The impact of phosphatidylserine exposure on cancer cell membranes on the activity of the anticancer peptide HB43

Abstract

HB43 (FAKLLAKLAKKLL) is a synthetic peptide active against cell lines derived from breast, colon, melanoma, lung, prostate, and cervical cancers. Despite its remarkable spectrum of activity, the mechanism of action at the molecular level has never been investigated, preventing further optimization of its selectivity. The alternation of charged and hydrophobic residues suggests amphipathicity, but the formation of alpha-helical structure seems discouraged by its short length and the large number of positively charged residues. Using different biophysical and in silico approaches we show that HB43 is completely unstructured in solution but assumes alpha-helical conformation in the presence of DPC micelles and liposomes exposing phosphatidylserine (PS) used as mimics of cancer cell membranes. Membrane permeabilization assays demonstrate that the interaction leads to the preferential destabilization of PS-containing vesicles with respect to PC-containing ones, here used as noncancerous cell mimics. ssNMR reveals that HB43 is able to fluidify the internal structure of cancer-cell mimicking liposomes while MD simulations show its internalization in such bilayers. This is achieved by the formation of specific interactions between the lysine side chains and the carboxylate group of phosphatidylserine and/or the phosphate oxygen atoms of targeted phospholipids, which could catalyze the formation of the alpha helix required for internalization. With the aim of better understanding the peptide biocompatibility and the additional antibacterial activity, the interaction with noncancerous cell mimicking liposomes exposing phosphatidylcholine (PC) and bacterial mimicking bilayers exposing phosphatidylglycerol (PG) is also described.

The Influence of Short Motifs on the Anticancer Activity of HB43 Peptide

“…The 1 H and 13 C chemical shift assignments of HB43 mutants are reported in Tables S1–S4 . As for the case of HB43 peptide [ 42 ], NMR data are consistent with the absence of a well-defined structure. This is demonstrated by (i) the limited chemical shift dispersion resulting in severe overlap among resonances of the same residue types, (ii) the large linewidth of amide protons which improves by lowering the temperature (experiments were performed at 278 K), probably due to the reduced exchange rate with the solvent, (iii) the chemical shift index (CSI) performed on Hα, Cα and Cβ atoms ( Figure 4 ) and (iv) CD spectra ( Figure S5 ).…”

“…Among the ACPs, we selected HB43 (also called FLAK50) for its wide spectrum of activity toward different kinds of cancers: breast, colon, skin, lung, and cervix cancer tissues according to the database, CancerPPD ( (accessed on January 2019)) [ 8 ]. Its versatility suggests that its mechanism of action is not related to a specific receptor but involves general traits of cancer cells, as also suggested by our previous studies [ 42 ].…”

“…It should be noted that at variance with mut1 and mut2 , mut3 and mut4 do not alter the amphipathic character of a hypothetical alpha-helix which has been found for HB43 when interacting with membrane models [ 42 ] ( Figure 2 ). Regarding mut3 , it is worth mentioning that the guanidinium group of arginine was shown to bind to the phosphate oxygen of phospholipids in a multidentate fashion, thus possibly improving the penetration into target membranes [ 79 , 80 ], although this might result in toxicity [ 81 ].…”

“…Isotropic bicelles for solution NMR were prepared as follows. A mixture of 33.3% DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and 66% DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) in chloroform was used to obtain a molar ratio of 0.5 (q = ([DMPC]/[DHPC]) thus ensuring a small almost spherical morphology [ 42 ]. The solvent was removed under a nitrogen stream and the samples were then lyophilized and resuspended in a 10 mM phosphate buffer at pH 6.6, to reach a final concentration of 1 M for the stock solution.…”

“…To better understand this effect, we decided to study the HB43 peptide (also called FLAK50). This peptide, with sequence FAKLLAKLAKKLL, has been shown to have an alpha structure and affinity for phosphatidylserine [ 42 ]. Furthermore, it has been reported to be active on skin, lung, cervical, breast, colon, and prostate cancer, according to the CancerPPD database [ 8 ].…”