Andrey Ivankin

A miniature mimic of host defense peptides with systemic antibacterial efficacy

Oligomers of acylated lysines (OAKs) are synthetic mimics of host defense peptides (HDPs) with promising antimicrobial properties. Here we challenged the OAK concept for its ability to generate both systemically efficient and economically viable lead compounds for fighting multidrug‐resistant bacteria. We describe the design and characterization of a miniature OAK composed of only 3 lysyls and 2 acyls (designated C12(ω7)K‐β12) that preferentially targets gram‐positive species by a bacteriostatic mode of action. To gain insight into the mechanism of action, we examined the interaction of OAK with various potential targets, including phospholipid bilayers, using surface plasmon resonance, and Langmuir monolayers, using insertion assays, epifluorescence microscopy, and grazing incidence X‐ray diffraction, in a complementary manner. Collectively, the data support the notion that C12(ω7)K β12 damages the plasma‐membrane architecture similarly to HDPs, that is, following a near‐classic 2‐step interaction including high‐affinity electrostatic adhesion and a subsequent shallow insertion that was limited to the phospholipid head group region. Notably, preliminary acute toxicity and efficacy studies performed with mouse models of infection have consolidated the potential of OAK for treating bacterial infections, including systemic treatments of methicillin‐resistant Staphylococcus aureus. Such simple yet robust chemicals might be useful for various antibacterial applications while circumventing potential adverse effects associated with cytolytic compounds.—Sarig, H., Livne, L., Held‐Kuznetsov, V., Zaknoon, F., Ivankin, A., Gidalevitz, D., Mor, A. A miniature mimic of host defense peptides with systemic antibacterial efficacy. FASEB J. 24, 1904–1913 (2010). www.fasebj.org

Role of the conformational rigidity in the design of biomimetic antimicrobial compounds.

A link between structural flexibility of biomimetic antimicrobials and their ability to penetrate into the hydrophobic core and disrupt the integrity of bacterial lipid model membranes has been established using liquid surface X-ray scattering techniques. Results indicate that the modes of interaction of flexible and conformationally restrained antimicrobials with the bacterial membranes are different.

A comparative study on the interactions of SMAP-29 with lipid monolayers

This work investigates the discrimination of lipid monolayers by the ovine antimicrobial peptide SMAP-29 and compares it to that of the human LL-37 peptide. Fluid phospholipid monolayers were formed in a Langmuir trough and subsequently studied with the X-ray scattering techniques of X-ray reflectivity and grazing incidence X-ray diffraction. Any changes in the phospholipid structure after injection of peptide under the monolayer were considered to be due to interactions between the peptides and lipids. The data show that SMAP-29 discriminates against negatively charged phospholipids in a similar way to LL-37. However, it is even more interesting to note that despite a higher concentration of SMAP-29 near the monolayer, ensured by its greater charge as compared to LL-37, the amount of SMAP-29 needed to observe monolayer disruption was around three and a half times the number of molecules of LL-37 used to see similar changes with the same system. This result suggests that the structure, amino acid sequence or size of the peptide may well be as important as electrical charge and therefore gives many implications for the further study of antimicrobial peptides with regards to novel drug design and development.

Antibacterial Properties and Mode of Action of a Short Acyl-Lysyl Oligomer

ABSTRACT We investigated the potency, selectivity, and mode of action of the oligo-acyl-lysine (OAK) NC12-2β12, which was recently suggested to represent the shortest OAK sequence that retains nonhemolytic antibacterial properties. A growth inhibition assay against a panel of 48 bacterial strains confirmed that NC12-2β12 exerted potent activity against gram-positive bacteria while exhibiting negligible hemolysis up to at least 100 times the MIC. Interestingly, NC12-2β12 demonstrated a bacteriostatic mode of action, unlike previously described OAKs that were bactericidal and essentially active against gram-negative bacteria only. The results of various experiments with binding to model phospholipid membranes correlated well with those of the cytotoxicity experiments and provided a plausible explanation for the observed activity profile. Thus, surface plasmon resonance experiments performed with model bilayers revealed high binding affinity to a membrane composition that mimicked the plasma membrane of staphylococci (global affinity constant [Kapp], 3.7 × 106 M−1) and significantly lower affinities to mimics of Escherichia coli or red blood cell cytoplasmic membranes. Additional insertion isotherms and epifluorescence microscopy experiments performed with model Langmuir monolayers mimicking the outer leaflet of plasma membranes demonstrated the preferential insertion of NC12-2β12 into highly anionic membranes. Finally, we provide mechanistic studies in support of the view that the bacteriostatic effect resulted from a relatively slow process of plasma membrane permeabilization involving discrete leakage of small solutes, such as intracellular ATP. Collectively, the data point to short OAKs as a potential source for new antibacterial compounds that can selectively affect the growth of gram-positive bacteria while circumventing potential adverse effects linked to lytic compounds.

Mechanism of membrane perturbation by the HIV-1 gp41 membrane-proximal external region and its modulation by cholesterol

Membrane-activity of the glycoprotein 41 membrane-proximal external region (MPER) is required for HIV-1 membrane fusion. Consequently, its inhibition results in viral neutralization by the antibody 4E10. Previous studies suggested that MPER might act during fusion by locally perturbing the viral membrane, i.e., following a mechanism similar to that proposed for certain antimicrobial peptides. Here, we explore the molecular mechanism of how MPER permeates lipid monolayers containing cholesterol, a main component of the viral envelope, using grazing incidence X-ray diffraction and X-ray reflectivity. Our studies reveal that helical MPER forms lytic pores under conditions not affecting the lateral packing order of lipids. Moreover, we observe an increment of the surface area occupied by MPER helices in cholesterol-enriched membranes, which correlates with an enhancement of the 4E10 epitope accessibility in lipid vesicles. Thus, our data support the view that curvature generation by MPER hydrophobic insertion into the viral membrane is functionally more relevant than lipid packing disruption.

Molecular Basis for Anticancer Activity of Host Defense Peptides

The high toxicity of most anticancer chemotherapeutic drugs and their deactivation by multidrug resistant phenotypes motivated an extensive search for drugs with new modes of action. Host defense peptides (HDPs) represent a promising class of natural-source drugs with a distinct mode of action, decreased likelihood of resistance development, and low intrinsic cytotoxicity. However, the development of antitumoral HDP-based compounds has been hindered by poor understanding of the molecular mechanisms of the HDPs’ selective killing of cancer cells. Accumulating evidence indicates that the outer leaflet of tumor cell plasma membranes is enriched in anionic lipids such as phosphatidylserine (PS) and various gangliosides that may render the cancer cells susceptible to cationic HDPs. Here we report results of a combined AFM-X-ray study aimed at the understanding an impact of elevated levels of PS and gangliosides GM3 and GD3 in cell membranes on the cytotoxic activity of HDPs. Lipid monolayers at the air-liquid interface and supported lipid bilayers composed of DPPC and GD3, GM3, or DPPS at different ratios were used to model plasma membrane of cancer cells. The electron density profiles across the films, derived from X-ray reflectivity data, demonstrate that HDPs penetrate into all DPPC/anionic lipid monolayers, however with a different propensity. The HDPs’ membrane-insertion propensity rises with increase in concentration of anionic lipids in the films and is the highest for GD3 and the lowest for DPPS. Grazing incidence X-ray diffraction data together with AFM indicate that HDPs can degrade effectively ordering of anionic lipids in the membranes. Our results suggest that the molecular mechanisms underlying the antibacterial and anticancer activities of HDPs may be the same.

Cholesterol as a Modulator of the HIV-1 gp41 Fusion Domain's Function

The early steps in HIV-1 infection of cells involve fusion of the envelope with the host cell membrane. The N-terminal fusion domain of the viral envelope glycoprotein 41 subunit is widely accepted to play a key role in the fusion process facilitating membrane anchoring, destabilization, and bending. Ability of the fusion domain to perform these essential functions depends strongly on the structural and mechanical properties of the host cell membrane, which are defined by the lipid composition and cholesterol content. Here we present results of an X-ray study aimed at the understanding the effect of cholesterol concentration in model lipid membranes on the activity of the viral fusion domain. Lipid monolayers at the air-liquid interface composed of DPPC and cholesterol were used to model an approximate environment where the fusion domain comes into contact with the host cell membrane. The electron density profiles across the films, derived from X-ray reflectivity data, demonstrate that the fusion domain penetrates into all DPPC/cholesterol monolayers. The depth of membrane insertion and orientation/conformation of the fusion domain within the film, as well as lipid-to-fusion peptide ratio, depend strongly on the membrane cholesterol concentration. Distinct insertion modes also suggest that the fusion domain-induced membrane curvature is considerably different in bilayers with low and high cholesterol content. Finally, using grazing incidence X-ray diffraction we have demonstrated that the viral fusion domain possesses limited membrane destabilizing effect and is incapable to degrade the gel phase in the DPPC/cholesterol films.