Minimalist Hybrid Ligand/Receptor-Based Pharmacophore Model for CXCR4 Applied to a Small-Library of Marine Natural Products Led to the Identification of Phidianidine A as a New CXCR4 Ligand Exhibiting Antagonist Activity

Here, we present a minimal hybrid ligand/receptor-based pharmacophore model (PM) for CXCR4, a chemokine receptor deeply involved in several pathologies, such as HIV infection, rheumatoid arthritis, cancer development/progression, and metastasization. This model, considerably simpler than those thus far proposed for this receptor, has been used to search for new CXCR4 inhibitors in a small marine natural product library available at ICB-CNR Institute (Pozzuoli, NA, Italy), since natural products, with their naturally selected chemical and functional diversity, represent a rich source of bioactive scaffolds; computational approaches allow searching for new scaffolds with a minimal waste of possibly precious natural product samples; and our “stripped-down” model substantially increases the probabilities of identifying potential hits even in small-sized libraries. This search, also validated by a systematic virtual screening of the same library, has led to the identification of a new CXCR4 ligand, phidianidine A (PHIA). Docking studies supported PHIA activity and suggested its possible binding modes to CXCR4. Using the CXCR4-expressing/CXCR7-negative GH4C1 cell line we show that PHIA inhibits CXCL12-induced DNA synthesis, cell migration, and ERK1/2 activation. The specificity of these effects was confirmed by the lack of PHIA activity in GH4C1 cells, in which siRNA highly reduces CXCR4 expression and the lack of cytoxicity of PHIA was also verified. Thus, PHIA represents a promising lead for a new family of CXCR4 modulators with wide margins of improvement in potency and specificity offered by the small and very simple underlying PM

Figure S5 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

Current density/voltage relationships of CLIC1 current in CSCs</p

Figure S1 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

IAA-94 blockade induces G1 cell cycle arrest in GBM cancer stem cells.</p

Figure S3 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

Quantitative real-time PCR for CLIC5 protein.</p

Figure S6 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

Effect of IAA on apoptosis in GB CSCs.</p

Figure S2 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

CLIC1 silencing western blot.</p

Figure S4 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

Current density/voltage relationships of CLIC1 current in CSCs</p

Supplementary Data from Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas

Supplementary Data from Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenoma

Table_1_Inhibition of Chloride Intracellular Channel 1 (CLIC1) as Biguanide Class-Effect to Impair Human Glioblastoma Stem Cell Viability.docx

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50 values significantly lower than metformin, (range 0.054–0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.</p

Additional file 1 of Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives

Additional file 1: Figure S1. Immunohistochemical distribution of CLIC1 in GBM. Figure S2. A) CLIC1 expression in GSCs isolated from 14 human GBMs, evaluated by RNA-seq, and expressed as counts per million reads mapped (CPM). Only GBM 39 (arrow) displayed low CLIC1 mRNA content. The Table reports the corresponding codes of the GSC cultures used in this study and the RNA-seq data deposited at NCBI Geo data set. B) Upper panel: CLIC1 expression evaluated by WB, in total cell lysates from selected GSC cultures. Membranes were re-probed with α-tubulin antibody after stripping and used as a reference for protein loading. Lower panel: Histograms report CLIC1/α-tubulin ratio of densitometric values and expressed in arbitrary units (A.U.) as mean ± S.D. Figure S3. Dose-response curves of novel biguanide derivatives and metformin on individual GSC cultures. The average response is reported in the Fig. 1A of the manuscript. Figure S4. Dose-response curves of novel biguanide derivatives and metformin on individual non-stem differentiated GBM cell cultures (GBM D). The average response is reported in the Fig. 1B of the manuscript. Figure S5. Dose-response curves of metformin and novel biguanide derivatives on individual ucMSC cultures. The average response is reported in the Fig. 1C of the manuscript. Figure S6. Kaplan-Mayer curves of Q48, Q54, and metformin (MET) depicting the effect of supramaximal concentration of these compounds on zebrafish embryos survival. Limited toxicity, not different from controls, was observed for all the compounds up to 5 days of treatment. Experiments were repeated twice, n = 20 per experimental group. Q48: log rank test for trend p = 0.62; Q54: log rank test for trend p = 0.64; metformin: log rank test for trend p = 0.38. Figure S7. Effect of different doses of metformin and Q54 on chick embryo survival after 10 days of incubation. Experiments were performed by Inovotion (La Tronche, France). No toxicity was observed for these compounds up to 3 mM. Experiments were repeated twice, n = 18 per experimental group. Figure S8. A. Dose-response curves of Q46, Q48, Q54 and metformin on rat astrocyte cultures. Limited toxicity is observed for all the novel compounds. Only metformin reduced astrocyte viability (− 50%) at the highest concentration tested (30 mM). Data are expressed as average of experiments preformed in quadruplicate and repeated twice. B. Table reports IC50 values in non-malignant rat astrocytes and GSCs and the calculated selectivity indices for each compound. According to the “selectivity criteria” all biguanides are considered selective compounds against GSCs (selectivity index > 10) (see reference [62]). Figure S9. A. CLIC1 expression in GBM19 GSCs carrying siRNA for both Luciferase (siLuc, silencing control) and CLIC1 (siCLIC1). B. Cell proliferation of GBM19 siLuc and GBM19 siCLIC1, treated with Q48 and Q54 (100 μM) for 48 h and evaluated by counting live cells. Data represent the mean ± S.E.M. ***p < 0.001 vs. respective control (CTR). Figure S10. Upper panels: Western blot depicting phospho-ERK1/2 (pERK) levels in GSC cultures in control conditions (CTR), after 5 min stimulation with bFGF (40 ng/ml) and after treatment of bFGF-stimulated cells with with Q48 (100 μM), Q54 and Q46 (300 μM), and metformin (10 mM) for 24 h. α-tubulin was used as loading reference. M.W. = molecular weight markers. Lower panels: densitometric analysis of pERK normalized for α-tubulin levels, * = p < 0.05; ** = p < 0.01. Figure S11. Comparison of CD44 (A) and MAP2 (B) mRNA expression in GBM3 GSCs cultivated as 2D monolayer or grown as 3D organoids, for 15, 21, and 30 days. Data are obtained by quantitative RT-PCR experiments. Figure S12. Kaplan-Meier analysis of the relationship between overall survival and CLIC1 expression. The statistical difference between the curves is measured by log-rank test. The prognostic effect of CLIC1 mRNA level in GBM according to The Cancer Genome Atlas (TGCA) databasea nalyzed using the GEPIA (Gene Expression Profiling Interactive Analysis database) software (A) and Chinese Glioma Genome Atlas (CGCA) (B). TPM: transcripts of per million; n(high): samples with expression level higher than the median of TPM; n(low): samples with expression level lower than the median of TPM. Figure S13. Expression stem cell-related markers (CD44, CD133, CD15, integrin-α6 ITGA6, Olig2, SOX2, S100A4, and nestin) in GSCs isolated from 14 human GBMs, evaluated by RNA-seq, and expressed as counts per million reads mapped (CPM). RNA-seq have been data deposited at NCBI Geo data set (see Figure S2 for the relative codes). Low CLIC1-expressing cells (GBM39) do not display differences as compared to high expressing GBMs. Figure S14. A) Dihydrofolate reductase (DHFR) expression in 14 human GSC cultures evaluated by RNA-seq, and expressed as counts per million reads mapped (CPM). Comparable expression was detected in cells derived from all the GBM analyzed. RNA-seq have been data deposited at NCBI Geo data set (see Figure S2 for the relative codes). B) Effect of Q48, Q54, Q46 and metformin (MET) on DHFR activity, incubating the compounds with purified enzyme. Methotrexate (MTX) was used as positive control. Q48, Q54, and MET, but not Q46, inhibited DHFR activity with an efficacy comparable to MTX. * p < 0.05. C) Effect of Q48, Q54, and metformin, on DHFR activity in living cells. Q48 and metformin, but not Q54, caused a moderate inhibition of enzyme activity in a time-dependent manner. * p < 0.05. Table S1. Patients’ and tumors’ characteristics. Table S2. Elemental analysis of biguanide derivatives. Table S3. Acidity constant (pKa) and distribution constant (LogD) at pH 7 of the novel biguanide derivatives and reference drugs (metformin and cycloguanil). Table S4. Statistical analysis of the effects of novel biguanide derivatives and metformin on GSCs. Table S5. Statistical analysis of the effects of novel biguanide derivatives and metformin on differentiated non-stem GBM cells. Table S6. Statistical analysis of the effects of novel biguanide derivatives and metformin on ucMSCs. Table S7. Statistical analysis of the effects of novel biguanides on low CLIC1-expressing GSCs (GBM39 and GBM44) and the average results from high CLIC1-expressing GSCs (GBM3, 5, 19, 23, and 37)