, colon-rectal, bladder, prostate and ovary cancers [16]. In a study performed by Asteriti et al. [17], analysis of a cancer microarray database demonstrated the overexpression of the AURKA regulator TPX2 in 27% of the classes of tumors assessed when compared to the corresponding normal tissues, making TPX2 one of the top-ten genes overexpressed in cancer. Though it has been not fully elucidated how this kinase promotes tumorigenesis, the interruption of the cell cycle checkpoints is a relevant factor. As stated, AURKA regulates the processes related to spindle assembly and stability, which are essential for the proper division of the cytoplasm of a cell into the daughter ones during cytokinesis. Therefore, AURKA overexpression may promote multinucleation concomitant with centrosome amplification, which would arise from defects in the mitotic progression and cell division, causing tetraploidization. In the presence of a functional p53 checkpoint, these cells will be arrested in the G1 phase or eliminated but, in a p53−/− background, the cells progress to extra rounds of DNA and centrosome duplication, giving rise to chromosomal instability and aneuploidy [18].

3. The 3-D structure of AURKA Human AURKA is composed of 403 amino acid residues and the first solved three-dimensional structure for this enzyme was experimentally achieved in 2002 [21]. Currently, there are more than 50 Human AURKA crystal structures in complexes with ATP, ADP, ANP (an ATP analog), in addition to several allosteric and active site synthetic inhibitors in more than 150 crystallographic complexes [2,11,22–24]. However, most of the human AURKA structures have coordinates only for the residues 127 to 388 since the first 123 amino acids of AURKA are intrinsically disordered, as well as the last 16 residues of the C-terminal region [24]. Experimental studies confirm such observations since, even for structures constructed with residues from 100 to 403, the first ordered residue in any model is always Ser123, while the last ordered residue refers to Gln394 [2]. As the other serine-threonine kinases, the auroras have two domains: The N-terminal, which includes the regulatory elements, and the catalytic C-terminal domain, with the two domains linked by a hinge (Fig. 1). The kinase site is located at the junction of these two domains and is composed by the amino acid residues ranging from 133 to 383 [7]. The N-terminal domain is responsible for the binding of adenosine triphosphate (ATP), which provides energy as a donor of phosphate groups in the reactions catalyzed by the Aurora kinases [25]. Auroras are activated by the phosphorylation of two threonine residues, which are fundamental for their activities, and are inactivated mainly by the degradation promoted by other proteins that recognize specific sites in Aurora kinases, the A-box and D-box [1,5]. The Nterminal regulatory domain varies significantly between the families while the C-terminal catalytic domain displays 71% of homology between the Human Auroras A and B. In this concern, the high degree of conservation of the sequences on the surface of the catalytic domain of AURKA must be considered when one analyzes the specificity for the substrates or aims designing specific inhibitors for the enzyme [3]. The domains of AURKA share the same structural characteristics of other kinase proteins, with the N-terminal region composed of a fivestranded β-sheet, one α-helix named “C-helix” and a short additional αhelix, the “B-helix”, which is perpendicular the C-helix (Fig. 2). The Cterminal domain displays seven α-helices and two β-sheets, which detains the catalytic aspartic acid of the HRD motif (triad His254-Arg255Asp256). It also contains a mobile activation loop, composed by the residues 274–299, beginning with the DFG motif (Asp274-Phe275-Gly276) 2

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TPX2. While AURKA is activated by phosphorylation in the activation loop to perform its centrosomal functions, it is activated through binding to TPX2 in the in the mitotic spindle [27]. There are three AURKA structures complexed with TPX2 (PDB entries 1OL5, 3E5A and 3HA6), one with ADP as a ligand (1OL5) and the others with inhibitors at the catalytic site [11,27]. Binding of TPX2 alters the conformation of the AURKA activation loop, bringing the phosphorylated Thr288 to a position where a salt bridge is formed with the Arg255 of the HRD motif, yielding an active kinase. In the absence of TPX2, Thr288 is oriented outwards and the activation loop is more extensive [11]. Nikonova et al. [2] grouped the catalytic and regulatory residues of AURKA into a structural spine, based on the structural alignment of AURKA with PKA and in a detailed analysis of PKA previously published by Kornev and Taylor (2010). In this concern, the regulatory spine of AURKA (Fig. 2) would consist of Gln185, Leu196, of the His254 from the HRD motif and the Phe275 from the DFG motif, while the catalytic spine would be composed of Val147, Ala160, Val218, Leu262–264, Leu318 and Phe322 [2]. This structure is called a spine because of the stacked arrangement of these residues and its integrity depends on the position of the DFG motif, whereas in an active kinase, the Phe275 residue is oriented towards the kinase domain, under the C-helix (DFG-in conformation). In addition, in the active conformation with TPX2, the side chain of Thr292 forms a hydrogen bond with the Asp256 residue from the HRD motif. In the structures in the DFG-in conformation but without TPX2, none of these interactions is observed. When the kinase is inactive, the Phe275 of the DFG motif is positioned below the ADP (DFG-out conformation) [2]. In addition to the DFG-in conformation, several crystalline structures of AURKA display an unusual DFG conformation, referred by Dodson et al. [24] as DFG-up. In the DFG-up conformation, Phe275 points upward in the N-terminal domain, being embedded between the C-helix and the βsheet of this domain. This position results in the disruption of a salt bridge between Lys162 of the β-sheet and Glu181 of the C-helix, which can be observed in the active AURKA [2,28]. Nikonova et al. [2] reports three somatic mutations in AURKA that are associated with cancer. One of these mutations refers to the exchange of Ser155 → Arg, which occurs at the interface between AURKA and TPX02. Mutation of this residue to an arginine disrupts the interaction with TPX2 due to the replacement of a small polar residue with a bulkier positively charged amino acid. The second mutation occurs at Val174 → Met, located at the junction of the B-/C-helix. This mutation leads to the constitutive activation of AURKA, possible in decorrence of the change in the patterns of interaction between the N/C domains [2].

Fig. 1. The 3-D structure of Aurora A-TPX2 complex (PDB code: 1OL5), highlighting the catalytic cleft between the N- and C-lobes, were an adenosine diphosphate (ADP) molecule and two magnesium ions are depicted. The N- and Clobes are connected by a hinge (highlighted in cyan). The activation loop and HRD motif at the C-lobe are highlighted, respectively, in blue and dark blue. The TPX2 interaction motifs are depicted as solid gray surfaces. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and ending with the sequence Pro297-Pro298-Glu299. The position and conformation of the activation loop determine whether a kinase is active or inactive [2]. AURKA is activated through phosphorylation at the residues Thr287 and Thr288 promoted by other kinases, the kinases activated by p21 (PAKs) and the protein A kinase (PAK) (which also phosphorylate Ser342 in vitro as an alternative site) or only at Thr287 by the atypical protein kinase C. The Auroras may assume different conformations depending on the binding of activators or inhibitors, as well as the phosphorylation of these residues. These conformations vary primarily at the positions of the activation loop and C-helix, but such variations may also occur at the N-terminal domain as a whole [26]. AURKA activation may also occurs after the binding of the mitotic spindle protein

Fig. 2. The 3-D structure of Aurora A-TPX2 complex (PDB code: 1OL5), highlighting the regulatory spine (in blue) composed by Gln185, Leu196, of the His254 (HRD motif) and Phe275 (DFG motif) and the elements of the catalytic spine (in green), which consist from Val147, Ala160, Val218, Leu262, Leu263, Leu264, Leu318 and Phe322. Some of the original α-helices and β-sheets were omitted to improve the visualization. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Binding modes of potentially selective type IV (A) and II (B) Aurora A inhibitors. Besides of performing interactions with the adenine-binding pocket, type II inhibitors also interacts with contiguous hydrophobic pocket (Leu194, Arg195, Leu196, Leu210 and Phe275) only accessible in the DFG-out conformation. The 2-D structures of these compounds and corresponding PDB codes for the crystallographic structures are listed in Table 1.

In the active conformation, Ser342 forms a hydrogen bond with Glu302 [RSer342-CH2Oδ-Hδ+ – –O(O)C-RGlu302], which is part of a segment contiguous to the activation loop of AURKA (residues 274–299). Therefore, the phosphorylation of Ser342 by PAK1 [RSer342-CH2PO42−] or the mutation of this residue to Ala/Asp [RAla342-CH3; RAsp342CH2C(O)O−] tend to disrupt the interaction with Glu302, then altering the conformation and the dynamics of the activation loop. In the phosphorylation, a destabilization in the conformation of the activation loop occurs, preventing the proper positioning for autophosphorylation and, consequently, the kinase activation. Consequently, subsequent dephosphorylation of Ser342 would be required to obtain an active kinase. However, the mutation to Asp342 would result in a permanent inactivation of the enzyme due to the repulsive interaction with Glu302 and the consequent poor positioning of the activation loop with respect to autophosphorylation [2]. Some studies also report the covalent attachment of small proteins (a process known as SUMOylation) to the Lys249 residue of AURKA in mice, which would correspond to Lys258 in human AURKA. The mutation of this residue to an arginine, which does not undergo SUMOylation, would result in the formation of defective and multipolar spindles and in the abnormal positioning of the mitotic spindle, but with the maintenance of kinase activity [29].

accessible after conformational changes at the activation loop resultant from the displacement of the Phe275 residue at the DFG motif. Since the amino acids at this site are less conserved than at the ATP site, interactions with this pocket in the DFG-out conformation is expected to yield more selective compounds than the Type I inhibitors [38,39]. The first Type II kinase inhibitor approved was Imatinib [39], but this class also comprise Axitinib [40] and Sorafenib [41]. Type III inhibitors bind to an allosteric site that is close to the ATPbinding cleft (e.g., TAK-733) and, therefore, as they do not hinder ATP binding, are considered steady-state noncompetitive or uncompetitive ATP inhibitors [42]. Type IV inhibitors also do not target the ATP binding cleft, but allosteric sites that are distant from the ATP site. Because Type II, but specially Type I inhibitors target the highly conserved ATP binding site in kinases, these inhibitors are expected to be promiscuous and, accordingly, type IV inhibitors represents an opportunity to achieve selective AURKA inhibition [43]. Though some Type IV inhibitors have been developed as GNF-2, an antagonist of BCR-Abl [44], only experimental AURKA type IV inhibitors are currently available. McIntyre et al. [11] investigated the interface of the AURKA/TPX2 complex using co-precipitation assays coupled with isothermal titration calorimetry, in order to quantify the energy contribution of each TPX2 residue to the interaction. Therefore, the Tyr8, Tyr10, Phe16 and Trp34 residues of TPX2 are crucial for the formation of tightly bound complexes, suggesting that this interaction could be abolished by the pharmacological blockade of any of the three cavities of AURKA (pockets Y, F and W) that accommodate such residues. The authors argue that phosphorylation of the AURKA residue Thr288 is also required for a high affinity binding to the AURKA/TPX2 complex. Therefore, the authors performed a screening of 1255 fragments against AURKA and identified 59 ligands. In all analyzes, the concomitant binding of ADP to the active site of the enzyme was observed. More than 75% of these hits (46 compounds) interacted specifically with the three pockets mentioned above (i.e., they are potential type IV inhibitors), demonstrating that the AURKA/TPX2 complex may be weakened if any disturbance occurs in one of these pockets. In this concern, the Y-pocket was particularly promising (35 fragments) followed by F-pocket (10 fragments). As expected for low molecular weight compounds, these fragments exhibited low affinity for the target, with IC50 values in the μM order [11]. A recent work published by Zang et al. [45] also focuses on the

4. Development of AURKA inhibitors for the treatment of cancer The oncogenic actions of AURKA, associated with its fundamental role in mitotic progression, makes the design of AURKA inhibitors a prominent strategy in the development of new cancer chemotherapies [5]. However, the design of selective and specific inhibitors of kinases is a challenge due to the high similarity in the kinase domains. Selective inhibitors of kinases can be sorted into four categories: I, II, III and IV [30–33]. Type I inhibitors mimic the ATP (i.e., they are competitive inhibitors of ATP) and bind to the active form of their protein kinase. The areas of interactions of the type I molecules can be divided into (1) hydrophobic, (2) adenine, (3) ribose or (4) phosphate-binding regions [34]. Some of the best-known type I inhibitors are vandetanib [35], ceritinib [36] and gefitinib [37]. Type II inhibitors bind to the inactive form of a protein kinase and, similarly to the Type I inhibitors, they compete with ATP by occupying the ATP binding cleft but they also interact with an adjacent hydrophobic pocket (Leu194, Arg195, Leu196, Leu210 and Phe275), only 4

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Table 1 Potentially selective type II and IV Aurora A inhibitors. Compound

Hyd2 N Hb1 N HN Hb1 Hyd3

O S

N N

Hb2 O

Hyd1 N Hb1

4ZTR: 6-[(4-{[(2Z,5Z)-2-[(4-ethylphenyl)imino]-3-methyl −4-oxo-1,3-thiazolidin-5-ylidene]methyl}pyridin-2-yl)amino]pyridine-3-carboxylic acid

II

H-bond: Ala213 (Hb1), Arg137 (Hb2); hydrophobic: Leu194 and Leu210 (Hyd1), Leu263 (Hyd2), Leu139 and Gly216 (Hyd3).

4ZTQ: (2Z,5Z)-2-[(4-ethylphenyl)imino]-3-(2-methoxyethyl)-5-(pyridin-4ylmethylidene)-1,3-thiazolidin-4-one

II

H-bond: Ala213 (Hb1); hydrophobic: Leu263 (Hyd1), Leu210 and Phe275 (Hb2); π–π stacking: Phe275 (Pi1).

2F4J: N-[4-({4-[(3-methyl-1H-pyrazol-5-yl)amino]-6-(4-methylpiperazin-1-yl) pyrimidin-2-yl}sulfanyl) phenyl]cyclopropane carboxamide

II

H-bond: Val218 (Hb1) and Asp294 (Hb2); hydrophobic: Leu270 (Hyd1), Tyr148, Lys153 and Leu215 (Hyd2), Leu210 and Phe275 (Hyd3).

4JAI: N-{4-[(6-oxo-5,6-dihydrobenzo[c][1,8]naphthyridin-1-yl)amino]phenyl} benzamide

II

3W18: 2-{3-[3-(1H-1,3-benzodiazol-2-yl)-1H-indazol-6-yl]-1H-pyrazol-5-yl}-N-(3fluorophenyl) acetamide

II

H-bond: Ala213 (Hb1); hydrophobic: Leu139 (Hyd1), Leu147, Leu194 and Leu263 (Hyd2), Gly140 (Hyd2), Leu208, Leu210 and Phe275 (Hyd3). H-bond: Glu211 (Hb1), Ala213 (Hb2); hydrophobic: Val147, Leu194, Leu196 and Leu208 (Hyd1), Leu263 (Hyd2), Leu139 (Hyd3).

3W2C: 2-{4-[3-(1H-1,3-benzodiazol-2-yl)-1H-indazol −6-yl]-1H-pyrazol-1-yl}-N-(3-methylbutyl) acetamide

II

4UZD: ethyl (9S)-9-[3-(1H-1,3-benzodiazol-2-yloxy) phenyl]-8-oxo-2H,4H,5H, 6H,7H,8H,9H-pyrrolo[3,4-b]quinoline-3-carboxylate

II

Pi1

N

O

Hyd2 Hyd1

Hb1 HN N

N N H

N

Hb2 NH

N Hyd2 S

Hyd2

Hyd2 Hb1

Interactionsa

S N

N

Type

OH

Hyd1

O

PDB code and IUPAC name of the ligand

O

Hyd3

Hb1 N Hyd2

HN O HN

NH Hyd2

O Hyd3

Hyd1

O F

Hb1 H N N Hb2

HN N

NH Hb2

N Hyd1 H

Hyd2

N

Hyd3

Hb1 H N N Hb2

Pi1 O

N N

N H

NH N

Hyd1

Hyd2

Hb1 O

Hb2 H N

Hb4 N O

O

N H Hyd2 Hb3

HN

Hyd1

O Hb3

H-bond: Glu211 (Hb1), Ala213 (Hb1); hydrophobic: Phe144, Val147 and Leu194 (Hyd1), Leu139, Leu210, Gly216 and Leu263 (Hyd2); π–π stacking: Phe144 (Pi1). H-bond: Ala213 (Hb1), Glu211 (Hb2), Lys162 (Hb3), Gln185 (Hb4); hydrophobic: Val147 (Hyd1), Leu162, Leu194, Leu208, Leu210

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Table 1 (continued) Compound

PDB code and IUPAC name of the ligand

Hb1 O

Hb2 H N

O

Type

O OH Hb3

Hyd1

S

Pi1

II

5ORN: 4,5-dihydro-6-(thiophen-2-yl)pyridazin-3(2H)-one

IV (Fpocket)

5ORO: 3-(4-chlorophenyl)-5,6-dihydroimidazo[2,1-b][1,3]thiazole

IV (Fpocket)

Hydrophobic: Phe157 and Tyr197 (Hyd1); π–π stacking: Phe157 and Tyr197 (Pi1).

5ORP: 1-[3-Chloro-5-(trifluoromethyl)pyridin-2-yl]-1,4-diazepane

IV (Fpocket)

5OSD: 5-(4-chlorophenyl) furan-2-carbohydrazide

IV (Fpocket)

hydrophobic: Trp128, Phe133, Glu152, Leu159 and Ile209 (Hyd1); π–π stacking: Phe157 (Pi1). hydrophobic: Trp128 and Leu159 (Hyd1); π–π stacking: Tyr197 (Pi1), Phe157 (Pi2).

5OSF: 2-(4-ethylphenoxy)-1-(piperidin-1-yl)ethanone

IV (Fpocket)

5OSE: methyl [5-(ethylsulfanyl)-1,3,4-thiadiazol-2-yl]carbamate

IV (Fpocket)

5ORY: 2,4-Difluoro-6-(1H-pyrazol-5-yl)phenol

IV (Ypocket)

hydrophobic: Arg179, Val182 and Tyr199 (Hyd1), Glu175 (Hyd2).

5ORZ: methyl 3-azanyl-5-thiophen-2-yl-thiophene-2-carboxylate

IV (Ypocket)

hydrophobic: Leu178 and Arg179 (Hyd1).

5OS0: 2-[4-(3-chlorophenyl) piperazin-1-ium-1-yl] ethanenitrile

IV (Ypocket)

hydrophobic: Leu178, Val182 and Tyr199 (Hyd1).

5OS1: 6-ethoxy-2-methyl-1,3-benzothiazole

IV (Ypocket)

5OS3: (1R)-1-(4-Ethoxyphenyl) ethanamine

IV (Ypocket)

hydrophobic: Val182 and Tyr199 (Hyd1), Leu178 (Hyd2), Lys166 (Hyd3). hydrophobic: Lys166 (Hyd1).

N Hyd2 H Hb3

O S Hyd1

HN N Pi1 S N

N

Cl Hyd1 Cl Pi

1

F

N

HN

F N Hyd1

F

Pi1

O

Pi2 O

HN NH2

Cl

Hyd1

Hyd1

N

O Pi1

O O O

Hyd1

N N N H

S

S F

HO Hyd2

Hyd1 F

N NH

Hyd1 S

O S

O NH2

Cl

Hyd1

N NH+

Hyd2 N Hyd1 O

N

Hyd3

S

Hyd1 O

and Gly276 (Hyd2). H-bond: Ala213 (Hb1), Glu211 (Hb2), Lys162 (Hb3), Gln185 (Hb4); hydrophobic: Val147 (Hyd1), Leu208, Leu210, Phe275 and Gly276 (Hyd2). hydrophobic: Phe157 and Leu159 (Hyd1); π–π stacking: Phe157 and Tyr197 (Pi1).

4UYN: ethyl (9S)-9-[5-(1H-1,3-benzodiazol-2-ylsulfanyl)furan-2-yl]-8-hydroxy2H,5H,6H,7H, 9H-pyrrolo[3,4-b]quinoline-3-carboxylate

Hb4 N

N

Interactionsa

hydrophobic: Trp128, Phe133 and Leu159 (Hyd1); π–π stacking: Phe157 (Pi1). hydrophobic: Trp128, Phe133, Glu152 and Leu159 (Hyd1).

NH2 (continued on next page)

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Table 1 (continued) Compound

O H

HN

PDB code and IUPAC name of the ligand

Type

Interactionsa

5OS4: (3Ar,5S,7aS)-5-phenyl-3a,4,5,6,7,7a-hexahydroisoindole −1,3-dione

IV (Ypocket)

hydrophobic: Val206 (Hyd1), Val182 and Tyr199 (Hyd2).

5ORV: [3,5-bis(methylsulfanyl)-1,2-thiazol-4-yl]methanol

IV (Ypocket)

cation–π: Leu178 (Cpi1).

5OBJ: 2-(3-fluorophenyl) quinoline-4-carboxylic acid

IV (Ypocket)

hydrophobic: Val206 (Hyd1), Lys166 and Glu175 (Hyd2); cation–π: Arg179 (Cpi1).

5DR2: 2-(3-bromophenyl) quinoline-4-carboxylic acid

IV (Ypocket)

hydrophobic: Val206 (Hyd1), Lys166 and Glu175 (Hyd1); cation–π: Arg179 (Cpi1).

5DOS, 5DT4, 5DN3: 2-(3-bromophenyl)-8-fluoro quinoline-4-carboxylic acid

IV (Ypocket)

hydrophobic: His201 (Hyd1), Lys166 and Glu175 (Hyd2); cation–π: Arg179 (Cpi1).

5ORR: 4-[4-(trifluoromethyl) phenyl]-1,2,3-thiadiazol-5-amine

IV (Ypocket)

5ORW: 3-(4-Fluorophenoxy)-1-thiomorpholin-4-ylpropan-1-one

IV (Ypocket)

6R4B: 6-(2,4-difluorophenyl)-N,N,4-trimethyl-2-oxo-1,2-dihydropyrimidine-5carboxamide

IV (Ypocket)

hydrophobic: Arg179, Val182 and Tyr199 (Hyd1); cation–π: Arg179 (Cpi1). hydrophobic: Arg179, Val182 and Val206 (Hyd1); π–π stacking: Tyr199 (Pi1). hydrophobic: Leu178 and Val206 (Hyd2); π–π stacking: His201 (Pi1).

5ORX: 6-(2,6-Dichlorophenoxy) pyridin-3-amine

IV (Ypocket)

5ORT: [3-(2,6-Dichlorophenyl)-5-methyl-1,2-oxazol-4-yl] methanol

IV (Ypocket)

5ORS: cyclobutyl-[4-(2-methoxyphenyl)piperidin-1-yl]methanone

IV (Ypocket)

Hyd1 Hyd2

O H

HO Cpi1 S

S

N S HO O

Cpi1 N F Hyd1

Hyd2

HO O

Cpi1 N Br Hyd1

Hyd2 HO O

Cpi1 N Br Hyd1

F Hyd

2

Cpi1

N N S

F F F Hyd1

NH2

S Hyd1

N O Pi1

O

F

O F

HN

N

O

N

Pi1

Hyd1 F Cl Hlb1 O

Hyd1 N Pi1

Cl

NH2

Cpi1 Hyd1 O N

Hb1 HO

Cl Hlb1

Cl

Hyd1 O

Pi1

N O

Hb1

Halogen bond: Glu175 (Hb1); hydrophobic: Leu178 (Hyd1); π–π stacking: Tyr199 (Pi1). H-bond: Arg179 (Hb1); Halogen bond: Glu175 (Hb1); hydrophobic: Val182 and Tyr199 (Hyd1); cation–π: Arg179 (Cpi1). H-bond: Lys166 (Hb1); hydrophobic: Leu178 and Val182 (Hyd1); π–π stacking: Tyr199 (Pi1).

(continued on next page)

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Table 1 (continued) Compound

Cpi1 NH2+ Hb1 Hyd1

Hyd2

S

Type

Interactionsa

5OS2: [2-[4-(hydroxymethyl) piperidin-1-yl]phenyl]methyl azanium

IV (Ypocket)

5OS5: 4-(4-hydroxyphenyl) sulfanylphenol

IV (Ypocket)

5OS6: (6-phenoxypyridin-3-yl)methanol

IV (Ypocket)

6R4A: 2-(1H-benzimidazol-1-yl)-N-(2-phenylethyl)acetamide

IV (Ypocket)

6R4C: ethyl [(2R)-1-(4-methylbenzyl)-3-oxopiperazin-2-yl]acetate

IV (Ypocket)

6R4D: (1S,10S)-12-cyclobutyl-1-hydroxy-5-methyl-10-(propan-2-yl)-9,12-diazatricyclo [8.2.1.02,7] trideca-2,4,6-trien-11-one

IV (Ypocket)

H-bond: His201 (Hb1), Tyr199 (Hb2); hydrophobic: Lys166 and Glu175 (Hyd1); cation–π: His201 (Cpi1). H-bond: Glu183 (Hb1); hydrophobic: Arg179 and Tyr199 (Hyd1), Leu178 and Val206 (Hyd2). H-bond: Glu183 (Hb1), Arg179 (Hb1); hydrophobic: Tyr199 (Hyd1), Leu178 (Hyd2). H-bond: Arg179 (Hb1); hydrophobic: Arg179 (Hyd1), Leu178 and Arg179 (Hyd2); cation–π: Arg179 (Cpi1). H-bond: Tyr199 (Hb1); hydrophobic: Tyr199 (Hyd1), Lys166, Leu169, Glu170 and Val206 (Hyd2); cation–π: His201 (Cpi1). H-bond: Lys166 (Hb1); hydrophobic: Lys166, Glu170, Val206 (Hyd1), Leu178, Val182, Tyr199 and Val206 (Hyd2); cation–π: His201 (Cpi1).

Hb2 OH

N

Hyd1

PDB code and IUPAC name of the ligand

Hb1 HO

OH

Hb2 N O HO Hb1

Hyd2

Hyd1

O Hb1 Hyd2 N

HN

Hyd1 N Pi1 Hb1 HN O O NHCpi1 O

Hyd1 Hyd2

Hyd1 Hb1 O H3C

N

H3C H2N Cpi1 a

OH Hyd2 CH3

For the sake of comparison, some of the numbering denoting the position of equivalent amino acid residues in different PDB complexes were normalized.

interaction between AURKA and TPX2 since the disruption of the interaction between the kinase and its activator represents a potential therapeutic strategy to explore the increased sensitivity of neoplastic cells to the mitotic stress. The authors synthesized a set of 20 fragments based on saturated nitrogen heterocycles that bear at least two carbonbased substituents. They also purchased other 60 shape-diverse fragments from the ZINC database and the resulting 80 fragments were screened against AURKA by high-throughput protein X-ray crystallography. Four fragments (3 commercial and one synthesized by the authors) bound to an AURKA Y-pocket and could be further developed to yield clinically useful AURKA allosteric inhibitors. Using a clever strategy, Asteriti et al. [46] mapped some of the TPX2 residues that interact with the AURKA F- and W-pockets to build a pharmacophore model, which was used to screen drug-like and leadlike compounds from the ZINC database. Four of the screened compounds demonstrated AURKA inhibition rates (Kd) in the low micromolar range through competition with TPX2. Using immunofluorescence assays, the authors showed that two of these compounds also promoted spindle pole defects in cultured osteosarcoma cells. Based on the classification proposed by McIntyre et al. [11], we performed a detailed analysis focusing on the AURKA inhibitors deposited in the Research Collaboratory for Structural Bioinformatics

Protein Data Bank (RCSB PDB). We must highlight, as most of the currently available AURKA inhibitors only target the ATP binding cleft (i.e., are Type I inhibitors) and, accordingly, are nonselective among kinases [43], we focused our analyses in Type II and IV inhibitors, which look more promising from the clinical perspective. Starting from 176 crystallographic structures of human AURKAs in the RCSB PDB, we identified 30 crystallographic complexes with allosteric Type IV inhibitors, with six ligands performing interactions with the amino acid residues from the F-pocket and the remaining 24 compounds interacting with the residues from the Y-pocket (Fig. 3, A). No inhibitor targeting the W-pocket was identified in the RCSB PDB during our analyses. Regarding the ATP-competitive inhibitors, a total of 76 compounds were identified and the majority (68 ligands) were Type I inhibitors, with only eight compounds performing interactions with the residues from the hydrophobic pocket only accessible in the DFG-out conformation of the enzyme (Fig. 3, B) (Table 1). Therefore, the design of Type II and IV AURKA inhibitors seems underexplored when compared to the traditional Type I ligands, in spite of the potential gains in terms of specificity. Concerning the binding modes of these AURKA inhibitors (Table 1), the ATP-competitive inhibitors usually display aromatic/ 8

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heteroaromatic rings in order to interact with the hydrophobic pocket just accessible in the DFG-out conformation; the only notable exception is the inhibitor identified under the PDB code 2F4J, where the interaction is performed by the cyclopropyl ring of the ligand (Table 1). The type IV allosteric inhibitors that act by hindering the interaction of TPX2 with the F-pocket always performs hydrophobic or hydrophobic plus π–π stacking interactions. A very similar profile is observed by the Y-pocket inhibitors but, some of these compounds (as shown in the last eight entries of Table 1) also display hydrogen bonds and, therefore, tend to be stronger inhibitors and more interesting leads. Hydrogen bonds adjacent to hydrophobic contacts tend to mutually reinforce each other because, while the H-bond will hold the hydrophobic group more firmly against the hydrophobic pocket wall of the target, the non-polar group will also increase the strength of the hydrogen bond via geometry stabilization [47].

[5] [6] [7] [8] [9] [10] [11]

5. Conclusions AURKA emerges as promising anticancer drug target because of its prominent role in the control of the cell cycle and in cellular division. However, because of the conformational changes promoted by the phosphorylation of some of its key residues and after TPX2 binding, the structural knowledge necessary for designing specific AURKA inhibitors is not trivial. Previous studies have shown that the displacement of the Phe275 residue from the DFG motif in the inactive form of AURKA (DFGout) may provide access to an adjacent hydrophobic pocket (Leu194, Arg195, Leu196, Leu210 and Phe275) to the catalytic cleft, allowing the design of more selective AURKA inhibitors. However, only few crystallographic structures of such inhibitors are currently available, then limiting the use of in silico methods (e.g., pharmacophore modelling) for the discovery of new leads. The allosteric inhibitors of AURKA look very promising from the clinical perspective because of the possibility of designing selective inhibitors, then avoiding nonspecific kinase inhibition. They can even be innocuous for the other Aurora subtypes considering that, according to Bayliss et al. [48], TPX2 regulates Aurora-A activity through binding at a site that is almost completely conserved on Aurora-B but, a single amino-acid difference (Gly198 in AURKA → Asn142 in AURKB) enables AURKB to interact with INCENP instead of TPX2 in vivo and to phosphorylate AURKB substrates. The same would apply to AURKC, which has INCENP (and not TPX2) as a binding partner [49]. However, as only crystallographic complexes of AURKA with small fragments are now available, more studies are still necessary to define specific pharmacophores for each of the three interaction sites with TPX2.

[12] [13]

[14]

[15]

[16]

[17] [18] [19]

[20]

Declaration of Competing Interest

[21]

Nothing declared. [22]

Acknowledgements The authors thank the Brazilian funding agency FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant N. 2018/08585-1 and 2018/16391-2) and the PRP-UNICAMP (Pró-Reitoria de Pesquisa da Unicamp) for the support.

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