Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization.

The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.

Production of NOS2 and inflammatory cytokines is reduced by selected protein kinase inhibitors with partial repolarization of HL-60 derived and human blood macrophages.

JAK/STAT pathway plays a well-known role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to identify new signaling pathways and repolarize macrophages by selected protein kinase inhibitors. HL-60 derived macrophages were chosen as model cells and human blood macrophages were used for comparison. M1 and M2 polarization of HL60 derived and human blood macrophages was promoted by LPS + IFNγ (LIF) and IL-4 treatments, respectively. In HL-60 derived macrophages, M1 polarization was mediated by Erk1/2 and p38 phosphorylation, while HSP27 phosphorylation was involved in M2 polarization. The inhibition of both MAPK and JAK/STAT pathways reduced the expression of NOS2, IP-10 and TNFα, IL-8 production was decreased by the inhibition of AMPK and PKD, the upstream kinase of HSP27. HSP27 phosphorylation was inhibited by NB 142, a PKD inhibitor. The expression of CD80 (M1 marker) was reduced by MAPK and JAK/STAT inhibitors, without increasing CD206 (M2 marker). On the other hand, CD206 was reduced by PKD and AMPK inhibitors, without increasing CD80 marker. Phagocytic capacity of HL-60 derived macrophages was higher in M1 macrophages and decreased by trametinib and a p38 inhibitor, while in human blood macrophages, where AT 9283, a JAK/STAT inhibitor also caused a significant decrease in M1 polarized macrophages, no difference was observed between M1 and M2 macrophages. Our results suggest that the repolarization of macrophages cannot be achieved by inhibiting their signaling pathways; nevertheless, the expression of certain polarization markers was decreased, therefore a "depolarization" could be observed both in M1 and M2 polarized cells. Selected protein kinase inhibitors of M1 polarization, decreasing NOS 2 and inflammatory cytokines may be potential candidates for therapeutical trials against inflammatory diseases.

HER2 Mediates PSMA/mGluR1-Driven Resistance to the DS-7423 Dual PI3K/mTOR Inhibitor in PTEN Wild-type Prostate Cancer Models.

Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor being very common in this cancer type. Extensive evaluation has been performed on the therapeutic potential of PI3K/AKT/mTOR inhibitors and the resistance mechanisms arising in patients with PTEN-mutant background. However, in patients with a PTEN wild-type phenotype, PI3K/AKT/mTOR inhibitors have not demonstrated efficacy, and this remains an area of clinical unmet need. In this study, we have investigated the response of PTEN wild-type prostate cancer cell lines to the dual PI3K/mTOR inhibitor DS-7423 alone or in combination with HER2 inhibitors or mGluR1 inhibitors. Upon treatment with the dual PI3K/mTOR inhibitor DS-7423, PTEN wild-type prostate cancer CWR22/22RV1 cells upregulate expression of the proteins PSMA, mGluR1, and the tyrosine kinase receptor HER2, while PTEN-mutant LNCaP cells upregulate androgen receptor and HER3. PSMA, mGluR1, and HER2 exert control over one another in a positive feedback loop that allows cells to overcome treatment with DS-7423. Concomitant targeting of PI3K/mTOR with either HER2 or mGluR1 inhibitors results in decreased cell survival and tumor growth in xenograft studies. Our results suggest a novel therapeutic possibility for patients with PTEN wild-type PI3K/AKT-mutant prostate cancer based in the combination of PI3K/mTOR blockade with HER2 or mGluR1 inhibitors.

EGFR Alterations Influence the Cetuximab Treatment Response and c-MET Tyrosine-Kinase Inhibitor Sensitivity in Experimental Head and Neck Squamous Cell Carcinomas.

Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

Synthesis and Structure-Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH.

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.

Discovery and optimization of novel benzothiophene-3-carboxamides as highly potent inhibitors of Aurora kinases A and B.

Aurora kinases as regulators of cell division have become promising therapeutic targets recently. Here we report novel, low molecular weight benzothiophene-3-carboxamide derivatives designed and optimized for inhibiting Aurora kinases. The most effective compound 36 inhibits Aurora kinases in vitro in the nanomolar range and diminishes HCT 116 cell viability blocking cytokinesis and inducing apoptosis. According to western blot analysis, the lead molecule inhibits Aurora kinases equipotently to VX-680 (Tozasertib) and similarly synergizes with other targeted drugs.

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma.

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).

Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.

Discovery and Biological Evaluation of Novel Dual EGFR/c-Met Inhibitors.

Activating mutations in the epidermal growth factor receptor (EGFR) have been identified in a subset of non-small cell lung cancer (NSCLC), which is one of the leading cancer types worldwide. Application of EGFR tyrosine kinase inhibitors leads to acquired resistance by secondary EGFR mutations or by amplification of the hepatocyte growth factor receptor (c-Met) gene. Although several EGFR and c-Met inhibitors have been reported, potent dual EGFR/c-Met inhibitors, which can overcome this latter resistance mechanism, have hitherto not been published and have not reached clinical trials. In the present study we have identified dual EGFR/c-Met inhibitors and designed novel N-[4-(quinolin-4-yloxy)-phenyl]-biarylsulfonamide derivatives, which inhibit the c-Met receptor and both the wild-type and the activating mutant EGFR kinases in nanomolar range. We have demonstrated by Western blot analysis that compound 10 inhibits EGFR and c-Met phosphorylation at cellular level and effectively inhibits viability of the NSCLC cell lines.

[Developing FGFR inhibitors as potential anti-cancer agents]. / Potenciális antitumor hatású FGFR inhibitorok fejlesztése.

Fibroblast Growth Factor Receptor (FGFR) family is a sequentially highly related subgroup of membrane proteins consisting of four tyrosine kinase type enzyme: FGFR1, FGFR2, FGFR3 and FGFR4. These are kinases of great interest in a wide spectrum of physiological processes such as tissue repair via controlling cell proliferation. As initiatiors of cell proliferation, in some cases they have leading roles in several types of cancer, eg. breast cancer, pancreas cancer, gastric tumors and multiple myeloma via overexpression and/or mutation. This phenomenon makes them promising targets for drug development in order to develop signal transduction therapies based on small molecule FGFR inhibitors. We have developed two main groups of lead molecules: compounds with benzotiophene and oxindole cores utilizing numerous methods from in silico modelling via in vitro biochemichal assays and testing on relevant cell lines to cytotoxicity assays.

[Development and biochemical characterization of EGFR/c-Met dual inhibitors]. / EGFR/c-Met kettosgátlók fejlesztése és biokémiai vizsgálata.

The epidermal growth factor receptor (EGFR) family has been well-known for more than ten years as the target of non-small lung carcinoma (NSCLC) which is one of the leading cause of mortality among the cancer types. The receptor tyrosine kinase inhibitors (gefitinib, erlotinib, lapatinib) which have been applied in the therapy, are not able to inhibit the progression of this disease perfectly because of resistance. It has been demonstrated that the amplification of mesenchymal-epithelial transition factor (c-Met) or secondary mutation of EGFR kinase causes the resistance against EGFR inhibitors in 18-20 percent of the cases. Clinical candidates inhibiting both of EGFR and c-Met kinases are unknown in the literature. We have developed quinoline-based inhibitors in our research project, which inhibit both kinases in submicromolar range in enzymatic assays, moreover we have demonstrated by western blot analysis that these compounds inhibit the autophosphorylation in vivo. The binding of the effective compounds was examined by in silico and docking simulations.

Targeting of a platinum-bound sunitinib analog to renal proximal tubular cells.

BACKGROUND: Activated proximal tubular cells play an important role in renal fibrosis. We investigated whether sunitinib and a kidney-targeted conjugate of sunitinib were capable of attenuating fibrogenic events in tubulointerstitial fibrosis. METHODS: A kidney-targeted conjugate was prepared by linkage of a sunitinib analog (named 17864) via a platinum-based linker to the kidney-specific carrier lysozyme. Pharmacological activity of 17864-lysozyme was evaluated in human kidney proximal tubular cells (HK-2); the capability of the kidney-directed conjugate to accumulate in the kidneys was studied in mice. Potential antifibrotic effects of a single-dose treatment were evaluated in the unilateral ureteral obstruction (UUO) model in mice. RESULTS: The 17864-lysozyme conjugate and its metabolites strongly inhibited tyrosine kinase activity. Upon intravenous injection, 17864-lysozyme rapidly accumulated in the kidneys and provided sustained renal drug levels for up to 3 days after a single dose. Renal drug level area under the curve was increased 28-fold versus an equimolar dose of sunitinib malate. Daily treatment of UUO mice with a high dose of sunitinib malate (50 mg/kg) resulted in antifibrotic responses, but also induced drug-related toxicity. A single dose of 17864-lysozyme (equivalent to 1.8 mg/kg sunitinib) was safe but showed no antifibrotic effects. CONCLUSION: Multikinase inhibitors like sunitinib can be of benefit in the treatment of fibrotic diseases, provided that their safety can be improved by strategies as presented in this paper, and sustained renal levels can be achieved.

Dendrimer-based macromolecular conjugate for the kidney-directed delivery of a multitargeted sunitinib analogue.

The development of a macromolecular conjugate of a multitargeted tyrosine kinase inhibitor is described that can be used for renal-specific delivery into proximal tubular cells. A novel sunitinib analogue, that is, 17864, is conjugated to a NH(2) -PAMAM-G3 dendrimer via the platinum (II)-based Universal Linkage System (ULS™). The activity of 17864 is retained after coordination to the ULS linker alone or when coupled to NH(2) -PAMAM-G3. 17864-UlS-NH(2) -PAMAM-G3 is non-toxic to proximal tubular cells in vitro. After intravenous administration to mice, 17864-UlS-NH(2) -PAMAM-G3 rapidly and efficiently accumulates in the kidneys. These results are encouraging for future studies focusing on the development of novel therapeutics for the treatment of renal diseases.

Development of a cell-selective and intrinsically active multikinase inhibitor bioconjugate.

Multikinase inhibitors are potent anticancer drugs that simultaneously intervene in multiple related signaling cascades, thus being capable of blocking salvage pathways that may play a role in the development of drug resistance. Multikinase inhibitors are increasingly evaluated for indications other than cancer, but long-term safety risks dictated by off-organ toxicities of these agents may prevent their safe and effective use. Here, we describe a new approach in which platinum coordination chemistry is applied for the development of a cell-selective multikinase inhibitor bioconjugate. The platinum(II) kinase inhibitor bioconjugate was designed to be active with the linker attached to the inhibitor and displayed improved activity by enhanced cell specificity as well as enhanced intracellular retention, thereby prolonging its pharmacological activity. In addition, the utilized platinum-based linkage technology potentiated the inhibitory activity of the multikinase inhibitor. These features in combination with carrier-mediated uptake in the target cells may revolutionize dosing regimens and safety profiles of (multi)kinase inhibitors.

A closer look at the structure of sterically stabilized liposomes: a small-angle X-ray scattering study.

The evaluation of the radial electron density profile of a drug containing a sterically stabilized liposomal system is described. Using synchrotron small-angle X-ray scattering, we were able to characterize the hydrophilic shell of the polyethylene glycol chains. Using a Gaussian model for describing the electron density profile along the normal of the bilayer, we got an asymmetric distribution of PEGylated lipids in accordance with theoretical considerations. Moreover, we used anomalous X-ray scattering to study the localization of a hydrophobic drug (a kinase inhibitor), which revealed that these molecules are mainly located in the hydrocarbon chain region of the phospholipid bilayer.

Inhibition of c-Met with the specific small molecule tyrosine kinase inhibitor SU11274 decreases growth and metastasis formation of experimental human melanoma.

The hepatocyte growth factor/scatter factor (HGF/SF) tyrosine kinase (TK) receptor c-Met plays a crucial role in the development of the invasive phenotype of tumors and thus represents an attractive candidate for targeted therapies in a variety of malignancies, including human malignant melanoma (MM). In contrast to what has been shown previously, we were not able to detect any genetic alterations, either in the juxtamembrane- or in the TK-domain of c-Met, in the studied MM cell lines. Nevertheless, c-Met was constitutively active in these cell lines without exogenous HGF/SF stimulation. The active receptor was localized to the adhesion sites of the cells. Addition of the c-Met TK inhibitor SU11274 specifically decreased the phosphotyrosine signal at the focal adhesions sites, which was accompanied by a decrease in cell proliferation as well as an increase in apoptotic cells. In addition, non-apoptotic concentrations of SU11274 significantly reduced the in vitro migratory capacity of MM cells in the modified Boyden-chamber assay. Administration of SU11274 significantly decreased primary tumor growth as well as the capacity for liver colony formation of MM cells in SCID mice. Our study provides the first evidence for an in vivo antitumor activity of SU11274 in a human melanoma xenograft model, and suggests c-Met as a valid target for the therapy of MM. Consequently, SU11274 treatment might represent a useful strategy for controlling melanoma progression and metastasis in patients with MM.

A novel drug discovery concept for tuberculosis: inhibition of bacterial and host cell signalling.

The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.