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.

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).

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.

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.

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.

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.

Consequences of Tacticity on the Growth and Permeability of Hyperthin Polyelectrolyte Multilayers.

A series of polyanions and polycations have been synthesized from atactic, syndiotactic, and isotactic forms of poly(methyl methacrylate) and used to construct polyelectrolyte multilayers (PEMs). Polymer tacticity has been found to have a large influence on film growth but only a slight effect on H2/N2 permeation selectivities and no significant influence on CO2/N2 permeation selectivities. The permeances of H2, CO2, and N2 across those PEMs exhibiting the highest H2/N2 selectivities were found to vary by as much as factors of 3, 6, and 5, respectively, depending on the tacticities employed. A simple model that accounts for the strong dependency of film growth on tacticity is presented.

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.

Peptide Recognition of Cholesterol in Fluid Phospholipid Bilayers.

N-Acetyl-LWYIKC-amide, a cholesterol recognition/interaction amino acid consensus (CRAC) peptide, has been found capable of recognizing an exchangeable form of cholesterol in liquid-disordered (l(d)) bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In sharp contrast, such recognition is barely detectable in analogous cholesterol-rich, liquid-ordered (l0) bilayers. These findings represent the first evidence for a peptide favoring a sterol as a nearest-neighbor in fluid bilayers. They also reveal that such recognition can be strongly dependent on the degree of compactness of the membrane.

Push and pull forces in lipid raft formation: the push can be as important as the pull.

Nearest-neighbor recognition measurements have been made using exchangeable mimics of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the liquid-ordered (lo) and liquid-disordered (ld) states. In the ld phase, the net interaction between these two lipids is repulsive. In the lo phase, their interactions are neither attractive nor repulsive. These results, together with previous nearest-neighbor measurements, imply that the overall driving force for lipid domain formation in bilayers composed of high-melting lipids, low-melting lipids, and cholesterol, corresponds to a strong pull (attraction) between the high-melting lipids and cholesterol, a significant push (repulsion) between the low-melting and high-melting lipids, and a significant push between the low-melting lipids and cholesterol. In a broader context, these results provide strong support for the notion that repulsive forces play a major role in the formation of lipid rafts.

Taming Amphotericin B.

A strategy is introduced for enhancing the cellular selectivity of Amphotericin B (AmB) and other classes of membrane-disrupting agents. This strategy involves attaching the agent to a molecular umbrella to minimize the disruptive power of aggregated forms. Based on this approach, AmB has been coupled to a molecular umbrella derived from one spermidine and two cholic acid molecules and found to have antifungal activities approaching that of the native drug. However, in sharp contrast to AmB, the hemolytic activity and the cytotoxcity of this conjugate toward HEK293 T cells have been dramatically reduced.

The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts.

CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been proposed for cholesterol's condensing effect: (i) an umbrella mechanism in which the acyl chains and cholesterol become more tightly packed as cholesterol content increases because they share limited space under phospholipid headgroups and (ii) a template mechanism whereby cholesterol functions as a planar hydrophobic template at the membrane surface, thereby maximizing hydrophobic interactions and the hydrophobic effect. Specifically, our NNR experiments rule out the umbrella mechanism and provide strong support for the template mechanism. Similar NNR measurements have also allowed us to address the question of whether the interactions between low-melting kinked phospholipids and cholesterol can play a significant role in the formation of lipid rafts. Specifically, these NNR measurements have led to our discovery of a new physical principle in the lipids and membranes area that must be operating in biological membranes, that is, a "push-pull" mechanism, whereby cholesterol is pushed away from low-melting phospholipids and pulled toward high-melting lipids. Thus, to the extent that lipid rafts play a role in the functioning of cell membranes, low-melting phospholipids must be active participants.

Exchangeable Mimics of DPPC and DPPG Exhibiting Similar Nearest-Neighbor Interactions in Fluid Bilayers.

The interactions between an exchangeable mimic of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Phos(±), with an exchangeable mimic of cholesterol, Chol, have been analyzed in fluid bilayers by means of nearest-neighbor recognition measurements. These interactions have been found to be very similar to those of an exchangeable mimic of 1,2-dipalmitoyl-sn-glycerol-3-phospho-(1'rac-glycerol) (DPPG), Phos(-), interacting with Chol. Thus, both phospholipids have a similar preference for becoming nearest-neighbors of Chol in the liquid-ordered (l0) phase, and both mix, ideally, with Chol in the liquid-disordered (ld) phase. These findings, together with the almost negligible screening effects found for the latter, provide strong evidence that electrostatic forces play a minor role in the preference that both phospholipids have in becoming a favored nearest-neighbor of Chol. They also imply that the main driving force for forming the liquid-ordered phase, and for defining the lateral organization of this phase, is an intrinsic affinity that high-melting lipids and cholesterol have for each other.

Molecular umbrella-amphotericin B conjugates.

A tetrawalled and an octawalled molecular umbrella conjugate of amphotericin B (AmB) have been synthesized. Both conjugates exhibit high water solubility, a low tendency to aggregate, negligible hemolytic activity at 100 µM, and an ability to release a derivative of AmB under reducing conditions that exhibits high antifungal activity. Whereas the larger, octawalled conjugate shows slight adsorption to liposomal membranes and an ability to cross them by passive transport, the tetrawalled analogue shows significant adsorption and much lower bilayer transport activity. The potential of molecular umbrella-AmB conjugates as therapeutic agents is briefly discussed.