Isoform Selectivity Profile Research Articles

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Comparative analysis of YAP/TEAD inhibitors in 2D and 3D cultures of primary human hepatocytes reveals a novel non-canonical mechanism of CYP induction

Induction of cytochrome P450 (CYP) genes constitutes an important cause of drug–drug interactions and preclinical evaluation of induction liability is mandatory for novel drug candidates. YAP/TEAD signaling has emerged as an attractive target for various oncological indications and multiple chemically distinct YAP/TEAD inhibitors are rapidly progressing towards clinical stages. Here, we tested the liability for CYP induction of a diverse set of YAP/TEAD inhibitors with different modes of action and TEAD isoform selectivity profiles in monolayers and 3D spheroids of primary human hepatocytes (PHH). We found that YAP/TEAD inhibition resulted in broad induction of CYPs in 2D monolayers, whereas, if at all, only marginal induction was seen in spheroid culture. Comprehensive RNA-Seq indicated that YAP/TEAD signaling was increased in 2D culture compared to spheroids, which was paralleled by elevated activities of the interacting transcription factors LXR and ESRRA, likely at least in part due to altered mechanosensing. Inhibition of this YAP/TEAD hyperactivation resulted in an overall reduction of hepatocyte dedifferentiation marked by increased hepatic functionality, including CYPs. These results thus demonstrate that the observed induction is due to on-target effects of the compounds rather than direct activation of xenobiotic sensing nuclear receptors. Combined, the presented data link hepatocyte dedifferentiation to YAP/TEAD dysregulation, reveal a novel non-canonical pathway of CYP induction and highlight the advantage of organotypic 3D cultures to predict clinically relevant pharmacokinetic properties, particularly for atypical induction mechanisms.

Design, synthesis and biological evaluation of dual HDAC and VEGFR inhibitors as multitargeted anticancer agents.

Herein, a novel series of dual histone deacetylase (HDAC) and vascular endothelial growth factor receptor (VEGFR) inhibitors were designed, synthesized and biologically evaluated based on previously reported pazopanib-based HDAC and VEGFR dual inhibitors. Most target compounds showed significant HDAC1, HDAC6 and VEGFR2 inhibition, which contributed to their potent antiproliferative activities against multiple cancer cell lines and significant antiangiogenic potencies in both human umbilical vein endothelial cell (HUVEC) tube formation assays and rat thoracic aorta ring assays. Further HDAC selectivity evaluations indicated that hydroxamic acids 5 and 9epossessed HDAC isoform selectivity profiles similar to that of the approved HDAC inhibitor suberoylanilide hydroxamic acid(SAHA), while hydrazide12 presented an HDAC isoform selectivity profilesimilar to that of the clinical HDAC inhibitor MS-275. The VEGFR inhibition profiles of 5, 9e and 12were similar to that of the approved VEGFR inhibitor pazopanib. The intracellular target engagements of Compounds 5 and 12were confirmed by western blot analysis. The metabolic stabilities of 5, 9e and 12 in mouse liver microsomes were inferior to that of pazopanib. These dual HDAC and VEGFR inhibitors provide lead compounds for further structural optimization to obtainpolypharmacological anticancer agents.

AVID200: A Novel TGF-β Inhibitor for the Treatment of Anemia Associated with Myelodysplastic Syndromes

AVID200 is an engineered TGF-β ligand trap comprised of TGF-β receptor ectodomains fused to a human Fc domain. AVID200 has antibody-like properties and is 1,000 times more selective for neutralizing TGF-β1 and TGF-β3 (pM potency), compared to TGF-b2. Since TGF-β1 and TGF-β3 are negative regulators of hematopoiesis, while TGF-b2 is a positive regulator of hematopoiesis, the unique isoform selectivity profile of AVID200 makes it an attractive agent for the treatment of Myelodysplastic syndrome (MDS) associated anemia. As TGF-β2 is implicated in normal cardiovascular function and tumor cell dormancy, the specificity profile of AVID200 also minimizes the potential for cardiotoxicity and promotion of metastasis, which have been observed with pan-neutralizing TGF- β agents.MDS consist of a group of disorders that lead to cytopenia through ineffective hematopoiesis. One critical factor that is commonly associated with the occurrence of these disorders is the pathological overproduction of TGF-β. Increased TGF-β results in the arrest of myeloprogenitor cells preventing hematopoiesis. This disruption of the differentiation program and resulting cytopenia can lead to development of acute myeloid leukemia (AML) in one-third of MDS patients.A critical feature of MDS is an increased level of non-homologous end-joining (NHEJ) activity, a low fidelity form of DNA damage repair, as compared to high-fidelity homologous recombination (HR) repair. The increased level of NHEJ repair in MDS cells results in a hypersensitivity to DNA damaging agents such as mitomycin C (MMC). Previously, in a model of MDS, treatment with TGF-β inhibitors was shown to restore the ratio of NHEJ:HR repair to normal, resulting in more accurate DNA repair. Therefore, the effect of increased TGF-β in patients with MDS is deleterious in two ways. First, elevated levels of TGF-β suppress erythropoiesis leading to anemia. Second, the shift from HR to NHEJ allows for less precise DNA repair and the development of mutations which could result in AML formation.Using a TGF-β responsive AML line, we have shown that AVID200 prevents the growth inhibition induced by TGF-β1. Additionally, AVID200 reversed the hypersensitivity to MMC, implying an improvement in DNA damage repair. Currently, we are investigating the effectiveness of treating two MDS cell lines (MDS-92 and MDS-L) with AVID200 to determine its ability to not only prevent the growth inhibition associated with TGF-β1, but also to enhance DNA damage repair.The safety of AVID200 has been evaluated in several preclinical models. In a chick embryo model of metastasis, AVID200 did not promote the growth of metastatic foci, whereas a pan-neutralizing TGF-β inhibitor did. This result is important since a significant percentage of patients with MDS go on to develop AML and other cancers. The cardiac safety of AVID200 was evaluated in a single-dose pilot non-human primate study. No adverse cardiovascular events were observed at doses of up to 30 mg/kg, the highest dose tested.These results indicate that AVID200 is a promising new agent for the safe and effective treatment for MDS associated anemia.(MJT is a Mitacs Accelerate Program Postdoctoral Fellow) DisclosuresThwaites:Formation Biologics: Employment, Research Funding. Koropatnick:Formation Biologics, Inc.: Research Funding. Tremblay:Formation Biologics: Employment. O'Connor-McCourt:Formation Biologics: Employment.

Synthesis, biological evaluation and in silico modelling studies of 1,3,5-trisubstituted pyrazoles carrying benzenesulfonamide as potential anticancer agents and selective cancer-associated hCA IX isoenzyme inhibitors

Inhibition of carbonic anhydrases (CAs, EC 4.2.1.1) has clinical importance for the treatment of several diseases. They participate in crucial regulatory mechanisms for balancing intracellular and extracellular pH of the cells. Among CA isoforms, selective inhibition of hCA IX has been linked to decreasing of cell growth for both primary tumors and metastases. The discovery of novel CA inhibitors as anticancer drug candidates is a current topic in medicinal chemistry. 1,3,5-Trisubstituted pyrazoles carrying benzenesulfonamide were evaluated against physiologically abundant cytosolic hCA I and hCA II and trans-membrane, tumor-associated hCA IX isoforms by a stopped-flow CO2 hydrase method. Their in vitro cytotoxicities were screened against human oral squamous cell carcinoma (OSCC) cell lines (HSC-2) and human mesenchymal normal oral cells (HGF) via 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) test. Compounds 6, 8, 9, 11, and 12 showed low nanomolar hCA II inhibitory potency with Ki < 10 nM, whereas compounds 9 and 12 displayed Ki < 10 nM against hCA IX isoenzyme when compared with reference Acetazolamide (AZA). Compound 9, 4-(3-(hydrazinecarbonyl)-5-(4-nitrophenyl)-1H-pyrazol-1-yl)benzenesulfonamide, can be considered as the most selective hCA IX inhibitor over off-target cytosolic isoenzymes hCA I and hCA II with the lowest Ki value of 2.3 nM and selectivity ratios of 3217 (hCA I/hCA IX) and 3.9 (hCA II/hCA IX). Isoform selectivity profiles were also discussed using in silico modelling. Cytotoxicity results pointed out that compounds 5 (CC50 = 37.7 μM) and 11 (CC50 = 58.1 μM) can be considered as lead cytotoxic compounds since they were more cytotoxic than 5-Fluorouracil (5-FU) and Methotrexate (MTX).

Structure- and Ligand-Based Discovery of Chromane Arylsulfonamide Nav1.7 Inhibitors for the Treatment of Chronic Pain.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Discovery of Novel Pazopanib-Based HDAC and VEGFR Dual Inhibitors Targeting Cancer Epigenetics and Angiogenesis Simultaneously.

Herein a novel series of pazopanib hybrids as polypharmacological antitumor agents were developed based on the crosstalk between histone deacetylases (HDACs) and vascular endothelial growth factor (VEGF) pathway. Among them, one ortho-aminoanilide 6d and one hydroxamic acid 13f exhibited considerable total HDACs and VEGFR-2 inhibitory activities. The HDAC inhibitory activities endowed 6d and 13f with potent antiproliferative activities, which was not observed in the approved VEGFR inhibitor pazopanib. Compounds 6d and 13f possessed comparable HDAC isoform selectivity profiles to the clinical class I HDAC inhibitor MS-275 and the approved pan-HDAC inhibitor SAHA, respectively. 6d and 13f also exhibited uncompromised multiple tyrosine kinases inhibitory activities relative to pazopanib. The intracellular dual inhibition to HDAC and VEGFR of 6d and 13f was validated by Western blot analysis. In both HUVECs tube formation assay and rat thoracic aorta rings assay, 6d and 13f showed comparable antiangiogenic potencies to pazopanib. What's more, 6d possessed desirable pharmacokinetic profiles with the oral bioavailability of 72% in SD rats and considerable in vivo antitumor efficacy in a human colorectal adenocarcinoma (HT-29) xenograft model.

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception.

Na,K-ATPase α4 is a testis-specific plasma membrane Na+ and K+ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca2+, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.

Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

Development of N-Hydroxycinnamamide-Based Histone Deacetylase Inhibitors with an Indole-Containing Cap Group

A novel series of histone deacetylase inhibitors combining N-hydroxycinnamamide bioactive fragment and indole bioactive fragment was designed and synthesized. Several compounds (17c, 17g, 17h, 17j and 17k) exhibited comparable even superior total HDACs inhibitory activity and in vitro antiproliferative activities relative to the approved drug SAHA. A representative compound 17a with moderate HDACs inhibition was progressed to isoform selectivity profile, western blot analysis and in vivo antitumor assay. Although HDACs isoform selectivity of 17a was similar to that of SAHA, our western blot results indicated that intracellular effects of 17a at 1 μM were class I selective. It was noteworthy that the effect on histone H4 acetylation of SAHA decreased with time while the effect on histone H4 acetylation of 17a maintained even increased. Most importantly, compound 17a exhibited promising in vivo antitumor activity in a U937 xenograft model.

Histone Deacetylase (HDAC) Inhibitors with a Novel Connecting Unit Linker Region Reveal a Selectivity Profile for HDAC4 and HDAC5 with Improved Activity against Chemoresistant Cancer Cells

The synthesis and biological evaluation of new potent hydroxamate-based HDAC inhibitors with a novel alkoxyamide connecting unit linker region are described. Biological evaluation includes MTT and cellular HDAC assays on sensitive and chemoresistant cancer cell lines as well as HDAC profiling of selected compounds. Compound 19i (LMK235) (N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide) showed similar effects compared to vorinostat on inhibition of cellular HDACs in a pan-HDAC assay but enhanced cytotoxic effects against the human cancer cell lines A2780, Cal27, Kyse510, and MDA-MB231. Subsequent HDAC profiling yielded a novel HDAC isoform selectivity profile of 19i in comparison to vorinostat or trichostatin A (TSA). 19i shows nanomolar inhibition of HDAC4 and HDAC5, whereas vorinostat and TSA inhibit HDAC4 and HDAC5 in the higher micromolar range.

Abstract 4749: Insights into the molecular mechanism of HSP90 binding of methoxy-substituted resorcinylic isoxazole amide inhibitors reveal different isoform selectivity profiles

Abstract The molecular chaperone Heat Shock Protein 90 (HSP90) regulates the conformation, stability and activity of several oncogenic client proteins (eg ERBB2, ALK, BRAF, CRAF and AKT). Inhibition of HSP90 results in the degradation of these clients, leading to cell cycle arrest and apoptosis. The resorcinylic isoxazole amide HSP90 inhibitor NVP-AUY922 is currently in Phase I/II clinical trials and other related agents are in development. We previously reported that one or both of the phenolic groups on the resorcinol ring are important for HSP90 inhibitory activity. To further characterise the importance of the phenol groups on protein binding and biological activity, a series of mono- and dimethoxy-substituted resorcinylic 5′-ethyl isoxazole amides were synthesized and compared with the 2′,4′-dihydroxy compound by X-ray crystal structure and biological techniques. The most potent HSP90 inhibitors in terms of binding to the HSP90α target was the 2′,4′-dihydroxy-5′-ethyl isoxazole CCT239215 (Kd=11nM), followed by the 2′-hydroxy-4′-methoxy analogue CCT078722 (Kd=36nM). In contrast, the 2′-methoxy-4′-hydroxy and 2′,4′-dimethoxy derivatives showed no binding to HSP90. X-ray crystal structures showed that for CCT078722, the 4′-methoxy group of the resorcinol ring disrupts the hydrogen bonding that is seen with 2′,4′-dihydroxy CCT239215 and NVP-AUY922, and instead is involved in van der Waals contacts. CCT239215 also showed the greatest cellular growth inhibitory potency (GI50=5-16nM in a panel of human cancer cell lines), followed by CCT078722 (GI50=59-588nM). As predicted, CCT078723 and CCT078721 were inactive in the cells (GI50&amp;gt;10µM). This structure-activity relationship was also maintained with respect to biomarkers of HSP90 target inhibition, as measured by depletion of HSP90 client proteins and induction of HSP72. In HCT116 human colon cells, CCT239215 and CCT078722 caused a loss of HSP90 from immunoprecipitates of the HSP90 co-chaperone, demonstrating dissociation between P23 and HSP90, further confirming their on-target effects. No such dissociation was observed with the inactive analogs. These results explain how the hydroxy group at the 2′-position on the resorcinol ring is crucial for HSP90 inhibitory activity. Interestingly, the 2′-hydroxy-4′-methoxy-5′-ethyl isoxazole CCT078722 exhibited 55-fold less affinity for GRP94 when compared to HSP90α/α, indicating that these compounds show potential for different isoform selectivity profiles. Modelling studies suggest that IIe247 in GRP94 negatively impacts on the binding of CCT078722. Our findings offer insights into the molecular mechanism of HSP90 binding by resorcinylic isoxazole inhibitors, provide active/inactive probe compounds for mechanistic studies and suggest potential approaches to isoform selectivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4749. doi:1538-7445.AM2012-4749

Identification of Selective Class II Histone Deacetylase Inhibitors Using a Novel Dual-Parameter Binding Assay Based on Fluorescence Anisotropy and Lifetime

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.

Inhibitors of Class 1 Histone Deacetylases Reverse Contextual Memory Deficits in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by cognitive impairments that progress to dementia and death. The earliest symptoms of AD present as a relatively pure deficit in memory retrieval. Therefore, drug treatments that intervene in the early stages of AD by rescuing memory deficits could be promising therapies to slow, or even reverse progression of the disease. In this study, we tested the potential of systemic histone deacetylase inhibitor (HDACi) treatment to rescue cognitive deficits in a mouse model of AD. APPswe/PS1dE9 mice showed pronounced contextual memory impairments beginning at 6 months of age. Chronic HDACi injections (2-3 weeks) did not alter contextual memory formation in normal mice, but had profound effects in transgenic animals. Injections of sodium valproate, sodium butyrate, or vorinostat (suberoylanilide hydroxamic acid; Zolinza) completely restored contextual memory in these mutant mice. Further behavioral testing of the HDACi-treated transgenic mice showed that the newly consolidated memories were stably maintained over a 2-week period. Measurement of the HDAC isoform selectivity profile of sodium valproate, sodium butyrate, and vorinostat revealed the common inhibition of class I HDACs (HDAC1, 2, 3, 8) with little effect on the class IIa HDAC family members (HDAC4, 5, 7, 9) and inhibition of HDAC6 only by vorinostat. These preclinical results indicate that targeted inhibition of class I HDAC isoforms is a promising avenue for treating the cognitive deficits associated with early stage AD.

Small Molecule Inhibitors of Phosphoinositide 3-Kinase (PI3K) &amp;#948; and &amp;#947;

In recent years, pharmaceutical companies have increasingly focused on phosphoinositide 3-kinases delta (PI3Kdelta) and gamma (PI3Kgamma) as therapeutic targets for the treatment of inflammatory and autoimmune diseases. All class 1 PI3-kinases (alpha/beta/gamma/delta) generate phospholipid second messengers that help govern cellular processes such as migration, proliferation, and apoptosis. PI3K delta/ gamma lipid kinases are mainly restricted to the hematopoetic system whereas PI3K alpha/beta are ubiquitously expressed, thus raising potential toxicity concerns for chronic indications such as asthma and rheumatoid arthritis. Therefore, the challenge in developing a small molecule inhibitor of PI3K is to define and attain the appropriate isoform selectivity profile. Significant advances in the design of such compounds have been achieved by utilizing x-ray crystal structures of various inhibitors bound to PI3Kgamma in conjunction with pharmacophore modeling and high-throughput screening. Herein, we review the history and challenges involved with the discovery of small molecule isoform-specific PI3K inhibitors. Recent progress in the design of selective PI3Kdelta, PI3Kgamma, and PI3Kdelta/gamma dual inhibitors will be presented.

Salmonella typhimuriumSipA-induced neutrophil transepithelial migration: involvement of a PKC-α-dependent signal transduction pathway

Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen. We previously showed that S. typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN. Here we sought to determine the specific PKC isoforms that are induced by the S. typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration. In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S. typhimurium and recombinant SipA induced activation of PKC-alpha, -delta, and -epsilon. To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles. Inhibitors selective for PKC-alpha (Gö-6976 and 2,2',3,3',4,4'-hexahydroxyl-1,1'-biphenyl-6,6'-dimethanoldimethyl ether) markedly reduced S. typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-delta (rottlerin) or PKC-epsilon (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN. These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy. Our results are the first to show that the S. typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-alpha is involved in the signal transduction cascade leading to PMN transepithelial migration.