Short Amphipathic Peptides Research Articles

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 640.

Abstract 7237: Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy

Abstract BRCA1 and BRCA-2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers (10% of BC) and from epigenetically silenced BRCA-1 (30% of sporadic BC) are associated with a lack of expression and/or function of BRCA-1, which induces genomic instability. Herein, we describe a new potent strategy combining mRNAs with a tumor selective peptide nanocarrier, to selectively rescue BRCA-1 function as potential therapeutic approach in cancers. Proprietary BRCA-1 derived mRNA were complexed with proprietary short amphipathic peptides and the resulting nanoparticles were evaluated on several breast cancer cells, harboring different BRCA-1 alterations. Sensitivity to Veliparib (PARPi) following the nanoparticle treatments were evaluated on PARPI resistant and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of varying sequences of BRCA-1 mRNA-peptide nanoparticles (2.0 mg/kg, weekly, IV; ADGN-901, -902, -903) and veliparib (50mg/kg, daily, oral) were evaluated on breast cancer (HCC1937-BRCA-1 deficient, PARPi resistant) mouse xenografts. We showed that ADGN-901 inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to 60% and ADGN-903 inhibits proliferation >60% of all cells harboring defects in BRCA-1. Interestingly, ADGN-901 and ADGN-903 were able to resensitize the PARPi resistant cells to veliparib inhibition. We demonstrated that ADGN-903 restores BRCA-1 function in homologous recombination. ADGN-903 prevents RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. IV administration of ADGN-901 and ADGN-903 (2.0 mg/kg) had a strong inhibitory effect on HCC-1937 tumor growth while ADGN-902 and veliparib were inactive. ADGN-901 reduced tumor growth by 95% and ADGN-903 abolished tumor growth with strong tumor regressions. ADGN-901 and ADGN-903, were also evaluated on large tumors of about 500-600 mm3 and were highly effective in preventing further progression and achieving shrinkage of large tumors. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. BRCA-1 mRNA-peptide nanoparticles are effective in rescuing BRCA-1 function and inducing tumor regressions in BRCA-1 altered breast cancer cells and tumors. Our study provides a proof-of-concept that restoration of wild type BRCA-1 expression could be a potent therapeutic strategy for BRCA-1 altered tumors whether or not they are PARPi resistant. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Neil Desai. Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7237.

Cytotoxic Staphylococcus aureus PSMα3 inhibits the aggregation of human insulin in vitro.

Phenol-soluble modulins (PSMs) are extracellular short amphipathic peptides secreted by the bacteria Staphylococcus aureus (S. aureus). They play an essential role in the bacterial lifecycle, biofilm formation, and stabilisation. From the PSM family, PSMα3 has been of special interest recently due to its cytotoxicity and highly stable α-helical conformation, which also remains in its amyloid fibrils. In particular, PSMα3 fibrils were shown to be composed of self-associating "sheets" of α-helices oriented perpendicular to the fibril axis, mimicking the architecture of canonical cross-β fibrils. Therefore, they were called cross-α-fibrils. PSMα3 was synthesised and verified for identity with wild-type sequences (S. aureus). Then, using several experimental techniques, we evaluated its propensity for in vitro aggregation. According to our findings, synthetic PSMα3 (which lacks the N-terminal formyl groups found in bacteria) does not form amyloid fibrils and maintains α-helical conformation in a soluble monomeric form for several days of incubation. We also evaluated the influence of PSMα3 on human insulin fibrillation in vitro, using a variety of experimental approaches in combination with computational molecular studies. First, it was shown that PSMα3 drastically inhibits the fibrillation of human insulin. The anti-fibrillation effect of PSMα3 was concentration-dependent and required a concentration ratio of PSMα3: insulin equal to or above 1 : 100. Molecular modelling revealed that PSMα3 most likely inhibits the production of insulin primary nuclei by competing for residues involved in its dimerization.

Abstract C102: mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers

Abstract BRCA1 and BRCA2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers, up to 10% BC, are associated with a lack of expression and/or function of BRCA-1 protein, which induces genomic instability. Considering the major role of BRCA1 gene in DNA repair process, therapeutic approaches using cytotoxic agents such as the platinum and PARP inhibitors are used as treatments. However, hypersensitivity to PARPi is limited in the clinic by acquired resistance after restoration of HR. We have developed a new potent strategy combining mRNAs with a tumor selective nanocarrier, to selectively rescue BRCA-1 functions and restore PARPi sensitivity, as potential therapeutic approach in cancers. ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-901, -902 , -903 nanoparticles containing mRNA derived from BRCA-1 were evaluated on breast cancer cells, harboring different BRCA-1 deletion and mutations. Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry and Tunel assay. BRCA-1 and RAD51 protein levels were evaluated by western blot. Sensitivity to Veliparib (PARPi) following ADGN-mRNA treatment was evaluated on PARPI resistance and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of IV-administered ADGN-BRCA NPs (2.0 mg/kg) was evaluated on breast (HCC1937-BRCA-1 deficient, PARPi resistant) cancer mouse xenografts. We have selected specific mRNA sequences encoding for different domains of BRCA-1 protein. We showed than ADGN-901 mediated rescue of BRCA-1 wild type expression inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to-60% and ADGN-903 markedly delay (>60%) the growth of all cells harboring defect in BRCA-1. In contrast, no significant cytotoxicity related to vehicle was observed. ADGN-901 and ADGN-903 resensitize the resistant cells to PARPi Veliparib inhibition. We demonstrated that the expression of domain of BRCA-1 encoded by ADGN-903 mRNA, appears to prevent RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. Intravenous-administration ADGN-901 and ADGN-903 (2.0mg/kg) prevented HCC-1937 tumor growth. ADGN-901 reduced tumor growth by 95% and ADGN-903 resulted in a tumor regression of 50%. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. ADGN-BRCA nanoparticles are effective in rescuing BRCA-1 functions in Breast cancer cells. Our study provides a proof-of-concept that restoration of the expression of the wild type form of BRCA-1 could be combined together with PARP inhibition for potent combinatorial cancer treatment. Citation Format: Gilles DIVITA, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Khadidja Boularache, Veronique Josserand, Neil Desai. mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C102.

Abstract 550: p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors

Abstract The p53 tumor suppressor mutations resulting in loss of function control tumor proliferation in 50% of human cancers. TP53 missense and nonsense mutations are present in about 50% and 7-12% of the p53 driven cancer, respectively. To date there is no effective treatment for rescuing p53 function in cancers harboring p53 nonsense mutations. We have developed a new potent strategy combining mRNA with a tumor selective nanocarrier, to rescue of p53 tumor suppressor functions as potential therapeutic approach in cancers. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides that form stable neutral nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring different types of p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (weekly, 0.5 & 1.0mg/kg) was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and lung H358 (p53 null/KRASG12C) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment was evaluated on PARPi resistant SUM-149PT and OVCAR-8 and PARPi sensitive MDA-MB436 cells. ADGN-531/AMG-510 synergy was evaluated on H358 and M-PACA cells and H358 mouse xenograft. ADGN-531 markedly delay the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. ADGN-531 mediated p53 function rescue is directly correlated to the type of p53 mutation in the cancer cells. ADGN-531 induces 80% cell proliferation inhibition in p53 null/nonsense cells and 20 to 50% in p53 missense cells. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition. ADGN-531 (1.0mg/kg) induces 40-60% tumor regression SaOs2, SW403 and H358 xenografted mouse model. We demonstrated a synergistic combination between ADGN-531/AMG-510 a KRASG12C inhibitor in human lung carcinoma. ADGN-531 (0.5mg/kg, weekly)/AMG-510 (10mg/kg, daily) induces about 80% tumor regression, higher than with either single agents. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to Veliparib. ADGN-531 treatments are well tolerated, no sign of clinical toxicity or inflammatory response was detected after single or repeated administrations. ADGN-531 is effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy or in combination together with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Khadidja Boularache, Melanie Guidetti, Veronique Josserand, Neil Desai. p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 550.

Abstract 1717: KRAS mutant gene editing prevents tumor growth in vivo and overcomes acquired resistance to KRASG12C inhibitor

Abstract Mutations on KRAS codon12 are among the most commonly observed mutations in pancreatic, colorectal and lung cancers. To date, only treatment targeting KRASG12C mutation have been successful in the clinic and approved. However, it is hampered by adaptive resistance, mainly associated with the emergence of other KRAS mutations and high amplification of KRASG12C allele. We have developed a new potent strategy combining gene editing with a tumor selective nanocarrier, to impair cancer cell proliferation by directly targeting G12D, G12V,and G12C mutations of the KRAS oncogene. ADGN-121, ADGN-122 and ADGN-123 are gene-editing complexes containing proprietary sgRNA targeting KRASG12D, KRASG12V or KRASG12C with mRNACas9 complexed with proprietary short amphipathic peptides that form stable neutral nanoparticles. The complexes were evaluated on pancreas, colorectal and lung cancer cells harboring KRASG12D, V, or C mutations. KRAS mutant indel frequency was evaluated by T7E1 method. In-vivo efficacy of IV-administered ADGN/mRNACas9gRNA (0.25-1.0mg/kg, 2 injections at day 1 & 7) was evaluated in Panc1 (KRASG12D) and SW403 (KRASG12V) mouse xenografts. ADGN-123 in combination with AMG-510 (KRASG12C inhibitor) was evaluated on H358, PACA2 and SW837 cells, exhibiting an acquired resistance to AMG-510. ADGN-121,-122 and -123 efficiently and selectively silenced KRASG12D, KRASG12V KRASG12C , respectively, in colorectal, pancreatic and lung cancer cells resulting in reduction of cell proliferation by 80% and inhibition of ERK and/or AKT phosphorylation. Only two IV-administrations of ADGN-121 containing gRNAG12D abolished Panc1 tumor growth in a dose dependent manner, resulting in tumor regression of 60-70% at 1.0mg/kg. ADGN-122 containing gRNAG12V abolished SW403 tumor growth with a tumor regression of 70% at 1.0 mg/kg. In contrast, no effect on tumor growth was observed with nonspecific gRNA. The combinations of ADGNs with drugs currently used on clinic have been evaluated. We demonstrated a synergistic combination ADGN-121/Abraxane in pancreatic carcinoma and ADGN-122/Capecitabine in colon Adenocarcinoma. We showed, that ADGN-123 containing gRNAG12C can effectively reduce the proliferation and inhibit ERK and AKT phosphorylation of AMG-510 acquired resistance cells and that no resistance occurs after ADGN-123 treatment. ADGN treatments are well tolerated, no sign of clinical toxicity, inflammatory response or emergence of other KRAS mutation was detected after single or repeated administrations. ADGNs were effective in targeting selectively mutated KRAS both in vitro and in vivo. Our study provides a proof-of-concept that ADGN can be applied to target driver mutations of cancers in vivo and permanently disrupt the oncogenic alleles, leading to major tumor regression. ADGN-123 can be used as a strategy to overcome resistance associated to small molecule inhibitors of KRASG12C. Citation Format: Gilles Divita, Veronica Guzman-Gonzales, Audrey Grunenberger, Elodie Czuba, Melanie Guidetti, Veronique Josserand, Neil Desai. KRAS mutant gene editing prevents tumor growth in vivo and overcomes acquired resistance to KRASG12C inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1717.

Synthetic amino acids-based short amphipathic peptides exhibit antifungal activity by targeting cell membrane disruption.

Availability of a limited number of antifungal drugs created a necessity to develop new antifungals with distinct mode of action. Investigation on a new series of peptides led us to identify Boc-His-Trp-His[1-(4-tert-butylphenyl)] (10g) as the most promising inhibitor exhibiting IC50 value of 4.4 µg/mL against Cryptococcus neoformans. Analog 10g exhibit high selectivity to fungal cells and was nonhemolytic and noncytotoxic at its minimum inhibitory concentration. 10g produced fungicidal effect on growing cryptococcal cells and displayed synergistic effect with amphotericin B. Overall cationic character of 10g resulted in interaction with negatively charged fungal membrane while hydrophobicity enhanced penetration inside the cryptococcal cells causing hole(s) formation and disruption to the membrane as evident by the scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy analyses. Flow cytometric investigation revealed rapid death of fungal cells by apopotic pathway.

Anticryptococcal activity and mechanistic studies of short amphipathic peptides.

Cryptococcus neoformans, an opportunistic fungal pathogen, causes cryptococcosis in immunocompromised persons. A series of modified L-histidines-containing peptides are synthesized that exhibit promising activity against C. neoformans. Analog 11d [L-His(2-adamantyl)-L-Trp-L-His(2-phenyl)-OMe] produced potency with an IC50 of 3.02 µg/ml (MIC = 5.49 µg/ml). This peptide is noncytotoxic and nonhaemolytic at the MIC and displays synergistic effects with amphotericin B at subinhibitory concentration. Mechanistic investigation of 11d using microscopic tools indicates cell wall and membrane disruption of C. neoformans, while flow cytometric analysis confirms cell death by apoptosis. This study indicates that 11d exhibits antifungal potential and acts via the rapid onset of action.

62 (PB052) - p53 mRNA rescue of tumor suppressor function prevents tumor growth and restores PARPi sensitivity in p53-deficient cancers in vitro and in vivo

Background: Loss of function in tumor suppressor genes is commonly associated with the onset/progression of cancer and the development of therapeutic resistance. The p53 tumor suppressor mutations resulting in loss of function are common across variety of tumors and control tumor cell proliferation in more than 50% of cancers. P53 null mutations are present in about 10% of p53 driven cancers. However, to date there is no effective treatment for rescuing p53 function in cancers. We have developed a new potent strategy combing p53 mRNA with a tumor selective nanocarrier, to rescue p53 tumor suppressor function as potential therapeutic approach in cancers. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-531 nanoparticles containing full length p53-mRNA were evaluated on 20 different cancer cell lines (osteosarcoma, pancreas, colorectal, ovarian, lung, breast and prostate cancers) harboring different types of p53 mutations (null, deletion, nonsense, missense). Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry, Elisa and Tunel assay. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (0.5 mg/kg) was evaluated on colorectal SW403 (p53 deleted) and osteosarcoma SaOs2 (p53 null) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment in vitro was evaluated on PARPI resistant SUM-149PT and OVCAR-8 cells and on PARPi sensitive MDA-MB436 cells. Results: ADGN-531 NPs markedly delay the growth of a large panel of cancer cells harboring p53-null or nonsense mutations, by inducing cell cycle arrest in G1 due to p21 upregulation and apoptosis following PUMA activation. We demonstrated that ADGN-531 mediated p53 function rescue is directly correlated to the type and level of p53 mutation in the cancer cells. ADGN-531 induces 60% to 80% cell proliferation inhibition in p53 null or nonsense mutated cells and 20 to 50% in p53 missense mutated cells. Intravenous-administration ADGN-531 containing wild type p53-mRNA (0.5 mg/kg) resulted in tumor growth inhibition of 90% and 70% in SaOs2 and SW403 xenografted mouse model, respectively. ADGN-531 NPs treatments are well tolerated, without inducing clinical toxicity or inflammatory response. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to the PARPi veliparib. Conclusions: ADGN-531 nanoparticles are effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by the ADGN-531 NP targeted delivery strategy could be combined together with other therapies for potent combinatorial cancer treatment. No conflict of interest.

Formulating RALA/Au nanocomplexes to enhance nanoparticle internalisation efficiency, sensitising prostate tumour models to radiation treatment

BackgroundGold nanoparticles (AuNP) are effective radiosensitisers, however, successful clinical translation has been impeded by short systemic circulation times and poor internalisation efficiency. This work examines the potential of RALA, a short amphipathic peptide, to enhance the uptake efficiency of negatively charged AuNPs in tumour cells, detailing the subsequent impact of AuNP internalisation on tumour cell radiation sensitivity.ResultsRALA/Au nanoparticles were formed by optimising the ratio of RALA to citrate capped AuNPs, with assembly occurring through electrostatic interactions. Physical nanoparticle characteristics were determined by UV–vis spectroscopy and dynamic light scattering. Nano-complexes successfully formed at w:w ratios > 20:1 (20 µg RALA:1 µg AuNP) yielding positively charged nanoparticles, sized < 110 nm with PDI values < 0.52. ICP-MS demonstrated that RALA enhanced AuNP internalisation by more than threefold in both PC-3 and DU145 prostate cancer cell models, without causing significant toxicity. Importantly, all RALA-AuNP formulations significantly increased prostate cancer cell radiosensitivity. This effect was greatest using the 25:1 RALA-AuNP formulation, producing a dose enhancement effect (DEF) of 1.54 in PC3 cells. Using clinical radiation energies (6 MV) RALA-AuNP also significantly augmented radiation sensitivity. Mechanistic studies support RALA-AuNP nuclear accumulation resulting in increased DNA damage yields.ConclusionsThis is the first study to demonstrate meaningful radiosensitisation using low microgram AuNP treatment concentrations. This effect was achieved using RALA, providing functional evidence to support our previous imaging study indicating RALA-AuNP nuclear accumulation.Graphic abstract

Abstract 1826: CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy

Abstract Background: KRASG12V and KRASG12D mutations are among the most commonly observed mutations in pancreatic (30% and 51%) and colorectal (27% and 45%) adenocarcinomas and have been associated with poor prognosis. Activating mutations in KRAS play potent roles in cancer initiation, propagation, and maintenance. Although they represent important therapeutic targets, no effective treatment for direct inhibition of KRASG12V and KRASG12D mutants have been reported. We have developed a new potent strategy combing the potency of CRISPR for gene editing with a tumor selective nanocarrier, to impair cancer cell proliferation by directly targeting G12V and G12D mutations of the KRAS oncogene. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with CRISPR/Cas9. ADGN nanoparticles containing Cas9mRNA and gRNA targeting either KRASG12D or KRASG12V were evaluated on pancreas (PANC1, PK-45H) and colorectal (LS513, SW480, SW403) cancer cells harboring KRASG12D or KRASG12V mutations. KRAS mutant indel frequency was evaluated by T7E1 method and compared to KRASWT (HT29, HS-69) cells. Cell proliferation was determined by flow cytometry. In-vivo efficacy of IV-administered ADGN/mRNACas9/gRNA complexes was evaluated in PANC1-LUC (KRASG12D) and SW403 (KRASG12V) mouse xenografts. The phosphorylation of ERK and AKT was measured by western blot, KRAS mutation level in the tumor and variation of housekeeping genes were estimated by deep sequencing. Results: We have developed highly specific gRNAs targeting KRASG12V and KRASG12D mutants complexed with ADGN-nanoparticles. These complexes efficiently and selectively silenced KRASG12V and KRASG12D in colorectal and pancreatic cancer cells in vitro and in vivo. Silencing KRAS mutations reduced the cell proliferation by 70% and the phosphorylation of the downstream effector pathways ERK and AKT. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12D abolished PANC1 tumor growth and induced KRASG12D gene editing in the tumor with an indel frequency of 76%. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12V abolished SW403 tumor growth and induced KRASG12V gene editing in the tumor with an indel frequency of 82%. In contrast, no effect on tumor growth were observed with ADGN-nanoparticles containing non-specific gRNA or gRNA targeting other KRAS mutations. ADGN/mRNACas9/gRNA targeting KRASG12D or KRASG12V abolished pancreatic and colorectal tumor growth respectively in vivo, without inducing any significant toxicity, emergence of off target effects or other KRAS mutations. Conclusions: ADGN-nanoparticle containing mRNACas9/gRNA targeting KRASG12D or KRASG12V were effective in selectively targeting mutated KRAS both in vitro and in vivo. Our study provides a proof-of-concept that ADGN/CRISPR can be applied to target driver mutations of cancers in vivo and permanently disrupt the oncogenic alleles, leading to inhibition of tumor growth. We proposed a new strategy for targeting KRAS oncogenes that can be useful in treatment of pancreatic and colorectal cancers. Citation Format: Gilles Divita, Elodie Czuba, Melanie Guidetti, Audrey Gunenberger, Leslie Tempremant, Veronique Josserand, Neil Desai. CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1826.

The monomers, oligomers, and fibrils of amyloid-β inhibit the activity of mitoBKCa channels by a membrane-mediated mechanism

A causative agent of Alzheimer's disease (AD) is a short amphipathic peptide called amyloid beta (Aβ). Aβ monomers undergo structural changes leading to their oligomerization or fibrillization. The monomers as well as all aggregated forms of Aβ, i.e., oligomers, and fibrils, can bind to biological membranes, thereby modulating membrane mechanical properties. It is also known that some isoforms of the large-conductance calcium-activated potassium (BKCa) channel, including the mitochondrial BKCa (mitoBKCa) channel, respond to mechanical changes in the membrane. Here, using the patch-clamp technique, we investigated the impact of full-length Aβ (Aβ1–42) and its fragment, Aβ25–35, on the activity of mitoBKCa channels. We found that all forms of Aβ inhibited the activity of the mitoBKCa channel in a concentration-dependent manner. Since monomers, oligomers, and fibrils of Aβ exhibit different molecular characteristics and structures, we hypothesized that the inhibition was not due to direct peptide-protein interactions but rather to membrane-binding of the Aβ peptides. Our findings supported this hypothesis by showing that Aβ peptides block mitoBKCa channels irrespective of the side of the membrane to which they are applied. In addition, we found that the enantiomeric peptide, D-Aβ1–42, demonstrated similar inhibitory activity towards mitoBKCa channels. As a result, we proposed a general model in which all Aβ forms i.e., monomers, oligomers, and amyloid fibrils, contribute to the progression of AD by exerting a modulatory effect on mechanosensitive membrane components.

Abstract 3948: Simultaneous in vivo PTEN rescue and KRAS silencing in cancer therapy

Abstract Background : The tumor suppressor PTEN and the KRAS oncogene play essential roles in tumorigenesis. PTEN mutations and resulting loss of function are common across variety of tumors and influence tumor cell proliferation. KRAS mutations occur in &amp;gt; 40% of cancer and have been associated with increased tumorigenicity. In numerous cancers, cooperative interactions between KRAS mutations and PTEN loss have been reported to promote metastatic development. However, to date there is no effective treatment for inhibiting KRAS activity or rescuing PTEN function in cancers. We investigated a new technique for simultaneous PTEN rescue and silencing of KRAS that may have therapeutic potential in PTEN/KRAS mutated pancreatic and other cancers. Methods : ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles through non-covalent electrostatic and hydrophobic interactions with a large panel of nucleic acids. Self-assembled ADGN/nucleic acid nanoparticles remain stable over time in serum and plasma conditions. ADGN peptide complexes with wt PTEN mRNA or siRNA targeting KRAS were evaluated on pancreas (PANC1), ovarian (SKOV3), prostate (PC3) and glioblastoma (U25) cell lines. PTEN, KRAS and AKT phosphorylation were evaluated by western blot. Cell proliferation and apoptosis activation were determined by flow cytometry and Tunel assay. In-vivo Efficacy of IV-administered ADGN-peptides complexed with PTEN mRNA (0.25 mg/kg) and/or KRAS siRNA (0.5 mg/kg) was tested in PANC1-LUC mouse xenografts. Cytokine response to ADGN-nucleic acid complexes were measured in blood samples using Luminex cytokine 20 plex. Results : ADGN-mediated delivery of wt PTEN mRNA, rescued PTEN function in the cell-lines tested. Restoring PTEN resulted in ~90% reduction of cell proliferation, 3-8 fold activation of cell apoptosis, reduction of AKT phosphorylation and cell cycle arrest in G1. ADGN-nanoparticles containing wt PTEN mRNA resulted in tumor growth inhibition (TGI) of 80%. ADGN-mediated delivery of siRNA targeting KRAS, induced knock-down of KRAS expression by 60-70% in the cell-lines and significantly reduced viability by 50-70%. ADGN-nanoparticles containing siRNA KRAS resulted in a TGI of 50%. Combination mRNA PTEN and siRNAs KRAS ADGN nanoparticles, resulted in a TGI of 90% in PANC1 xenografts and also slowed development of distant metastates. No nonspecific cytokine response was observed following administration of ADGN-nucleic acid complexes.Conclusions: ADGN-peptides complexed with mRNA and/or siRNA were effective in targeting mutated PTEN and KRAS both invitro and invivo. This suggests a new strategy for targeting both tumor suppressors and oncogenes. Citation Format: Gilles Divita, Veronique Josserand, Melanie Guidetti, Neil Desai. Simultaneous in vivo PTEN rescue and KRAS silencing in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3948.

Antimicrobial Metallopeptides.

Antimicrobial peptides are short amphipathic peptides that are produced by the innate immune system in order to protect a host from pathogens. They have been shown to have broad-spectrum antimicrobial activity toward Gram-positive and Gram-negative bacteria, as well as antifungal, antiprotozoan, and antiviral activity. These peptides are able to exert their activity through a variety of mechanisms that include inhibiting DNA and RNA replication, inhibiting protein synthesis, permeabilizing the cell membrane, disrupting proton and ion transmembrane gradients, and inhibiting cell wall biosynthesis. Certain antimicrobial peptides are able to utilize metals to modulate their activity through structural changes upon metal binding, metal sequestration, and redox chemistry. This work aims to provide a review of the current literature regarding the influence of metals on the activity of antimicrobial metallopeptides and their uses in drug delivery and the treatment of implant-associated infections.

Optimisation of vectorisation property: A comparative study for a secondary amphipathic peptide

RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation and intracellular delivery. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. In this context, the secondary amphipathic peptide CADY has shown to form stable siRNA complexes and to improve their cellular uptake independent of the endosomal pathway. In the present work, we have described the parameters influencing CADY nanoparticle formation (buffers, excipients, presence of serum, etc.), and have followed in details the CPP:siRNA self-assembly. Once optimal conditions were determined, we have compared the ability of seven different CADY analogues to form siRNA-loaded nanoparticles compared to CADY:siRNA. First of all, we were able to show by biophysical methods that structural polymorphism (α-helix) is an important prerequisite for stable nanoparticle formation independently of occurring sequence mutations. Luciferase assays revealed that siRNA complexed to CADY-K (shorter version) shows better knock-down efficiency on Neuro2a-Luc+ and B16-F10-Luc+ cells compared to CADY:siRNA. Altogether, CADY-K is an ideal candidate for further application especially with regards to ex vivo or in vivo applications.

Protein vaccination with HPV16 E7/Pep-1 nanoparticles elicits a protective T-helper cell-mediated immune response.

Two human papillomavirus (HPV) viral oncoproteins, E6 and E7 represent ideal targets for development of a therapeutic HPV vaccine. It is important to reduce the rate of HPV-associated malignancies through improvement of vaccine modalities. In this study, we used a short amphipathic peptide carrier, Pep-1, for delivery of the full-length HPV16 E7 protein into mammalian cells and evaluated immune responses and protective effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7/Pep-1 protein form stable nanoparticles through noncovalent binding with an average size of 120 to 250 nm. The efficient delivery of E7 protein by Pep-1 at molar ratio of 1:20 was detected in HEK-293T cell line for 1 h and 3 h post-transfection. Immunization with E7/Pep-1 nanoparticles at a ratio of 1:20 induced a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein in a murine tumor model. These data suggest that Pep-1 peptide would indicate promising applications for improvement of HPV therapeutic vaccines. © 2016 IUBMB Life, 68(6):459-467, 2016.

Mixed α/β-Peptides as a Class of Short Amphipathic Peptide Hydrogelators with Enhanced Proteolytic Stability.

Peptide hydrogels are a highly promising class of materials for biomedical application, albeit facing many challenges with regard to stability and tunability. Here, we report a new class of amphipathic peptide hydrogelators, namely mixed α/β-peptide hydrogelators. These mixed α/β-gelators possess good rheological properties (high storage moduli) and form transparent self-supporting gels with shear-thinning behavior. Infrared spectroscopy indicates the presence of β-sheets as the underlying secondary structure. Interestingly, self-assembled nanofibers of the mixed α/β-peptides display unique structural morphologies with alteration of the C-terminus (acid vs amide) playing a key role in the fiber formation and gelation properties of the resulting hydrogels. The incorporation of β3-homoamino acid residues within the mixed α/β-peptide gelators led to an increase in proteolytic stability of the peptides under nongelating conditions (in solution) as well as gelating conditions (as hydrogel). Under diluted conditions, degradation of mixed α/β-peptides in the presence of elastase was slowed down 120-fold compared to that of an α-peptide, thereby demonstrating beneficial enzymatic resistance for hydrogel applications in vivo. In addition, increased half-life values were obtained for the mixed α/β-peptides in human blood plasma, as compared to corresponding α-peptides. It was also found that the mixed α/β-peptides were amenable to injection via needles used for subcutaneous administrations. The preformed peptide gels could be sheared upon injection and were found to quickly reform to a state close to that of the original hydrogel. The shown properties of enhanced proteolytic stability and injectability hold great promise for the use of these novel mixed α/β-peptide hydrogels for applications in the areas of tissue engineering and drug delivery.

C5A Protects Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge.

A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate.

Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.