Photo Cross-Linking Probes Containing ϵ-N-Thioacyllysine and ϵ-N-Acyl-(δ-aza)lysine Residues.

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ϵ-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the ϵ-N-glutaryllysine and ϵ-N-myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

Functionalized Helical ß-Peptoids.

Peptidomimetic foldamers adopting well-defined three-dimensional structures while being stable toward proteolysis are of interest in biomedical research, chemical biology, and biomimetic materials science. Despite their backbone flexibility, ß-peptoids containing N-( S)-1-(1-naphthyl)ethyl ( Ns1npe) side chains can fold into unique triangular prism-shaped helices. We report herein the successful introduction of amino groups onto robustly folded ß-peptoid helices by construction and incorporation of novel chiral building blocks. This is the first example of an X-ray crystal structure of a linear ß-peptoid containing more than one type of side chain. We thus present a unique foldamer design comprising a robustly folded core with functionalized side chains protruding perpendicular to the helical axis to provide a highly predictable display of functional groups. This work paves the way for development of ß-peptoid foldamers with a desired function, such as catalytic properties or as scaffolds enabling polyvalent display.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

A Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH.

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+)as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuinsin vitro To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C1) to palmitoyl (C16) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, "long chain" acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1-3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD(+)-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuinsin vivo These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD(+)/NADH ratio.

ß-Peptoid Foldamers at Last.

For a long time, peptides were considered unsuitable for drug development due to their inherently poor pharmacokinetic properties and proteolytic susceptibility. However, this paradigm has changed significantly in the past decade with the approval of numerous antibodies and proteins as drugs. In parallel, research in the field of synthetic molecules that are able to mimic or complement folding patterns exhibited by biopolymers, but are not recognized by proteases, have received considerable attention as well. Such entities were coined "foldamers" by Professor Gellman in an Account published in this journal in the late 1990s. Oligomers of N-alkylated 3-aminopropionic acid residues have been called ß-peptoids due to their structural similarity to ß-peptides and peptoids (N-alkylglycines), respectively. Because bona fide foldamer behavior has been demonstrated for both parent architectures, we wondered if the ß-peptoids could serve as a successful addition to the known ensemble of peptidomimetic foldamers. When we entered this field, only the seminal description of libraries of ß-peptoid dimers and trimers by Hamper et al. had been published a number of years earlier [ J. Org. Chem. 1998 , 63 , 708 ]. Perhaps somewhat naïvely in retrospect, we envisioned that elongation of chain length combined with introduction of bulky α-chiral side chains would deliver folded structures as reported for the α-peptoid counterparts. Initially, we, and others, were unsucessful in obtaining stable secondary structures of ß-peptoid oligomers, and instead, these residues were either incorporated in cyclic structures or in combination with other types of residues to give peptidomimetic constructs with heterogeneous backbones. Amphiphilic architectures with various membrane-targeting activities, such as mimics of antimicrobial peptides or cell-penetrating peptides, have thus been particularly successful. Introduction of ß-peptoid residues in histone deacetylase inhibitors mimicking nonribosomal cyclotetrapeptides have also been reported. In the present Account, we will sketch the scientific journey that ultimately delivered robustly folded ß-peptoid oligomers. Contributions involving biological evaluation of peptidomimetic constructs containing ß-peptoid residues, as mentioned above, which were investigated leading up to these recently reported high-resolution helical structures, will thus be discussed. On the basis of the work described in this Account, we envision that ß-peptoids will find future utility as peptidomimetics for biomedical investigation containing both heterogeneous and homogeneous backbones. The recent demonstration of control over the secondary structure of a homogeneous ß-peptoid backbone now enables structure-based design of scaffolds with predictable display of desired functionalities in three dimensions.

Triangular prism-shaped ß-peptoid helices as unique biomimetic scaffolds.

ß-Peptoids are peptidomimetics based on N-alkylated ß-aminopropionic acid residues (or N-alkyl-ß-alanines). This type of peptide mimic has previously been incorporated in biologically active ligands and has been hypothesized to be able to exhibit foldamer properties. Here we show, for the first time, that ß-peptoids can be tuned to fold into stable helical structures. We provide high-resolution X-ray crystal structures of homomeric ß-peptoid hexamers, which reveal right-handed helical conformations with exactly three residues per turn and a helical pitch of 9.6-9.8 Å between turns. The presence of folded conformations in solution is supported by circular dichroism spectroscopy showing length- and solvent dependency, and molecular dynamics simulations provide further support for a stabilized helical secondary structure in organic solvent. We thus outline a framework for future design of novel biomimetics that display functional groups with high accuracy in three dimensions, which has potential for development of new functional materials.

Effects of thionation and fluorination on cis-trans isomerization in tertiary amides: an investigation of N-alkylglycine (peptoid) rotamers.

Peptoids constitute a class of peptidomimetics with potential as protease resistant, biologically active ligands. To harness the full potential of such compounds, however, detailed predictive insight into their propensity to adopt well-defined secondary structures is highly desirable. In this work we present an investigation of the effects of thioamides and/or fluorides in peptoid monomer model systems using chemical synthesis, NMR spectroscopy, and X-ray crystallography. We find that the steric environment surrounding the tertiary amide bonds is the key promoter of conformational preference, and X-ray crystallographic interrogation of our model systems did not suggest the presence of stabilizing n → π* interactions unless the carbonyls were altered electronically by α-halogenation or thioamide formation. In addition to the function as an investigative tool, these two types of modification may thus be utilized as stabilizers of secondary structure in future oligomer designs, such as the cis-amide-based polypeptoid helices that resemble the polyproline type-I helix.

Guanidino groups greatly enhance the action of antimicrobial peptidomimetics against bacterial cytoplasmic membranes.

Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide-ß-peptoid chimeras. Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.

Cis-trans amide bond rotamers in ß-peptoids and peptoids: evaluation of stereoelectronic effects in backbone and side chains.

Non-natural peptide analogs have significant potential for the development of new materials and pharmacologically active ligands. One such architecture, the ß-peptoids (N-alkyl-ß-alanines), has found use in a variety of biologically active compounds but has been sparsely studied with respect to folding propensity. Thus, we here report an investigation of the effect of structural variations on the cis-trans amide bond rotamer equilibria in a selection of monomer model systems. In addition to various side chain effects, which correlated well with previous studies of α-peptoids, we present the synthesis and investigation of cis-trans isomerism in the first examples of peptoids and ß-peptoids containing thioamide bonds as well as trifluoroacetylated peptoids and ß-peptoids. These systems revealed an increase in the preference for cis-amides as compared to their parent compounds and thus provide novel strategies for affecting the folding of peptoid constructs. By using NMR spectroscopy, X-ray crystallographic analysis, and density functional theory calculations, we present evidence for the presence of thioamide-aromatic interactions through C(sp(2))-H···S(amide) hydrogen bonding, which stabilize certain peptoid conformations.