Drug Design: Do Not Forget the Supramolecular Factor.

A major challenge currently facing medicinal chemists is designing agents that can selectively destroy drug resistant fungi and bacteria that have begun to emerge. One factor that has been overlooked by virtually all drug discovery/development approaches is the supramolecular factor, in which aggregated forms of a drug candidate exhibit low selectivity in destroying targeted cells while the corresponding monomers exhibit high selectivity. This Perspective discusses how we were led to the supramolecular factor through fundamental studies with simple model systems, how we reasoned that the selectivity of monomers of the antifungal agent amphotericin B should be much greater than the selectivity of the corresponding aggregates, and how we confirmed this hypothesis using derivatives of amphotericin B. In a broader context, these findings provide a strong rationale for considering the supramolecular factor in the design of new drug candidates and the testing of virtually all of them.

Creating Hyperthin Membranes for Gas Separations.

Interest in creating membranes that can separate gases has intensified in recent years owing, in large part, to climate change. Specifically, the need for separating CO2 and N2 from flue gas in an economically viable fashion is now considered urgent. This Perspective highlights two recent developments from my laboratory─defect repair of polyelectrolyte multilayers (PEMs) using micellar solutions of sodium dodecyl sulfate (SDS) and the surface modification of a highly permeable polymer, poly[1-(trimethylsilyl) propyne] (PTMSP)─which I believe have significant implications not only for this CO2/N2 problem but also for the ever-growing area of layer-by-layer (LbL) thin films. A brief mention is also made of past efforts that have been aimed at creating hyperthin membranes from porous surfactants and from PEMs with a view toward gas separations.

Increased CO2/N2 selectivity of PTMSP by surface crosslinking.

The surface crosslinking of poly[1-(trimethylsilyl)-1-propyne] (PTMSP) membranes by dithiothreitol under thiol-ene click reaction conditions has yielded membranes having CO2/N2 selectivities in excess of 30 with CO2 permeances in excess of 300 GPU (gas permeation units). The simplicity of this surface crosslinking strategy together with these permeation results suggests that PTMSP that is modified in such ways could lead to useful materials for the separation of CO2/N2 from flue gas and for certain other gaseous mixtures.

Cholesterol's Condensing Effect: Unpacking a Century-Old Mystery.

The ability of cholesterol to uncoil (i.e., condense) the acyl chains of phospholipids has been known for a century. Despite extensive studies of the interactions between cholesterol and phospholipids, a molecular-level understanding of this uncoiling phenomenon has remained elusive. Equally unclear has been whether cholesterol's two different faces (i.e., its relatively smooth α face and its relatively rough ß face) contribute to its condensing power. Because cholesterol's condensing effect is believed to play a major role in controlling the fluidity, structure, and functioning of all animal cell membranes, its biological importance cannot be overstated. This Perspective focuses on experimental evidence that addresses (i) the credibility of a popular "umbrella" mechanism that has been used to account for cholesterol's condensing effect, (ii) the credibility of an alternate "template" mechanism, (iii) the importance of cholesterol two-faced character with respect to its condensing power, and (iv) the viability of a surface occupancy model.

Membrane-Disrupting Molecules as Therapeutic Agents: A Cautionary Note.

Mechanistic studies have shown that aggregates of a common membrane disrupting molecule, Triton X-100, destroy the integrity of cholesterol-rich phospholipid bilayers via a catastrophic rupture process. In sharp contrast, attack on such membranes by monomers of Triton X-100 destroys their integrity through mild leakage events. This discovery of duplicity in the destruction of membrane integrity by a membrane-disrupting molecule has led to the design of derivatives of Amphotericin B that exhibit a lower tendency to aggregate and antifungal and hemolytic activities that are well-separated. An animal study with one such derivative has shown that its efficacy is similar to that of Amphotericin B but with substantially reduced toxicity. A related in vitro study of a series of derivatives of l-phenylalanine has revealed that monomers possess significant antibacterial activity, while aggregates of these same molecules exhibit hemolytic as well as antibacterial activity. Taken together, these experimental findings point to the need for paying special attention to differences in the selectivity between monomeric and aggregated forms of membrane-disrupting molecules as therapeutic agents, where monomers are expected to be the more selective species. Whether improving the selectivity of antimicrobial peptides and other antimicrobial agents is also possible by reducing their tendency to aggregate, and whether membrane-disrupting molecules can be created that exploit differences in the lipid composition between coronaviruses and mammalian cells, are two important questions that remain to be answered.

Defect Repair of Polyelectrolyte Bilayers Using SDS: The Action of Micelles Versus Monomers.

Defects within single, double, and triple polyelectrolyte bilayers derived from poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethyammonium chloride) (PDDA) have been repaired using aqueous solutions of sodium dodecyl sulfate (SDS), as evidenced by a reduction in their permeability and an increase in their permeation selectivity. In contrast to the use of monomer solutions of SDS, which were moderately effective in repairing only double and triple bilayers, micellar solutions proved highly effective for all three assemblies. Evidence for intact micelles or micellar fragments being deposited on the surface of single bilayers of PSS/PDDA has been obtained from a combination of atomic force microscopy, X-ray photoelectron spectroscopy, ellipsometry, and contact angle measurements. Observed CO2 permeances of ca. 200 GPU and CO2/N2 selectivities of ca. 30 for SDS-repaired, single bilayers of PSS/PDDA suggest that further development of such assemblies could have the practical potential for the separation of CO2 from N2 in the flue gas.

Improving the Cellular Selectivity of a Membrane-Disrupting Antimicrobial Agent by Monomer Control and by Taming.

Antimicrobial resistance represents a significant world-wide health threat that is looming. To meet this challenge, new classes of antimicrobial agents and the redesign of existing ones will be required. This review summarizes some of the studies that have been carried out in my own laboratories involving membrane-disrupting agents. A major discovery that we made, using a Triton X-100 as a prototypical membrane-disrupting molecule and cholesterol-rich liposomes as model systems, was that membrane disruption can occur by two distinct processes, depending on the state of aggregation of the attacking agent. Specifically, we found that monomers induced leakage, while attack by aggregates resulted in a catastrophic rupture of the membrane. This discovery led us to design of a series of derivatives of the clinically important antifungal agent, Amphotericin B, where we demonstrated the feasibility of separating antifungal from hemolytic activity by decreasing the molecule's tendency to aggregate, i.e., by controlling its monomer concentration. Using an entirely different approach (i.e., a "taming" strategy), we found that by covalently attaching one or more facial amphiphiles ("floats") to Amphotericin B, its aggregate forms were much less active in lysing red blood cells while maintaining high antifungal activity. The possibility of applying such "monomer control" and "taming" strategies to other membrane-disrupting antimicrobial agents is briefly discussed.

The Origin of Lipid Rafts.

The time-averaged lateral organization of the lipids and proteins that make up mammalian cell membranes continues to be the subject of intense interest and debate. Since the introduction of the fluid mosaic model almost 50 years ago, the "lipid raft hypothesis" has emerged as a popular concept that has captured the imagination of a large segment of the biomembrane community. In particular, the notion that lipid rafts play a pivotal role in cellular processes such as signal transduction and membrane protein trafficking is now favored by many investigators. Despite the attractiveness of lipid rafts, their composition, size, lifetime, biological function, and even the very existence remain controversial. The central tenet that underlies this hypothesis is that cholesterol and high-melting lipids have favorable interactions (i.e., they pull together), which lead to transient domains. Recent nearest-neighbor recognition (NNR) studies have expanded the lipid raft hypothesis to include the influence that low-melting lipids have on the organization of lipid membranes. Specifically, it has been found that mimics of cholesterol and high-melting lipids are repelled (i.e., pushed away) by low-melting lipids in fluid bilayers. The picture that has emerged from our NNR studies is that lipid mixing is governed by a balance of these "push and pull" forces, which maximizes the number of hydrocarbon contacts and attractive van der Waals interactions within the membrane. The power of the NNR methodology is that it allows one to probe these push/pull interaction energies that are measured in tens of calories per mole.

Clicking the Surface of Poly[1-(trimethylsilyl)propyne] (PTMSP) via a Thiol-Ene Reaction: Unexpected CO2/N2 Permeability.

The surface modification of poly[1-(trimethylsilyl)propyne] (PTMSP) film via a thiol-ene click reaction with sodium 3-mercapto-1-propanesulfonate has yielded membranes having a CO2 permeance as high as 530 GPU with a CO2/N2 selectivity of 21. This level of performance, together with the simplicity of this surface modification, suggests that such materials could become viable alternatives to some of the most promising membrane materials that are currently being explored for the practical capture of CO2 from flue gas.

Hyperthin Membranes for Gas Separations via Layer-by-Layer Assembly.

Thin film formation via the Layer-by-Layer method is now a well-established and broadly used method in materials science. We have been keenly interested in exploiting this technique in the area of gas separations. Specifically, we have sought to create hyperthin (<100 nm) polyelectrolyte-based membranes that have practical potential for the separation of CO2 from N2 (flue gas) and H2 from CO2 (syngas). In this personal account, we summarize recent studies that have been aimed at measuring the influence of a variety of factors that can affect the permeability and permeation selectivity of hyperthin polyelectrolyte multilayers (PEMs).

Layer-by-layer assembly of a polymer of intrinsic microporosity: targeting the CO2/N2 separation problem.

A polymer of intrinsic microporosity, 1, has been incorporated into a series of polyelectrolyte multilayers via the layer-by-layer deposition method. One of these assemblies [a 6 nm multilayer derived from 1 plus poly(diallyldimethylammonium chloride)] showed exceptional permeation properties with respect to CO2 and N2. Specifically, this film exhibited a CO2 permeance of 130 GPU and a CO2/N2 selectivity of 33.

Net Interactions That Push Cholesterol Away from Unsaturated Phospholipids Are Driven by Enthalpy.

The exchangeable unsaturated phospholipids c1-Phos and c3-Phos, which bear one and three permanent kinks, respectively, in their acyl chains, are mimics of the biologically important, low-melting phosphatidylcholines (PCs) having one and three cis double bonds in their sn-2 chains (i.e., 16:0,18:1 PC and 16:0,18:3 PC, respectively). The net interaction of an exchangeable form of cholesterol (Chol) with c1-Phos and with c3-Phos has been investigated using the nearest-neighbor recognition method. These interactions were found to be unfavorable in both cases having a positive free energy, ω, for replacing like by unlike nearest-neighbor contacts. The values for this free energy (or interaction parameter) were 165 cal/mol between Chol and c1-Phos and 395 cal/mol between Chol and c3-Phos. We now report the temperature dependence of these interactions in liquid-disordered bilayers. Their experimentally determined temperature dependencies, in combination with Monte Carlo simulations, revealed that the interaction parameter ω is dominated in both cases by enthalpy. These findings have important implications for the distribution of lipids in natural membranes and for the formation of lipid rafts.

A plug and socket approach for tightening polyelectrolyte multilayers.

A plug and socket approach for tightening polyelectrolyte multilayers is introduced based on the use pendant ß-cyclodextrin groups. Prototypical multilayers derived from poly(sodium 4-styrene sulfonate) and ß-cyclodextrin-containing poly(4-vinylbenzyltrimethylammonium chloride) are described. Evidence for tightened multilayers has been obtained from gas permeation, swelling and quartz crystal microbalance with dissipation (QCM-D) measurements.

pKa-Dependent Facilitated Transport of CO2 across Hyperthin Polyelectrolyte Multilayers.

Hyperthin (ca. 20-30 nm thick) polyelectrolyte multilayers have been fabricated that are capable of facilitated transport of CO2. These membranes were fabricated from polycations bearing pendant groups of varying basicity plus poly(sodium 4-styrenesulfonate) as a polycounterion. A strong dependency of such transport on the basicity of the pendant groups (i.e., fixed carrier sites) has been found, where pKa values in the range of ca. 5-7 appear optimal.

Lipid Raft Formation: Key Role of Polyunsaturated Phospholipids.

The forces that drive lipid raft formation are poorly understood. To date, most of the attention has focused on attractive interactions between cholesterol and high-melting lipids. Remarkably little attention has been paid to repulsive forces. Here, we show that repulsive interactions between an exchangeable mimic of cholesterol and an exchangeable mimic of a low-melting phospholipid in liquid-disordered bilayers can be much stronger than the attractive forces between this same sterol and an exchangeable mimic of a high-melting phospholipid in liquid-ordered bilayers. We conclude that polyunsaturated phospholipids have been largely overlooked as major players in lipid raft formation. This knowledge should stimulate considerable interest in controlling the levels of polyunsaturated phospholipids for the proper functioning of cell membranes.

Simple Strategy for Taming Membrane-Disrupting Antibiotics.

A strategy has been devised for increasing the cellular selectivity of membrane-disrupting antibiotics based on the attachment of a facially amphiphilic sterol. Using Amphotericin B (AmB) as a prototype, covalent attachment of cholic acid bound to a series of α,ω-diamines has led to a dramatic reduction in hemolytic activity, a significant reduction in toxicity toward HEK293T cells, and significant retention of antifungal activity.

Evidence for Surface Recognition by a Cholesterol-Recognition Peptide.

Two cholesterol recognition/interaction amino-acid consensus peptides, N-acetyl-LWYIKC-amide, and N-acetyl-CLWYIK-amide, have been coupled to exchangeable mimics of Chol (cholesterol) and Phos (1,2-dipalmitoyl-sn-glycerol-3-phospho-(1'rac-glycerol)) via disulfide bond formation. Equilibration between Chol and Phos via thiolate-disulfide interchange reactions has revealed that both peptides favor Chol as a nearest-neighbor in liquid-disordered (ld) bilayers to the same extent. In contrast, no Chol- or Phos-recognition could be detected by these peptides in analogous liquid-ordered (lo) bilayers. Fluorescence measurements of the tryptophan moiety have shown that both peptides favor the membrane-water interface. Taken together, these results provide strong evidence that the recognition behavior of the LWYIK motif is, fundamentally, a surface phenomenon but that partial penetration into the bilayer is also necessary.

Gas Permeability of Hyperthin Polyelectrolyte Multilayers Having Matched and Mismatched Repeat Units.

A series of polyelectrolyte multilayers (PEMs) has been fabricated using polyanions and polycations that have repeat units (i) similar in structure and composition (matched), (ii) partially similar in structure and composition (semimatched), and (iii) very different in structure and composition (mismatched). The primary aim of this investigation was to determine whether the matching of the polyelectrolytes can significantly influence the permeability properties of hyperthin PEMs. While matching, per se, was not found to be a key factor in defining membrane permeability, large differences in permeability were observed (the permeances of N2 varied by a factor of 20), which were correlated with the concentration of pendant aryl groups present, i.e., the greater the concentration of these groups, the higher the permeability. Analysis by AFM indentation measurements further revealed that high-permeability PEMs tend to be more compliant than low-permeability PEMs. These findings underscore the need for considering a broad range of polyelectrolyte combinations when optimizing a particular functional property of PEMs.