Gilad Y, 1,3 Firer MA, 2 Albeck A 3 and Gellerman G 1

1. "Switch off/switch on" regulation of drug cytotoxicity by conjugation to a cancer cell targeting peptide Gilad Y,1,3 Firer MA,2AlbeckA3 and Gellerman G1 1 Department of Biological Chemistry, Ariel University, Ariel, 40700, Israel 2Department of Chemical Engineering, Ariel University, Ariel, 40700, Israel 3The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel ABSTRACT Targeted delivery of chemotherapeutic agents is one of the most important and challenging issues in modern chemotherapy. Targeted drug delivery using peptides with the capability of recognizing unique/over-expressed receptors on a cancer cell surface, is a powerful and much inquired technique (1). The conjugation of a number of anti-cancer therapeutics to one targeting peptide , through an amino acid platforms, is an opportunity for an efficient and economical utilization of a limited amount of these receptors. In addition, such conjugation can create a multi pro-drug system, in which drugs' toxicity is reduced. Therefore, a 'second chance' is given to candidates that failed advanced biological trials due to their over-potency. We previously have shown a facile synthesis of an anticancer agent - bis-9-anilinoacridine - peptidyle conjugates using Fmoc SPOS (2). Here we show a synthesis of more sophisticated Bi-nuclei amino acid scaffolds, their farther conjugation to a targeting peptide and finally in-vitro tests of these conjugates:This report demonstrates that murine B-cell leukemic cells, previously resistant to a chemotherapeutic chlorambucil, can be made sensitive to that drug so long as it is conjugated to a targeting peptide (3). Another result which is shown therein, is that the linkage of chemotherapeutics to a platform [DNA alkylator CLB and Topo I inhibitor camptothecin(CAMP) were linked to Lys] and subsequent conjugation of this platform to the carrier peptide, gains "switch off/switch on" capabilities specifically activating this "cocktail" of drugs in murine leukemic cells which are expressing antibodies to the carrier (4). Synthesis of Bi-nuclei AA-drug platforms and pMBP conjugate Specificity of p-MBP–CLB conjugates for target Target specificity of p-MBP–CLB conjugates for MBP cells Dose–response curves of myelin basic protein (MBP) and BCL-1 cells exposed to increasing doses of CLB or Melph Lysine based platforms Cells were cultured for 24 or 48 h together with drug (5–50 mmol/l). The cell suspensions were then centrifuged and the supernatants were replaced with fresh medium plus XTT reagent. The cells were reincubated for another 4 h and ODs in the wells were measured at both 480 and 680 nm. The difference between these measurements was used to calculate the % growth inhibition in test wells compared with control cells exposed to medium alone.* MBPor BCL-1 cells were exposed to a constant (50 mmol/l) concentration of p-MBP–FITC together with increasing (0–70 mmol/l) amounts of p-MBP–CLB and cultured for 2 h. The cell suspension was centrifuged, the cell pellet was washed thrice, and FITC fluorescence was measured using at 488 nm excitation and 530 nm emission. Inhibition of p-MBP–FITC binding was calculated for each concentration of added p-MBP–CLB by comparison with the control mixture (0 mmol/l p-MBP–Chlor).* Sensitivity of MBP cells exposed to MBP bioconjugates Reversal of drug resistance in target but not control cells (a) Fmoc-Lys(Alloc) -OH, DIPEA/DMF, rt, 90min (b) 20%piperidine/DMF; (c) CLB, PyBOP, DIPEA/DMF, rt, 90min; (d) Pd(PPh3)4, barbituric acid/DCM 3h; (e) TFA/H2O/EDT/TIPS/DCM (3:2:2:93), rt, 90min. Serine/Tyrosine based platforms Cells were cultured for 48 h with equivalent and increasing doses (0–50 mmol/l) of CLB conjugated as either single-copy (p-MBP–CLB1) or multicopyconjugates (p-MBP–CLB2 or p-MBP–CLB4). Control cells were also exposed to culture medium alone or medium containing 50 mmol/l melphalan. MBP (a) BCL-1 and normal mouse spleen cells (b) were cultured with equivalent and increasing drug concentrations of either free CLB or p-MBP–CLB. The % growth inhibition was calculated against the same, but untreated cells. *Each graph point represents the mean and SD of at least three measurements. CLB, chlorambucil (4-[bis(2-chloroethyl) amino]benzenebutanoic acid; MBP, myelin basic protein; FITC, fluorescein isothiocyanate. "Switch off/switch on" regulation of CLB+CAMP "cocktail" cytotoxicityby conjugation to a cell targeting peptide A B (a) Fmoc-(L)AA-OH, DIPEA/DMF, rt, 90min (b) 20%piperidine/DMF; (c) CLB, PyBOP, DIPEA/DMF, rt, 90min; (d) CLB, DCC, DMAP/DMF, rt, 3h; (e) TFA/H2O/EDT/TIPS/DCM (3:2:2:93), rt, 90min. Synthesis of pMBP-Drug Conjugate (a) Fmoc chemistry SPPS (b) Fmoc-Lys(Alloc)-OH, PyBOP, DIPEA/DMF, rt, 90min (c) 20%piperidine/DMF; (d) CLB, PyBOP, DIPEA/DMF, rt, 90min; (e) Pd(PPh3)4 (0.1eq), barbituric acid/DMF, 3h; (f) 1, DIPEA, rt, (90min x 2), (g) TFA/H2O/EDT/TIPS/DCM (3:2:2:93), 0oC-RT, 90min. m/4z m/3z A: Free or platform-linked drugs. B: MBP-conjugated drugs. The effect of free, platform-linked and peptide-conjugated drugs on cancer cells growth was studied. MBP or BCL-1 hybridoma cells were cultured for 48 h either alone or together with different compounds in increasing concentrations (5-50 µM). At the end of the culture period, cell growth was estimated by XTT assay. After 4 h incubation of the cells with the XTT reagent, optical density (OD) of the reduced XTT product was then measured at 480 and 680nm. Percentage of growth inhibition by a test compound was calculated by comparison of the treated culture versus a control culture (free of any compound). The result shown for each concentration point represents the mean +/- standard error calculated from 3 different experiments. In each experiment the compounds were tested in triplicates. HPLC Analysis of the pMBP conjugate LC-MS Analysis of the pMBP conjugate CONCLUSIONS Bearing functional side chains suitable for drug linkage, amino acids can form remarkable architectures with highly versatile and tunable drug linkage/release capabilities. Importantly, the developed facile SPOS is suitable for combinatorial synthesis of loaded MAAPs, significantly accelerating the discovery of favorable parameters matching essential for defining desired release profiles of the drugs from the platforms. The results of this study show that the use of multi-loaded dendrone linkers, that bear several covalently bound cytotoxic agents to one carrier molecule have enhanced efficacy for targeted cancer cells.In addition it was demonstrated that linkage of a cytotoxic drug cocktail consisting of DNA alkylator CLB and/or Topo I inhibitor CAMP to the Lys platform and subsequent conjugation to the MBP carrier gains "switch off/switch on" capabilities, specifically activating the cocktail in the target MBP hybridoma cells. In this study, a cocktail of free CLB and CAMP was non-specifically cytotoxic to hybridoma cell lines. Platform Lys(CLB)CAMP was not cytotoxic to any cell line (switch off), while MBP peptide – Lys(CBL)CAMP conjugate showed remarkable specific cytotoxicity (90% growth inhibition/1mM) on MBP hybridoma (switch on). Based on these results we are now constructing and assessing more sophisticated MAAPs bearing diverse chemotherapeutic "cocktails" in different linkage variations. These are being conjugated to peptide carriers of clinical significance for preclinical cancer therapy assessment. • REFRENCES • 1- Firer M, Gellerman G. Targeted drug delivery for cancer therapy: the other side of antibodies, J HematolOncol2012, 5(1):70 • 2- Brider T., Gilad Y., Gellerman G., A fast entry to the novel medicinally important 9-anilinoacridine peptidyls by solid phase organic synthesis (SPOS), Tetrahedron Lett., 2011, 52, 3640-3644. • 3- G. Gellerman, S. Baskin, L. Galia, Y. Gilad and M. A. Firer. Drug resistance to chlorambucil in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide,Anti-Cancer Drugs2013, 24:112-119. • 4- Gilad Y,Firer MA, Rozovsky A,Ragozin E,Redko B, Albeck A and Gellerman G. "Switch off/switch on" regulation of drug cytotoxicity by conjugation to a cell targeting peptide. Submitted.