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Kidney cancer (or renal cell carcinoma, RCC) is the sixth most common malignancy in the United States and one of the relatively few whose incidence is increasing. Because of the near universal resistance which occurs with the use of current treatment regimens, reprogrammed metabolic pathways are being investigated as potential targets for novel therapies of this disease. Borrowing from studies on other malignancies, we have identified the PAK4 and NAD biosynthetic pathways as being essential for RCC growth. We now show, using the dual PAK4/NAMPT inhibitor KPT-9274, that interference with these signaling pathways results in reduction of G2-M transit as well as induction of apoptosis and decrease in cell invasion and migration in several human RCC cell lines. Mechanistic studies demonstrate that inhibition of the PAK4 pathway by KPT-9274 attenuates nuclear β-catenin as well as the Wnt/β-catenin targets cyclin D1 and c-Myc. Furthermore, NAPRT1 downregulation, which we show occurs in all RCC cell lines tested, makes this tumor highly dependent on NAMPT for its NAD requirements, such that inhibition of NAMPT by KPT-9274 leads to decreased survival of these rapidly proliferating cells. When KPT-9274 was administered in vivo to a 786-O (VHL-mut) human RCC xenograft model, there was dose-dependent inhibition of tumor growth with no apparent toxicity; KPT-9274 demonstrated the expected on-target effects in this mouse model. KPT-9274 is being evaluated in a phase I human clinical trial in solid tumors and lymphomas, which will allow this data to be rapidly translated into the clinic for the treatment of RCC. Mol Cancer Ther; 15(9); 2119-29. ©2016 AACR.

Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules., Introduction Nucleophosmin (NPM1) is one of the most frequently mutated genes in de novo acute myeloid leukemia (AML) (~30% of cases) (1). Only approximately 50% of patients with NPM1-mutated AML [...]

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin a in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin a for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis. [ABSTRACT FROM AUTHOR]

Objective: To investigate the hypothesis that selective inhibitors of nuclear export (SINE compounds), recently approved for treatment of refractory plasma cell (PC) malignancy, may have potential in the treatment of lupus. Methods: Female NZB/NZW mice were treated with the SINE compound KPT‐350 or vehicle control. Tissue specimens were harvested and analyzed by flow cytometry, using standard markers. Nephritis was monitored by determining the proteinuria score and by histologic analysis of kidney specimens. Serum anti–double‐stranded DNA (anti‐dsDNA) levels were measured by enzyme‐linked immunosorbent assay, and total numbers of IgG‐secreting and dsDNA‐specific antibody‐secreting cells were assessed by enzyme‐linked immunospot assay. Results: KPT‐350 abrogated murine lupus nephritis at both early and late stages of the disease and rapidly impaired generation of autoreactive PCs in germinal centers (GCs). SINE compounds inhibited the production of NF‐κB–driven homeostatic chemokines by stromal cells, altering splenic B and T cell strategic positioning and significantly reducing follicular helper T cell, GC B cell, and autoreactive PC counts. KPT‐350 also decreased levels of cytokines and chemokines involved in PC survival and recruitment in the kidney of lupus‐prone mice. Exportin 1, the target of SINE compounds, was detected in GCs of human tonsils, splenic B cells of lupus patients, and multiple B cell subsets in the kidneys of patients with lupus nephritis. Conclusion: Collectively, our results provide support for the therapeutic potential of SINE compounds, via their targeting of several molecular and cellular pathways critical in lupus pathogenesis, including autoantibody production by plasma cells. [ABSTRACT FROM AUTHOR]

Selective mislocalization of E2F7 in head and neck cancers drives anthracycline resistance and can be reversed. Restoring balance in the nucleus: Despite recent advances in cancer treatment, resistance to cancer therapy and resulting mortality remain common in head and neck squamous cell carcinoma. In their search for the causes of treatment resistance, Saenz-Ponce et al. identified a mechanism dependent on the balance of two proteins that regulate transcription and these proteins' localization within cancer cells. Specifically, the authors discovered that a transcriptional inhibitor called E2F7 is frequently mislocalized to the cytoplasm in these tumors, whereas its transcription-activating counterpart, E2F1, remains in the nucleus and drives transcription of treatment resistance genes. The authors also identified an approved drug that can prevent the export of E2F7 from the nucleus and thereby restore the efficacy of anthracycline chemotherapy in head and neck cancer. Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)–dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer. [ABSTRACT FROM AUTHOR]

Background: Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton. Methodology/Principal Findings: We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I. Conclusions/Significance: Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is an aggressive disease with poor prognosis and limited therapeutic options. Recent advances in the immunotherapy field have enabled the development of new treatment strategies, among which the use of bispecific antibodies (BsAbs), able to redirect T cells against tumors, has shown promising results. In particular, a BsAb that uses TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) as a target was constructed and demonstrated good results in redirecting CD3+ T cells to kill TRAIL-R2-expressing TNBC cells. In the present study, we investigated whether treatment with selinexor, a selective inhibitor of nuclear export (SINE) targeting exportin-1/chromosome maintenance protein 1 (XPO1/CRM1), could potentiate the antitumor activity of this BsAb. In combination experiments, we found that selinexor-exposed TNBC cells exhibited greater growth inhibition when treated with the TRAIL-R2xCD3 BsAb than that expected by simple additivity. Similarly, the apoptosis rate in selinexor/TRAIL-R2xCD3 BsAb-treated TNBC cells was significantly higher than that observed after exposure to either single agent. Together, our results suggest that the combination of selinexor and TRAIL-R2xCD3 BsAb can be a viable anticancer strategy and indicate this treatment as a promising therapeutic option for TNBC patients. [ABSTRACT FROM AUTHOR]

Estrogen signaling plays important roles in diverse physiological and pathophysiological processes. However, the relationship between estrogen signaling and epigenetic regulation is not fully understood. Here, we explored the effect of estrogen signaling on the expression of Ten-Eleven Translocation (TET) family genes and DNA hydroxylmethylation in estrogen receptor alpha positive (ERα+) breast cancer cells. By analyzing the RNA-seq data, we identified TET2 as an estradiol (E2)-responsive gene in ERα+ MCF7 cells. RT-qPCR and Western blot analyses confirmed that both the mRNA and protein levels of TET2 gene were upregulated in MCF7 cells by E2 treatment. ChIP-seq and qPCR analyses showed that the enrichment of ERα and H3K27ac on the upstream regulatory regions of TET2 gene was increased in MCF7 cells upon E2 treatment. Moreover, E2 treatment also led to a significant increase in the global 5-hydroxymethylcytosine (5hmC) level, while knockout of TET2 abolished such E2-induced 5hmC increase. Conversely, treatment with ICI 182780, a potent and selective estrogen receptor degrader (SERD), inhibited TET2 gene expression and down-regulated the 5hmC level in MCF7 cells. Taken together, our study identified an ERα/TET2/5hmC epigenetic pathway, which may participate in the estrogen-associated physiological and pathophysiological processes. • E2 treatment promotes TET2 gene expression through activating the ERα/enhancer-mediated transcription. • E2 treatment upregulates 5hmC level in ERα-positive breast cancer cells, whereas TET2 depletion abolishes 5hmC increase. • Antiestrogen treatment down-regulates TET2 gene expression and 5hmC level in ERα-positive breast cancer cells. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common untreatable form of dementia. Identifying molecular biomarkers that allow early detection remains a key challenge in the diagnosis, treatment, and prognostic evaluation of the disease. Here, we report a novel experimental and analytical model characterizing epigenetic alterations during AD onset and progression. We generated the first integrated base-resolution genome-wide maps of the distribution of 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), and 5-formyl/carboxy-cytosine (5fC/caC) in normal and AD neurons. We identified 27 AD region-specific and 39 CpG site-specific epigenetic signatures that were independently validated across our familial and sporadic AD models, and in an independent clinical cohort. Thus, our work establishes a new model and strategy to study the epigenetic alterations underlying AD onset and progression and provides a set of highly reliable AD-specific epigenetic signatures that may have early diagnostic and prognostic implications.

Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC., Competing Interests: CONFLICTS OF INTEREST William Senapedis, Erkan Baloglu, Yosef Landesman, Michael Kauffman, Christian Argueta, Trinayan Kashyap, Hua Chang and Sharon Shacham are employees of Karyopharm Therapeutics and hold patents, equity and stocks and have received both major and minor remunerations from Karyopharm. All other authors have no potential conflict of interests.

Introduction: The goal of this study was to examine the effects of selinexor, an inhibitor of exportin-1 mediated nuclear export, on DNA damage repair and to evaluate the cytotoxic effects of selinexor in combination with DNA damaging agents (DDAs) in cancer cells., Results: Selinexor reduced the expression of DNA damage repair (DDR) proteins. This did not induce significant DNA damage in tested cell lines. Inhibition of DDR protein expression resulted in enhanced cancer cell death when cells were pretreated with DDAs. In contrast, enhanced cell death was not detected in cells that were pretreated with selinexor then with DDAs. In vivo , single-agent selinexor, docetaxel, or cisplatin treatment resulted in 66.7%, 51.5%, and 26.6% tumor growth inhibition (TGI), respectively, in an MDA-MB-231 xenograft model. Consequently, combination treatment with docetaxel or cisplatin followed by selinexor in vivo resulted in 93.9% and 103.4% TGI, respectively. Immunohistochemical staining and immunoblot analysis of tumor sections confirmed reduced expression of DDR proteins., Conclusion: Selinexor treatment inhibited DDR mechanisms in cancer cell lines and therefore potentiated DNA damage-based therapy. The sequential combination of DDAs followed by selinexor increased cancer cell death. This combination is superior to each individual therapy and has a mechanistic rationale as a novel anticancer strategy., Methods: Cancer cells treated with selinexor ± DDAs were analyzed using reverse phase protein arrays, immunoblots, quantitative PCR and immunofluorescence. Mice bearing MDA-MB-231 tumors were treated with subtherapeutic doses of selinexor, cisplatin, docetaxel and selinexor in combination with either cisplatin or docetaxel. Tumor growth was evaluated for 25 days., Competing Interests: CONFLICTS OF INTEREST The authors from Karyopharm Therapeutics are all full time Karyopharm employees and have no conflicts of interest to disclose. All authors have read and approved the manuscript for publication in “Oncotarget”.

Multiple myeloma (MM) is a plasma cell neoplasm that results in over 11,000 deaths in the United States annually. The backbone therapy for the treatment of MM patients almost always includes combinations with corticosteroids such as dexamethasone (DEX). We found that DEX in combination with selinexor, an inhibitor of exportin-1 (XPO1) activity, synergistically inhibits the mTOR pathway and subsequently promotes cell death in MM cells. Specifically, we show that selinexor induces the expression of the glucocorticoid receptor (GR) and when combined with dexamethasone increases GR transcriptional activity. Moreover, we found that key downstream targets of the mTOR pathway are deregulated by the combination and identified a mechanism in which GR enhances the expression of REDD1 in GR positive cells while suppressing mTOR activity and cell viability. While the single agent activity of selinexor in MM cells appears to be GR-independent, synergy with DEX depends on GR expression. These data suggest that patients with tumor cells that are GR positive will benefit substantially from the combination. The current findings are consistent with the beneficial therapeutic outcome in patients with MM when treated with the combination of selinexor and DEX. In addition, they provide a rationale for testing GR and REDD1 as predictive and prognostic markers of response, respectively, for patients treated with this beneficial combination., Competing Interests: CONFLICTS OF INTEREST CA, TK, BK, TJU, CG, SH, EB, ML, WS, SS and YL are stockholders in Karyopharm; SS is Officer of Karyopharm. The study was sponsored by Karyopharm Therapeutics. All authors reviewed the manuscript and vouch for the accuracy of the data.

Background and Aims: Docetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon., Material and Methods: Here, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives., Results and Conclusions: We show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients., Competing Interests: CONFLICTS OF INTEREST Yosef Landesman, Christian Argueta, Michael G Kauffman and Sharon Shacham are employees of Karyopharm Therapeutics, Newton, MA, USA. Other authors declare that they have no competing interests.

The nuclear export protein, exportin-1 (XPO1/CRM1), is overexpressed in many cancers and correlates with poor prognosis. Selinexor, a first-in-class Selective Inhibitor of Nuclear Export (SINE) compound, binds covalently to XPO1 and blocks its function. Treatment of cancer cells with selinexor results in nuclear retention of major tumor suppressor proteins and cell cycle regulators, leading to growth arrest and apoptosis. Recently, we described the selection of SINE compound resistant cells and reported elevated expression of inflammation-related genes in these cells. Here, we demonstrated that NF-κB transcriptional activity is up-regulated in cells that are naturally resistant or have acquired resistance to SINE compounds. Resistance to SINE compounds was created by knockdown of the cellular NF-κB inhibitor, IκB-α. Combination treatment of selinexor with proteasome inhibitors decreased NF-κB activity, sensitized SINE compound resistant cells and showed synergistic cytotoxicity in vitro and in vivo. Furthermore, we showed that selinexor inhibited NF-κB activity by blocking phosphorylation of the IκB-α and the NF-κB p65 subunits, protecting IκB-α from proteasome degradation and trapping IκB-α in the nucleus to suppress NF-κB activity. Therefore, combination treatment of selinexor with a proteasome inhibitor may be beneficial to patients with resistance to either single-agent.

A library of 91 heterocyclic compounds composed of 16 distinct scaffolds has been synthesized through a sequence of phosphine-catalyzed ring-forming reactions, Tebbe reactions, Diels-Alder reactions, and, in some cases, hydrolysis. This effort in diversity-oriented synthesis produced a collection of compounds that exhibited high levels of structural variation both in terms of stereochemistry and the range of scaffolds represented. A simple but powerful sequence of reactions thus led to a high-diversity library of relatively modest size with which to explore biologically relevant regions of chemical space. From this library, several molecules were identified that inhibit the migration and invasion of breast cancer cells and may serve as leads for the development of antimetastatic agents., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Raf kinase inhibitor protein (RKIP) interacts with a number of different proteins and regulates multiple signaling pathways. Here, we show that locostatin, a small molecule that covalently binds RKIP, not only disrupts interactions of RKIP with Raf-1 kinase, but also with G protein-coupled receptor kinase 2. In contrast, we found that locostatin does not disrupt binding of RKIP to two other proteins: inhibitor of κB kinase α and transforming growth factor β-activated kinase 1. These results thus imply that different proteins interact with different regions of RKIP. Locostatin's mechanism of action involves modification of a nucleophilic residue on RKIP. We observed that after binding RKIP, part of locostatin is slowly hydrolyzed, leaving a smaller RKIP-butyrate adduct. We identified the residue alkylated by locostatin as His86, a highly conserved residue in RKIP's ligand-binding pocket. Computational modeling of the binding of locostatin to RKIP suggested that the recognition interaction between small molecule and protein ensures that locostatin's electrophilic site is poised to react with His86. Furthermore, binding of locostatin would sterically hinder binding of other ligands in the pocket. These data provide a basis for understanding how locostatin disrupts particular interactions of RKIP with RKIP-binding proteins and demonstrate its utility as a probe of specific RKIP interactions and functions.