KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Novel Erlotinib-Chalcone Hybrids Diminish Resistance in Head and Neck Cancer by Inducing Multiple Cell Death Mechanisms.

In a search for novel therapeutic options for head and neck squamous cell carcinomas (HNSCCs) generally treated with limited therapeutic success, we synthesized a series of novel erlotinib-chalcone molecular hybrids with 1,2,3-triazole and alkyne linkers and evaluated them for their anticancer activity on Fadu, Detroit 562 and SCC-25 HNSCC cell lines. Time- and dose-dependent cell viability measurements disclosed a significantly increased efficiency of the hybrids compared to the 1:1 combination of erlotinib and a reference chalcone. The clonogenic assay demonstrated that hybrids eradicate HNSCC cells in low micromolar concentrations. Experiments focusing on potential molecular targets indicate that the hybrids trigger the anticancer effect by a complementary mechanism of action that is independent of the canonical targets of their molecular fragments. Confocal microscopic imaging and real-time apoptosis/necrosis detection assay pointed to slightly different cell death mechanisms induced by the most prominent triazole- and alkyne-tethered hybrids (6a and 13, respectively). While 6a featured the lowest IC50 values on each of the three HNSCC cell lines, in Detroit 562 cells, this hybrid induced necrosis more markedly compared to 13. The therapeutic potential indicated by the observed anticancer efficacy of our selected hybrid molecules validates the concept of development and justifies further investigation to reveal the underlying mechanism of action.

Protein Kinase D3 (PKD3) Requires Hsp90 for Stability and Promotion of Prostate Cancer Cell Migration.

Prostate cancer metastasis is a significant cause of mortality in men. PKD3 facilitates tumor growth and metastasis, however, its regulation is largely unclear. The Hsp90 chaperone stabilizes an array of signaling client proteins, thus is an enabler of the malignant phenotype. Here, using different prostate cancer cell lines, we report that Hsp90 ensures PKD3 conformational stability and function to promote cancer cell migration. We found that pharmacological inhibition of either PKDs or Hsp90 dose-dependently abrogated the migration of DU145 and PC3 metastatic prostate cancer cells. Hsp90 inhibition by ganetespib caused a dose-dependent depletion of PKD2, PKD3, and Akt, which are all involved in metastasis formation. Proximity ligation assay and immunoprecipitation experiments demonstrated a physical interaction between Hsp90 and PKD3. Inhibition of the chaperone-client interaction induced misfolding and proteasomal degradation of PKD3. PKD3 siRNA combined with ganetespib treatment demonstrated a specific involvement of PKD3 in DU145 and PC3 cell migration, which was entirely dependent on Hsp90. Finally, ectopic expression of PKD3 enhanced migration of non-metastatic LNCaP cells in an Hsp90-dependent manner. Altogether, our findings identify PKD3 as an Hsp90 client and uncover a potential mechanism of Hsp90 in prostate cancer metastasis. The molecular interaction revealed here may regulate other biological and pathological functions.

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.

Synthesis and Antiproliferative Activity of Novel Imipridone-Ferrocene Hybrids with Triazole and Alkyne Linkers.

Imipridones, including ONC201, ONC206 and ONC212 (which are emblematic members of this class of compounds developed by Oncoceutics) constitute a novel class of anticancer agents, with promising results in clinical trials. With the aim of increasing the ROS (reactive oxygen species) responsivity of the synthesized molecules, a set of novel ferrocene-imipridone hybrids were designed and synthesized. Our strategy was motivated by the documented interplay between the imipridone-triggered activation of TRAIL (the tumor necrosis factor-related apoptosis-inducing ligand) and mitochondrial ClpP (Caseinolytic protease P) and the ROS-mediated effect of ferrocene-containing compounds. In order to obtain novel hybrids with multitarget characters, the ferrocene moiety was tethered to the imipridone scaffold through ethynylene and 1,2,3-triazolyl linkers by using Sonogashira coupling of Cu(I)- and Ru(II)-catalyzed azide-alkyne cycloadditions. The biological activities of the new hybrids were examined by using in vitro cell viability assays on four malignant cell lines (PANC-1, A2058, EBC-1 and Fadu), along with colony formation assays on the most resistant PANC-1 cell line. Several hybrids caused a significantly greater drop in the cell viability compared to ONC201, and two of them completely overcame the resistance, with IC50 values comparable to those produced by ONC201. The two most potent hybrids, but not ONC201, induced apoptosis/necrosis in PANC-1 and A2058 cells after 24 h of treatment.

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.

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers.

Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure-activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

Novel Crizotinib-GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems.

Several promising anti-cancer drug-GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]-GnRH-I targeting peptide. Our most prominent crizotinib-GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]-GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]-GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib-GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets-the ATP-binding site of RTKs- and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib-GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug-GnRH conjugates.

The Potential Impact of Connexin 43 Expression on Bcl-2 Protein Level and Taxane Sensitivity in Head and Neck Cancers-In Vitro Studies.

The poor prognosis of head and neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable predictive markers. Connexin 43 (Cx43) protein and its cell-communication channels have been assigned tumor suppressor functions while the anti-apoptotic Bcl-2 (B-cell lymphoma-2) protein has been associated with negative prognostic significance in cancer. This study aimed to test the role of Cx43 protein on Bcl-2 expression, tumor progression and response to taxane-based treatment in HNSCC. Human papillomavirus (HPV) negative HNSCC cell lines were tested for paclitaxel sensitivity through measuring apoptosis induction, cell viability and changes in Cx43 and Bcl-2 levels using flow cytometry, cell viability assay, immunocytochemistry and western blot. Inhibition of Cx43 expression using siRNA increased Bcl-2 protein levels in SCC25 (tongue squamous cell carcinoma) cells, while forced Cx43 expression reduced Bcl-2 levels and supported paclitaxel cytotoxicity in FaDu (hypopharynx squamous cell carcinoma) cells. In vitro results were in line with protein expression and clinicopathological features tested in tissue microarray samples of HNSCC patients. Our data demonstrate that elevated Cx43 and reduced Bcl-2 levels may indicate HNSCC sensitivity to taxane-based treatments. On the contrary, silencing of the Cx43 gene GJA1 (gap junction protein alpha-1) can result in increased Bcl-2 expression and reduced paclitaxel efficiency. Clinical tumor-based analysis also confirmed the inverse correlation between Cx43 and Bcl-2 expression.

Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) have a high mortality rate, although several potential therapeutic targets have already been identified. Gonadotropin-releasing hormone receptor (GnRH-R) expression is less studied in head and neck cancers, hence, we investigated the therapeutic relevance of GnRH-R targeting in HNSCC patients. Our results indicate that half of the patient-derived samples showed high GnRH-R expression, which was associated with worse prognosis, making this receptor a promising target for GnRH-based drug delivery. Photodynamic therapy is a clinically approved treatment for HNSCC, and the efficacy and selectivity may be enhanced by the covalent conjugation of the photosensitizer to a GnRH-R targeting peptide. Several native ligands, gonadotropin-releasing hormone (GnRH) isoforms, are known to target GnRH-R effectively. Therefore, different 4Lys(Bu) modified GnRH analogs were designed and conjugated to protoporphyrin IX. The receptor binding potency of the novel conjugates was measured on human pituitary and human prostate cancer cells, indicating only slightly lower GnRH-R affinity than the peptides. The in vitro cell viability inhibition was tested on Detroit-562 human pharyngeal carcinoma cells that express GnRH-R in high levels, and the results showed that all conjugates were more effective than the free protoporphyrin IX.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues.

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues' cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.

Targeted tumour therapy is the focus of recent cancer research. Gonadotropin-releasing hormone (GnRH) analogues are able to deliver anticancer agents selectively into tumour cells, which highly express GnRH receptors. However, the effectiveness of different analogues as targeting moiety in drug delivery systems is rarely compared, and the investigated types of cancer are also limited. Therefore, we prepared selectively labelled, fluorescent derivatives of GnRH-I, -II and -III analogues, which were successfully used for drug targeting. In this manuscript, we investigated these analogues' solubility, stability and passive membrane permeability and compared their cellular uptake by various cancer cells. We found that these labelled GnRH conjugates provide great detectability, without undesired cytotoxicity and passive membrane permeability. The introduced experiments with these conjugates proved their reliable tracking, quantification and comparison. Cellular uptake efficiency was studied on human breast, colon, pancreas and prostate cancer cells (MCF-7, HT-29, BxPC-3, LNCaP) and on dog kidney cells (Madin-Darby canine kidney). Each of the three conjugates was taken up by GnRH-I receptor-expressing cells, but the different cells preferred different analogues. Furthermore, we demonstrated for the first time the high cell surface expression of GnRH-I receptors and the effective cellular uptake of GnRH analogues on human pharynx tumour (Detroit-562) cells. In summary, our presented results detail that the introduced conjugates could be innovative tools for the examination of the GnRH-based drug delivery systems on various cells and offer novel information about these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.