Potency of a small molecule that targets the molluscum contagiosum virus processivity factor increases when conjugated to a tripeptide.

We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.

BMaps: A Web Application for Fragment-Based Drug Design and Compound Binding Evaluation.

Fragment-based drug design uses data about where, and how strongly, small chemical fragments bind to proteins, to assemble new drug molecules. Over the past decade, we have been successfully using fragment data, derived from thermodynamically rigorous Monte Carlo fragment-protein binding simulations, in dozens of preclinical drug programs. However, this approach has not been available to the broader research community because of the cost and complexity of doing simulations and using design tools. We have developed a web application, called BMaps, to make fragment-based drug design widely available with greatly simplified user interfaces. BMaps provides access to a large repository (>550) of proteins with 100s of precomputed fragment maps, druggable hot spots, and high-quality water maps. Users can also employ their own structures or those from the Protein Data Bank and AlphaFold DB. Multigigabyte data sets are searched to find fragments in bondable orientations, ranked by a binding-free energy metric. The designers use this to select modifications that improve affinity and other properties. BMaps is unique in combining conventional tools such as docking and energy minimization with fragment-based design, in a very easy to use and automated web application. The service is available at https://www.boltzmannmaps.com.

An allosteric inhibitor of sirtuin 2 deacetylase activity exhibits broad-spectrum antiviral activity.

Most drugs used to treat viral disease target a virus-coded product. They inhibit a single virus or virus family, and the pathogen can readily evolve resistance. Host-targeted antivirals can overcome these limitations. The broad-spectrum activity achieved by host targeting can be especially useful in combating emerging viruses and for treatment of diseases caused by multiple viral pathogens, such as opportunistic agents in immunosuppressed patients. We have developed a family of compounds that modulate sirtuin 2, an NAD+-dependent deacylase, and now report the properties of a member of that family, FLS-359. Biochemical and x-ray structural studies show that the drug binds to sirtuin 2 and allosterically inhibits its deacetylase activity. FLS-359 inhibits the growth of RNA and DNA viruses, including members of the coronavirus, orthomyxovirus, flavivirus, hepadnavirus, and herpesvirus families. FLS-359 acts at multiple levels to antagonize cytomegalovirus replication in fibroblasts, causing modest reductions in viral RNAs and DNA, together with a much greater reduction in infectious progeny, and it exhibits antiviral activity in humanized mouse models of infection. Our results highlight the potential of sirtuin 2 inhibitors as broad-spectrum antivirals and set the stage for further understanding of how host epigenetic mechanisms impact the growth and spread of viral pathogens.

A small molecule that targets the processivity factor of molluscum contagiosum virus has therapeutic potential.

Molluscum contagiosum (MC) is an infectious disease that occurs only in humans with a tropism that is narrowly restricted to the outermost epidermal layer of the skin. Molluscum contagiosum virus (MCV) is the causative agent of MC which produces skin lesions that can persist for months to several years. MCV is efficiently transmitted by direct physical contact or by indirect contact with fomites. MC is most prevalent in children and immune compromised patients. The failure to develop a drug that targets MCV replication has been hampered for decades by the inability to propagate MCV in cell culture. To address this dilemma, we recently engineered a surrogate poxvirus expressing the MCV processivity factor (mD4) as the drug target. The mD4 protein is essential for viral replication by keeping the viral polymerase tethered to the DNA template. In this study we have designed and synthesized a lead compound (7269) that is able to prevent mD4 dependent processive DNA synthesis in vitro (IC50 = 6.8 µM) and effectively inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells (EC50 = 13.2 µM) with negligible cytotoxicity. In human liver microsomes, 7269 was shown to be stable for almost 2 h. When tested for penetration into human cadaver skin in a formulated gel, the level of 7269 in the epidermal layer was nearly 100 times the concentration (EC50) needed to inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells. The gel formulated 7269 was scored as a non-irritant on skin and shown to have a shelf-life that was completely stable after several months. In summary, 7269 is a potential Lead for becoming the first MCV anti-viral compound to treat MC and thereby, addresses this unmet medical need that has persisted for many decades.

Herpes Simplex Virus-1 infection in human primary corneal epithelial cells is blocked by a stapled peptide that targets processive DNA synthesis.

PURPOSE: Acyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus -1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. METHODS: A series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. RESULTS: The stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. CONCLUSIONS: Hydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

Fragment-based design of small molecule PCSK9 inhibitors using simulated annealing of chemical potential simulations.

PCSK9 is a protein secreted by the liver that binds to the low-density lipoprotein receptor (LDLR), causing LDLR internalization, decreasing the clearance of circulating LDL particles. Mutations in PCSK9 that strengthen its interactions with LDLR result in familial hypercholesterolemia (FH) and early onset atherosclerosis, while nonsense mutations of PCSK9 result in cardio-protective hypocholesterolemia. These observations led to PCSK9 inhibition for cholesterol lowering becoming a high-interest therapeutic target, with antibody drugs reaching the market. An orally-available small molecule drug is highly desirable, but inhibiting the PCSK9/LDLR protein-protein interaction (PPI) has proven challenging. Alternate approaches to finding good lead candidates are needed. Motivated by the FH mutation data on PCSK9, we found that modeling the PCSK9/LDLR interface revealed extensive electron delocalization between and within the protein partners. Based on this, we hypothesized that compounds assembled from chemical fragments could achieve the affinity required to inhibit the PCSK9/LDLR PPI if they were selected to interact with PCSK9 in a way that, like LDLR, also involves significant fractional charge transfer to form partially covalent bonds. To identify such fragments, Simulated Annealing of Chemical Potential (SACP) fragment simulations were run on multiple PCSK9 structures, using optimized partial charges for the protein. We designed a small molecule, composed of several fragments, predicted to interact at two sites on the PCSK9. This compound inhibits the PPI with 1 µM affinity. Further, we designed two similar small molecules where one allows charge delocalization though a linker and the other doesn't. The first inhibitor with charge delocalization enhances LDLR surface expression by 60% at 10 nM, two orders of magnitude more potent than the EGF domain of LDLR. The other enhances LDLR expression by only 50% at 1 µM. This supports our conjecture that fragments can have surprisingly outsized efficacy in breaking PPI's by achieving fractional charge transfer leading to partially covalent bonding.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor.

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4-19.7 µM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 µM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.

High-performance nanomaterials formed by rigid yet extensible cyclic ß-peptide polymers.

Organisms have evolved biomaterials with an extraordinary convergence of high mechanical strength, toughness, and elasticity. In contrast, synthetic materials excel in stiffness or extensibility, and a combination of the two is necessary to exceed the performance of natural biomaterials. We bridge this materials property gap through the side-chain-to-side-chain polymerization of cyclic ß-peptide rings. Due to their strong dipole moments, the rings self-assemble into rigid nanorods, stabilized by hydrogen bonds. Displayed amines serve as functionalization sites, or, if protonated, force the polymer to adopt an unfolded conformation. This molecular design enhances the processability and extensibility of the biopolymer. Molecular dynamics simulations predict stick-slip deformations dissipate energy at large strains, thereby, yielding toughness values greater than natural silks. Moreover, the synthesis route can be adapted to alter the dimensions and displayed chemistries of nanomaterials with mechanical properties that rival nature.

Hot-spot identification on a broad class of proteins and RNA suggest unifying principles of molecular recognition.

Chemically diverse fragments tend to collectively bind at localized sites on proteins, which is a cornerstone of fragment-based techniques. A central question is how general are these strategies for predicting a wide variety of molecular interactions such as small molecule-protein, protein-protein and protein-nucleic acid for both experimental and computational methods. To address this issue, we recently proposed three governing principles, (1) accurate prediction of fragment-macromolecule binding free energy, (2) accurate prediction of water-macromolecule binding free energy, and (3) locating sites on a macromolecule that have high affinity for a diversity of fragments and low affinity for water. To test the generality of these concepts we used the computational technique of Simulated Annealing of Chemical Potential to design one small fragment to break the RecA-RecA protein-protein interaction and three fragments that inhibit peptide-deformylase via water-mediated multi-body interactions. Experiments confirm the predictions that 6-hydroxydopamine potently inhibits RecA and that PDF inhibition quantitatively tracks the water-mediated binding predictions. Additionally, the principles correctly predict the essential bound waters in HIV Protease, the surprisingly extensive binding site of elastase, the pinpoint location of electron transfer in dihydrofolate reductase, the HIV TAT-TAR protein-RNA interactions, and the MDM2-MDM4 differential binding to p53. The experimental confirmations of highly non-obvious predictions combined with the precise characterization of a broad range of known phenomena lend strong support to the generality of fragment-based methods for characterizing molecular recognition.

Design, synthesis and biological evaluation of renin inhibitors guided by simulated annealing of chemical potential simulations.

We have applied simulated annealing of chemical potential (SACP) to a diverse set of ∼150 very small molecules to provide insights into new interactions in the binding pocket of human renin, a historically difficult target for which to find low molecular weight (MW) inhibitors with good bioavailability. In one of its many uses in drug discovery, SACP provides an efficient, thermodynamically principled method of ranking chemotype replacements for scaffold hopping and manipulating physicochemical characteristics for drug development. We introduce the use of Constrained Fragment Analysis (CFA) to construct and analyze ligands composed of linking those fragments with predicted high affinity. This technique addresses the issue of effectively linking fragments together and provides a predictive mechanism to rank order prospective inhibitors for synthesis. The application of these techniques to the identification of novel inhibitors of human renin is described. Synthesis of a limited set of designed compounds provided potent, low MW analogs (IC50s<100nM) with good oral bioavailability (F>20-58%).

Synthesis and evaluation of orally active small molecule HIV-1 Nef antagonists.

The HIV-1 Nef accessory factor enhances viral replication and promotes immune system evasion of HIV-infected cells, making it an attractive target for drug discovery. Recently we described a novel class of diphenylpyrazolodiazene compounds that bind directly to Nef in vitro and inhibit Nef-dependent HIV-1 infectivity and replication in cell culture. However, these first-generation Nef antagonists have several structural liabilities, including an azo linkage that led to poor oral bioavailability. The azo group was therefore replaced with either a one- or two-carbon linker. The resulting set of non-azo analogs retained nanomolar binding affinity for Nef by surface plasmon resonance, while inhibiting HIV-1 replication with micromolar potency in cell-based assays without cytotoxicity. Computational docking studies show that these non-azo analogs occupy the same predicted binding site within the HIV-1 Nef dimer interface as the original azo compound. Computational methods also identified a hot spot for inhibitor binding within this site that is defined by conserved HIV-1 Nef residues Asp108, Leu112, and Pro122. Pharmacokinetic evaluation of the non-azo B9 analogs in mice showed that replacement of the azo linkage dramatically enhanced oral bioavailability without substantially affecting plasma half-life or clearance. The improved oral bioavailability of non-azo diphenylpyrazolo Nef antagonists provides a starting point for further drug lead optimization in support of future efficacy testing in animal models of HIV/AIDS.

A computational chemistry perspective on the current status and future direction of hepatitis B antiviral drug discovery.

Computational chemical biology, applied to research on hepatitis B virus (HBV), has two major branches: bioinformatics (statistical models) and first-principle methods (molecular physics). While bioinformatics focuses on statistical tools and biological databases, molecular physics uses mathematics and chemical theory to study the interactions of biomolecules. Three computational techniques most commonly used in HBV research are homology modeling, molecular docking, and molecular dynamics. Homology modeling is a computational simulation to predict protein structure and has been used to construct conformers of the viral polymerase (reverse transcriptase domain and RNase H domain) and the HBV X protein. Molecular docking is used to predict the most likely orientation of a ligand when it is bound to a protein, as well as determining an energy score of the docked conformation. Molecular dynamics is a simulation that analyzes biomolecule motions and determines conformation and stability patterns. All of these modeling techniques have aided in the understanding of resistance mutations on HBV non-nucleos(t)ide reverse-transcriptase inhibitor binding. Finally, bioinformatics can be used to study the DNA and RNA protein sequences of viruses to both analyze drug resistance and to genotype the viral genomes. Overall, with these techniques, and others, computational chemical biology is becoming more and more necessary in hepatitis B research. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."

Thermodynamics of engineered gold binding peptides: establishing the structure-activity relationships.

Adsorption behavior of a gold binding peptide was experimentally studied to achieve kinetics and thermodynamics parameters toward understanding of the binding of an engineered peptide onto a solid metal surface. The gold-binding peptide, GBP1, was originally selected using a cell surface display library and contains 14 amino acid residues. In this work, single- and three-repeats of GBP1 were used to assess the effects of two parameters: molecular architecture versus secondary structure on adsorption on to gold substrate. The adsorption measurements were carried out using surface plasmon resonance (SPR) spectroscopy at temperatures ranging from 10 to 55 °C. At all temperatures, two different regimes of peptide adsorption were observed, which, based on the model, correspond to two sets of thermodynamics values. The values of enthalpy, ΔH(ads), and entropy, ΔS(ads), in these two regimes were determined using the van't Hoff approach and Gibbs-Helmholtz relationship. In general, the values of enthalpy for both peptides are negative indicating GBP1 binding to gold is an exothermic phenomenon and that the binding of three repeat gold binding peptide (3l-GBP1) is almost 5 times tighter than that for the single repeat (l-GBP1). More intriguing result is that the entropy of adsorption for the 3l-GBP1 is negative (-43.4 ± 8.5 cal/(mol K)), while that for the l-GBP1 is positive (10.90 ± 1.3 cal/(mol K)). Among a number of factors that synergistically contribute to the decrease of entropy, long-range ordered self-assembly of the 3l-GBP1 on gold surface is the most effective, probably through both peptide-solid and peptide-peptide intermolecular interactions. Additional adsorption experiments were conducted in the presence of 2,2,2-trifluoroethanol (TFE) to determine how the conformational structures of the biomolecules responded to the environmental perturbation. We found that the peptides differ in their conformational responses to the change in solution conditions; while l-GBP does not fold in the presence of TFE, 3l-GBP1 adopted two types of secondary structure (ß-strand, α-helix) and that peptide's binding to the solid is enhanced by the presence of low percentages of TFE solvent. Not only do these kinetics and thermodynamics results provide adsorption behavior and binding of genetically engineered peptides for inorganics (GEPI), but they could also provide considerable insights into fundamental understanding peptide molecular recognition and their selective specificity for the solids. Moreover, comprehensive work described herein suggests that multiple repeat forms of the solid binding peptides possess a conformational component that can be exploited to further tailor affinity and binding of a given sequence to a solid material followed by ordered assembly as a convenient tool in future practical applications.

Synthesis and characterization of cyclic peptides that are ß-helical in trifluoroethanol.

We show that three designed cyclic d,l-peptides are ß-helical in TFE-a solvent in which the archetypal ß-helical peptide, gA, is unstructured. This result represents an advance in the field of ß-helical peptide foldamers and a step toward achieving ß-helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG-PS composite resin prefunctionalized with the alkanesulfonamide 'SCL' linker and preloaded with glycine. Our new conditions avoided C-terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic ß-helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

A fragment-based approach to the SAMPL3 Challenge.

The success of molecular fragment-based design depends critically on the ability to make predictions of binding poses and of affinity ranking for compounds assembled by linking fragments. The SAMPL3 Challenge provides a unique opportunity to evaluate the performance of a state-of-the-art fragment-based design methodology with respect to these requirements. In this article, we present results derived from linking fragments to predict affinity and pose in the SAMPL3 Challenge. The goal is to demonstrate how incorporating different aspects of modeling protein-ligand interactions impact the accuracy of the predictions, including protein dielectric models, charged versus neutral ligands, ΔΔGs solvation energies, and induced conformational stress. The core method is based on annealing of chemical potential in a Grand Canonical Monte Carlo (GC/MC) simulation. By imposing an initially very high chemical potential and then automatically running a sequence of simulations at successively decreasing chemical potentials, the GC/MC simulation efficiently discovers statistical distributions of bound fragment locations and orientations not found reliably without the annealing. This method accounts for configurational entropy, the role of bound water molecules, and results in a prediction of all the locations on the protein that have any affinity for the fragment. Disregarding any of these factors in affinity-rank prediction leads to significantly worse correlation with experimentally-determined free energies of binding. We relate three important conclusions from this challenge as applied to GC/MC: (1) modeling neutral ligands--regardless of the charged state in the active site--produced better affinity ranking than using charged ligands, although, in both cases, the poses were almost exactly overlaid; (2) simulating explicit water molecules in the GC/MC gave better affinity and pose predictions; and (3) applying a ΔΔGs solvation correction further improved the ranking of the neutral ligands. Using the GC/MC method under a variety of parameters in the blinded SAMPL3 Challenge provided important insights to the relevant parameters and boundaries in predicting binding affinities using simulated annealing of chemical potential calculations.

Diverse fragment clustering and water exclusion identify protein hot spots.

Simulated annealing of chemical potential located the highest affinity positions of eight organic probes and water on eight static structures of hen egg white lysozyme (HEWL) in various conformational states. In all HELW conformations, a diverse set of organic probes clustered in the known binding site (hot spot). Fragment clusters at other locations were excluded by tightly-bound waters so that only the hot-spot cluster remained in each case. The location of the hot spot was correctly predicted irrespective of the protein conformation and without accounting for protein flexibility during the simulations. Any one of the static structures could have been used to locate the hot spot. A site on a protein where a diversity of organic probes is calculated to cluster, but where water specifically does not bind, identifies a potential small-molecule binding site or protein-protein interaction hot spot.

Vibrational circular-dichroism spectroscopy of homologous cyclic peptides designed to fold into ß helices of opposite chirality.

Cyclic ß-helical peptides have been developed as model structured biomolecules for examining peptide adsorption and conformation on surfaces. As a key prerequisite to circular-dichroism (CD) analysis of these model peptides on surfaces, their conformations and the corresponding vibrational spectra in the 1400-1800 cm⁻¹ range were analyzed by vibrational circular-dichroism (VCD) spectroscopy in solution. The two model peptides ("ß Leu and ß Val") were examined in chloroform, where they each fold into a homogeneous well-defined antiparallel double-stranded ß-helical species, as determined previously by NMR and electronic CD spectroscopy. Because the ß-helical conformations of ß Leu and ß Val are well characterized, the VCD spectra of these peptides can be unambiguously correlated with their structures. In addition, these two ß-helical peptides differ from one another in two key respects that make them uniquely advantageous for CD analysis--first, while their backbone conformations are topologically similar, ß Leu and ß Val form helices of opposite chiralities; second, the two peptides differ in their sequences, i.e., composition of the side chains attached to the backbone. The observed VCD spectra for ß Leu and ß Val are roughly mirror images of each other, indicating that the VCD features are dominated by the chirality and conformation of the peptide backbone rather than by the peptide sequence. Accordingly, spectra similarly characteristic of peptide secondary structure can be expected for peptides designed to be structural analogs of ß Leu and ß Val while incorporating a variety of side chains for studies of surface adsorption from organic and aqueous solvents.

Simulations of nanocylinders self-assembled from cyclic ß-tripeptides.

This paper examines the self-assembly of cyclic ß-tripeptides using density functional theory. On the basis of literature precedents, these cyclic peptides were expected to self-assemble into cylindrical structures by stacking through backbone-backbone hydrogen bonding. Our calculations show that such stacking is energetically favorable, that the association energy per cyclic peptide decreases (becomes more favorable), and that the overall macrodipole moment of the cylindrical assembly increases with the number of stacked rings, for up to eight rings. For a structure in which two peptide ring units are joined through a single side chain-side chain covalent linker, the association energy between the two rings is favorable, albeit less so than for the unlinked rings. Significantly, the association energy in the dimers is only weakly dependent on the length (above a certain minimum) and conformation of the covalent linkers. Finally, as a plausible route for controlling assembly/disassembly of nanocylinders, we show that, for a pair of rings, each bearing a single amino-functionalized side chain, protonation of the amino group results in a strongly positive (unfavorable) association energy between the two rings.

Characterization of the adhesive plaque of the barnacle Balanus amphitrite: amyloid-like nanofibrils are a major component.

The nanoscale morphology and protein secondary structure of barnacle adhesive plaques were characterized using atomic force microscopy (AFM), far-UV circular dichroism (CD) spectroscopy, transmission Fourier transform infrared (FTIR) spectroscopy, and Thioflavin T (ThT) staining. Both primary cement (original cement laid down by the barnacle) and secondary cement (cement used for reattachment) from the barnacle Balanus amphitrite (= Amphibalanus amphitrite) were analyzed. Results showed that both cements consisted largely of nanofibrillar matrices having similar composition. Of particular significance, the combined results indicate that the nanofibrillar structures are consistent with amyloid, with globular protein components also identified in the cement. Potential properties, functions, and formation mechanisms of the amyloid-like nanofibrils within the adhesive interface are discussed. Our results highlight an emerging trend in structural biology showing that amyloid, historically associated with disease, also has functional roles.

Engineering a beta-helical D,L-peptide for folding in polar media.

Beta helices--helices formed by alternating D,L-peptides and stabilized by beta-sheet hydrogen bonding--are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of beta-helical structure by presenting the first design and biophysical characterization of a hydrophilic D,L-peptide, 1, that forms a beta helix in methanol. The design of 1 is based on the beta-hairpin/beta helix--a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected beta-hairpin/beta-helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR-based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol (1 m) transitions cooperatively to an extended conformation (1 w) resembling a cyclic beta-hairpin; observation of an isodichroic point in the solvent-dependent CD spectra indicates that this transition is a two-state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 degrees C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the (1)H NMR C alphaH spectroscopic resonances of 1 m are downfield-shifted with respect to random-coil values, a hitherto unreported property for beta helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed beta-helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water-soluble beta helices.