# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 5 Mechanisms of Bioactivities
### Anticancer Activity  
Aside from the antimicrobial activity, nontrivial aspects on the mechanism of anticancer activity of diastereomeric HDPs have also been explored. A noteworthy mechanism study was carried out by [PERSON] and [PERSON] [66] where they tested anticancer diastereomeric HDPs against small unilamellar vesicles (SUVs) as well as live cells. The SUVs were composed of phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylserine and cholesterol (PC/SM/PE/PS/Cho) lipids and mimicked cancer cell membranes, which possessed approximately 3% phosphatidylserine (PS) content in the outer leaf and thus have a slight negative charge [67]. Utilizing a combination of surface plasmon resonance and confocal microscopy for binding analysis as well as fluorescence spectroscopy and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy for structure and membrane permeation analysis, it was found that the diastereomeric HDPs could lyse cancer cells in a manner very similar to that of antimicrobial diastereomeric peptides [66]. Particularly, the peptides first bind the cell membrane via electrostatic interactions (Figure S2) and then dissolved the membrane like a detergent (Figure S3). Additionally, the diastereomers had a distorted helical structure and remained on the surface of the membrane once bound, as also shown in previous studies of antimicrobial diastereomers [51; 57]. However, some important differences were found in the anticancer mechanism of diastereomers that may have profound implications for the different design of anticancer HDPs compared with that of antimicrobial ones. In the study, SUVs were loaded with varying amounts of negatively-charged PS ranging from 3% to 20%. A previous study demonstrated that antimicrobial diastereomeric HDPs had up to 100-fold stronger binding to SUVs that mimicked negatively-charged bacterial membranes than to SUVs that mimicked zwitterionic mammalian membranes [49]. Following this logic, one would expect the SUVs loaded with more PS to bind more of the tested anticancer diastereomers. However, the results showed that the diastereomers bind only slightly better (i.e., approximately 2-fold) to the negative model membranes that mimicked cancer cells than to those mimicking normal cells. Moreover, a calcein release assay to detect membrane lysis showed that the tested diastereomers had similar lytic activity on SUVs with 3%-9% PS and that the activity was even lower on vesicles containing 20% PS [66]. These results indicated that the negative charge of cancer cell membranes may not be the sole decisive factor for their recognition. This further challenges the possibility that the leakage of negatively-charged PS to the outer membrane leaflet may be the key element for diastereomer recognition of cancer cells. Particularly, the addition of positive charges at the N-terminus of the peptides did not significantly affect their activity towards live cancer cells. Such procedure is known to increase the antimicrobial activity of diastereomers [66; 68; 69]. Furthermore, it was found that inversion of the amino acid sequence of diastereomers, which should affect only the dipole moment and thus decrease the helical structure, exerted a profound effect on the anticancer activity. One of the original un-inverted diastereomeric peptides afforded the best discrimination between negatively-charged and neutral SUVs. Nevertheless, this peptide afforded the lowest selectivity toward live cells. In contrast, its corresponding inverted sequence exhibited little to no differentiation between the SUVs, yet it had the highest selectivity for live cells [66]. Thus, the presence of negatively-charged PS in the outer membrane leaflet of cancer cells could not entirely explain the strong selective toxicity exhibited by the anticancer diastereomers as there are more factors involved in the mechanism of action of anticancer diastereomeric HDPs.