Substituted pteridinones, pyrimidines, pyrrolopyrimidines, and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors: Pharmacophore modeling data.

The RSK2 kinase is a downstream effector of the Ras/Raf/MEK/ERK pathway that is aberrantly active in a range of cancer types and has been recognized an anticancer target. The inhibition of RSK2 kinase activity would disrupt multiple pro-cancer processes; however, there are few RSK2 inhibitors. The data have been obtained for a series of pteridinone-, pyrimidine-, purine-, and pyrrolopyrimidine-based compounds, developed to establish a structure-activity relationship for RSK inhibition. The compounds were docked into the ATP-binding site of the N-terminal domain of the RSK2 kinase using Glide. The binding conformations of these molecules was then used to generate a set of pharmacophore models to determine the structural requirements for RSK2 inhibition. Through the combination of these models, common features (pharmacophores) can be identified that can inform the development of further small molecule RSK inhibitors. The synthesis and evaluation of the pteridinone- and pyrimidine-based compounds was reported in the related articles: Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study (Casalvieri et al., 2020) and Molecular docking of substituted pteridinones and pyrimidines to the ATP-binding site of the N-terminal domain of RSK2 and associated MM/GBSA and molecular field datasets (Casalvieri et al., 2020). [1], [2]. The synthesis and evaluation of the purine- and pyrrolopyrimidine-based compounds was reported in the related research article: N-substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors (Casalvieri et al., 2021) [3].

Evaluation of Thymidine Phosphorylase Inhibitors in Glioblastoma and Their Capacity for Temozolomide Potentiation.

A number of studies have shown high levels of thymidine phosphorylase (TP) expression in glioblastoma (GBM), with trace or undetectable TP levels in normal developed brain tissue. TP catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate, maintaining nucleoside homeostasis for efficient DNA replication and cell division. The TP-mediated catabolism of thymidine is responsible for multiple protumor processes and can support angiogenesis, glycation of proteins, and alternative metabolism. In this study, we examined the effect of TP inhibition in GBM using the known nanomolar TP inhibitors 5-chloro-6-[1-(2'-iminopyrrolidin-1'-yl)methyl]uracil (TPI) and the analogous 6-[(2'-aminoimidazol-1'-yl)methyl]uracils. Although these TP inhibitors did not demonstrate any appreciable cytotoxicity in GBM cell lines as single agents, they did enhance the cytotoxicity of temozolomide (TMZ). This pontetiated action of TMZ by TP inhibition may be due to limiting the availability of thymine for DNA repair and replication. These studies support that TP inhibitors could be used as chemosensitizing agents in GBM to improve the efficacy of TMZ.

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors.

The RSK2 kinase is the downstream effector of the Ras/Raf/MEK/ERK pathway, that is often aberrantly activated in acute myeloid leukemia (AML). Recently, we reported a structure-activity study for BI-D1870, the pan-RSK inhibitor, and identified pteridinones that inhibited cellular RSK2 activity that did not result in concomitant cytotoxicity. In the current study, we developed a series of pyrrolopyrimidines and purines to replace the pteridinone ring of BI-D1870, with a range of N-substituents that extend to the substrate binding site to probe complementary interactions, while retaining the 2,6-difluorophenol-4-amino group to maintain interactions with the hinge domain and the DFG motif. Several compounds inhibited cellular RSK2 activity, and we identified compounds that uncoupled cellular RSK2 inhibition from potent cytotoxicity in the MOLM-13 AML cell line. These N-substituted probes have revealed an opportunity to further examine substituents that extend from the ATP- to the substrate-binding site may confer improved RSK potency and selectivity.

Substituted oxindol-3-ylidenes as AMP-activated protein kinase (AMPK) inhibitors.

AMP-activated protein kinase (AMPK) is a central metabolic regulator that promotes cancer growth and survival under hypoxia and plays a role in the maintenance of cancer stem cells. A major challenge to interrogating the potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity and has scope for modification. Here, we have designed and synthesized several series of oxindoles to determine the structural requirements for AMPK inhibition and to improve selectivity. We identified two potent, novel oxindole-based AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of AMPK, this key feature evades interaction with the common recptor tyrosine kinase targets of sunitinib. Cellular engagement of AMPK by these oxindoles was confirmed by the inhibition of phosphorylation of acetyl-CoA carboxylase (ACC), a known substrate of AMPK, in myeloid leukemia cells. Interestingly, although AMPK is highly expressed and activated in K562 cells these oxindole-based AMPK inhibitors did not impact cell viability or result in significant cytotoxicity. Our studies serve as a platform for the further development of oxindole-based AMPK inhibitors with therapeutic potential.

Molecular docking of substituted pteridinones and pyrimidines to the ATP-binding site of the N-terminal domain of RSK2 and associated MM/GBSA and molecular field datasets.

The data have been obtained for a series of substituted pteridinones and pyrimidines that were developed based on BI-D1870 to establish a structure-activity relationship for RSK inhibition. The 19 compounds, 12 of these with R- and S-isomeric forms, were docked into the ATP-binding site of the N-terminal domain of the RSK2 kinase using Schrodinger Glide. The binding conformations of these molecules and their interactions with RSK2 may inform the development of further small molecule RSK inhibitors. The molecular mechanics energies combined with the generalized Born and surface area continuum solvation (MM-BGSA) method was used to estimate the free energy of binding of the small molecules with RSK2. The molecular field characteristics of the docked confirmations of the inhibitors was examined using Cresset Forge software. The synthesis and evaluation of these compounds was reported in the related research article: Substituted pteridinones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors: a structure-activity study (Casalvieri et al., 2020).

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study.

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition. We have identified inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without potent cytotoxicity. The structure-activity data from this study will be used as a platform to develop novel RSK2 inhibitors.

2-Arylamino-6-ethynylpurines are cysteine-targeting irreversible inhibitors of Nek2 kinase.

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 µM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 µM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 µM (Nek2); GI50 (SKBR3) 2.2 µM] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 µM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 µM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

Development of Potent Pyrazolopyrimidinone-Based WEE1 Inhibitors with Limited Single-Agent Cytotoxicity for Cancer Therapy.

WEE1 kinase regulates the G2 /M cell-cycle checkpoint, a critical mechanism for DNA repair in cancer cells that can confer resistance to DNA-damaging agents. We previously reported a series of pyrazolopyrimidinones based on AZD1775, a known WEE1 inhibitor, as an initial investigation into the structural requirements for WEE1 inhibition. Our lead inhibitor demonstrated WEE1 inhibition in the same nanomolar range as AZD1775, and potentiated the effects of cisplatin in medulloblastoma cells, but had reduced single-agent cytotoxicity. These results prompted the development of a more comprehensive series of WEE1 inhibitors. Herein we report a series of pyrazolopyrimidinones and identify a more potent WEE1 inhibitor than AZD1775 and additional compounds that demonstrate that WEE1 inhibition can be achieved with reduced single-agent cytotoxicity. These studies support that WEE1 inhibition can be uncoupled from the potent cytotoxic effects observed with AZD1775, and this may have important ramifications in the clinical setting where WEE1 inhibitors are used as chemosensitizers for DNA-targeted chemotherapy.

Selective Targeting of RSK Isoforms in Cancer.

The p90 ribosomal S6 kinase family (RSK1-4) is a group of highly conserved Ser/Thr kinases that act as downstream effectors of the Ras/Raf/MEK/ERK signaling pathway. The RSKs phosphorylate a range of substrates involved in transcription, translation, cell cycle regulation, and cell survival. Although the RSKs have a high degree of sequence homology, their functional differences in cancer are of great interest. Current RSK inhibitors target more than one RSK isoform, and this may limit their efficacy as anticancer agents. Here, we review the structure and function of the RSK kinases, their role in cancer growth and survival, and their potential as modulators of chemoresistance. In addition, we summarize the development of current RSK inhibitors and their limitations.

Structure-guided design of purine-based probes for selective Nek2 inhibition.

Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 µM; Nek2 IC50 = 0.62 µM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 µM; Nek2 IC50 = 0.27 µM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.

Cyclin-Dependent Kinase (CDK) Inhibitors: Structure-Activity Relationships and Insights into the CDK-2 Selectivity of 6-Substituted 2-Arylaminopurines.

Purines and related heterocycles substituted at C-2 with 4'-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favor competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10-80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1'-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency toward CDK2 (IC50 0.044 µM) but was ∼2000-fold less active toward CDK1 (IC50 86 µM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor-kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket and that is preferred in CDK2 but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.

Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors.

In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors.

Targeting WEE1 Kinase in Cancer.

WEE1 kinase plays a crucial role in the G2-M cell-cycle checkpoint arrest for DNA repair before mitotic entry. Normal cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1-S checkpoint and depend on a functional G2-M checkpoint for DNA repair. WEE1 is expressed at high levels in various cancer types including breast cancers, leukemia, melanoma, and adult and pediatric brain tumors. Many of these cancers are treated with DNA-damaging agents; therefore, targeting WEE1 for inhibition and compromising the G2-M checkpoint presents an opportunity to potentiate therapy. In this review we summarize the current WEE1 inhibitors, the potential for further inhibitor development, and the challenges in the clinic for the WEE1 inhibitor strategy.

A novel substituted aminoquinoline selectively targets voltage-sensitive sodium channel isoforms and NMDA receptor subtypes and alleviates chronic inflammatory and neuropathic pain.

Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

A WEE1 Inhibitor Analog of AZD1775 Maintains Synergy with Cisplatin and Demonstrates Reduced Single-Agent Cytotoxicity in Medulloblastoma Cells.

The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition, we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775 displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-throughput screen. However, given the lack of structure-activity data for AZD1775, we developed a small series of analogs to determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma. Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects; therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the tumor and potentiate the effect of DNA-damaging agents such as cisplatin.

Strategies and Approaches of Targeting STAT3 for Cancer Treatment.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates the expression of genes related to cell cycle, cell survival, and immune response associated with cancer progression and malignancy in a number of cancer types. Once activated, STAT3 forms a homodimer and translocates to the nucleus where it binds DNA promoting the translation of target genes associated with antiapoptosis, angiogenesis, and invasion/migration. In normal cells, levels of activated STAT3 remain transient; however, STAT3 remains constitutively active in approximately 70% of human solid tumors. The pivotal role of STAT3 in tumor progression has promoted a campaign in drug discovery to identify small molecules that disrupt the function of STAT3. A range of approaches have been used to identify novel small molecule inhibitors of STAT3, including high-throughput screening of chemical libraries, computational-based virtual screening, and fragment-based design strategies. The most common approaches in targeting STAT3 activity are either via the inhibition of tyrosine kinases capable of phosphorylating and thereby activating STAT3 or by preventing the formation of functional STAT3 dimers through disruption of the SH2 domains. However, the targeting of the STAT3 DNA-binding domain and disruption of binding of STAT3 to its DNA promoter have not been thoroughly examined, mainly due to the lack of adequate assay systems. This review summarizes the development of STAT3 inhibitors organized by the approach used to inhibit STAT3, the current inhibitors of each class, and the assay systems used to evaluate STAT3 inhibition and offers an insight into future approaches for small molecule STAT3 inhibitor development.

Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors.

Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. No medical treatments are currently available. Using a combined genetic and genomic screen of individual human gonadotrope pituitary tumor samples, we recently identified the mammalian sterile-20 like kinase 4 (MST4) as a protumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. To identify novel inhibitors of the MST4 kinase for potential future clinical use, computational-based virtual library screening was used to dock the SelleckChem kinase inhibitor library into the ATP-binding site of the MST4 crystal structure. Several inhibitor candidates were identified with the potential to bind with high affinity. Using a TR-FRET in vitro recombinant kinase assay, hesperadin, initially described as an Aurora kinase inhibitor, exhibited potent inhibition of the MST4 kinase at nanomolar concentrations. The LßT2 gonadotrope pituitary cell hypoxic model was used to test the ability of this inhibitor to antagonize MST4 actions. Under short-term severe hypoxia (1% O2), MST4 protection from hypoxia-induced apoptosis was abrogated in the presence of hesperadin. Similarly, under chronic hypoxia (5%), hesperadin blocked the proliferative and colony-forming actions of MST4 as well as the ability to activate specific downstream signaling and hypoxia-inducible factor-1 effectors. Together, these data identify hesperadin as the first potent, selective inhibitor of the MST4 kinase with the capacity to block pituitary tumor cell growth in a hypoxic microenvironment.

RSK3: A regulator of pathological cardiac remodeling.

The family of p90 ribosomal S6 kinases (RSKs) are pleiotropic effectors for extracellular signal-regulated kinase signaling pathways. Recently, RSK3 was shown to be important for pathological remodeling of the heart. Although cardiac myocyte hypertrophy can be compensatory for increased wall stress, in chronic heart diseases, this nonmitotic cell growth is usually associated with interstitial fibrosis, increased cell death, and decreased cardiac function. Although RSK3 is less abundant in the cardiac myocyte than other RSK family members, RSK3 appears to serve a unique role in cardiac myocyte stress responses. A potential mechanism conferring the unique function of RSK3 in the heart is anchoring by the scaffold protein muscle A-kinase anchoring protein ß (mAKAPß). Recent findings suggest that RSK3 should be considered as a therapeutic target for the prevention of heart failure, a clinical syndrome of major public health significance.

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.