Peptide conjugates with polyaromatic hydrocarbons can benefit the activity of catalytic RNAs.

Early stages of life likely employed catalytic RNAs (ribozymes) in many functions that are today filled by proteins. However, the earliest life forms must have emerged from heterogenous chemical mixtures, which included amino acids, short peptides, and many other compounds. Here we explored whether the presence of short peptides can help the emergence of catalytic RNAs. To do this, we conducted an in vitro selection for catalytic RNAs from randomized sequence in the presence of ten different peptides with a prebiotically plausible length of eight amino acids. This in vitro selection generated dozens of ribozymes, one of them with ∼900-fold higher activity in the presence of one specific peptide. Unexpectedly, the beneficial peptide had retained its N-terminal Fmoc protection group, and this group was required to benefit ribozyme activity. The same, or higher benefit resulted from peptide conjugates with prebiotically plausible polyaromatic hydrocarbons (PAHs) such as fluorene and naphthalene. This shows that PAH-peptide conjugates can act as potent cofactors to enhance ribozyme activity. The results are discussed in the context of the origin of life.

One-pot chemical pyro- and tri-phosphorylation of peptides by using diamidophosphate in water.

Protein (pyro)phosphorylation is emerging as a post-translational modification (PTM) in signalling pathways involved in many cellular processes. However, access to synthetic pyrophosphopeptides that can serve as tools for understanding protein pyrophosphorylation is quite limited. Herein, we report a chemical phosphorylation method that enables the synthesis of pyrophosphopeptides in aqueous medium without the need for protecting groups. The strategy employs diamidophosphate (DAP) in a one-pot sequential phosphorylation-hydrolysis of mono-phosphorylated peptide precursors. This operationally simple method exploits the intrinsic nucleophilicity of a phosphate moiety installed on serine, threonine or tyrosine residues in complex peptides with excellent chemoselectivity and good yields under mild conditions. We demonstrate the installation of the pyrophosphate group within a wide range of model peptides and showcase the potential of this methodology by selectively pyrophosphorylating the highly functionalized Nopp140 peptide fragment. The potential to produce higher (poly)phosphorylated peptides was demonstrated as a proof-of-principle experiment where we synthesized the triphosphorylated peptides using this one-pot strategy.

Water-Based Dynamic Depsipeptide Chemistry: Building Block Recycling and Oligomer Distribution Control Using Hydration-Dehydration Cycles.

The high kinetic barrier to amide bond formation has historically placed narrow constraints on its utility in reversible chemistry applications. Slow kinetics has limited the use of amides for the generation of diverse combinatorial libraries and selection of target molecules. Current strategies for peptide-based dynamic chemistries require the use of nonpolar co-solvents or catalysts or the incorporation of functional groups that facilitate dynamic chemistry between peptides. In light of these limitations, we explored the use of depsipeptides: biorelevant copolymers of amino and hydroxy acids that would circumvent the challenges associated with dynamic peptide chemistry. Here, we describe a model system of N-(α-hydroxyacyl)-amino acid building blocks that reversibly polymerize to form depsipeptides when subjected to two-step evaporation-rehydration cycling under moderate conditions. The hydroxyl groups of these units allow for dynamic ester chemistry between short peptide segments through unmodified carboxyl termini. Selective recycling of building blocks is achieved by exploiting the differential hydrolytic lifetimes of depsipeptide amide and ester bonds, which we show are controllable by adjusting the solution pH, temperature, and time as well as the building blocks' side chains. We demonstrate that the polymerization and breakdown of the depsipeptides are facilitated by cyclic morpholinedione intermediates, and further show how structural properties dictate half-lives and product oligomer distributions using multifunctional building blocks. These results establish a cyclic mode of ester-based reversible depsipeptide formation that temporally separates the polymerization and depolymerization steps for the building blocks and may have implications for prebiotic polymer chemical evolution.

Thioesters provide a plausible prebiotic path to proto-peptides.

It is widely assumed that the condensation of building blocks into oligomers and polymers was important in the origins of life. High activation energies, unfavorable thermodynamics and side reactions are bottlenecks for abiotic peptide formation. All abiotic reactions reported thus far for peptide bond formation via thioester intermediates have relied on high energy molecules, which usually suffer from short half-life in aqueous conditions and therefore require constant replenishment. Here we report plausible prebiotic reactions of mercaptoacids with amino acids that result in the formation of thiodepsipeptides, which contain both peptide and thioester bonds. Thiodepsipeptide formation was achieved under a wide range of pH and temperature by simply drying and heating mercaptoacids with amino acids. Our results offer a robust one-pot prebiotically-plausible pathway for proto-peptide formation. These results support the hypothesis that thiodepsipeptides and thiol-terminated peptides formed readily on prebiotic Earth and were possible contributors to early chemical evolution.

Mutually stabilizing interactions between proto-peptides and RNA.

The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.

Directed remodeling of the mouse gut microbiome inhibits the development of atherosclerosis.

The gut microbiome is a malleable microbial community that can remodel in response to various factors, including diet, and contribute to the development of several chronic diseases, including atherosclerosis. We devised an in vitro screening protocol of the mouse gut microbiome to discover molecules that can selectively modify bacterial growth. This approach was used to identify cyclic D,L-α-peptides that remodeled the Western diet (WD) gut microbiome toward the low-fat-diet microbiome state. Daily oral administration of the peptides in WD-fed LDLr-/- mice reduced plasma total cholesterol levels and atherosclerotic plaques. Depletion of the microbiome with antibiotics abrogated these effects. Peptide treatment reprogrammed the microbiome transcriptome, suppressed the production of pro-inflammatory cytokines (including interleukin-6, tumor necrosis factor-α and interleukin-1ß), rebalanced levels of short-chain fatty acids and bile acids, improved gut barrier integrity and increased intestinal T regulatory cells. Directed chemical manipulation provides an additional tool for deciphering the chemical biology of the gut microbiome and might advance microbiome-targeted therapeutics.

Prebiotic Peptides: Molecular Hubs in the Origin of Life.

The fundamental roles that peptides and proteins play in today's biology makes it almost indisputable that peptides were key players in the origin of life. Insofar as it is appropriate to extrapolate back from extant biology to the prebiotic world, one must acknowledge the critical importance that interconnected molecular networks, likely with peptides as key components, would have played in life's origin. In this review, we summarize chemical processes involving peptides that could have contributed to early chemical evolution, with an emphasis on molecular interactions between peptides and other classes of organic molecules. We first summarize mechanisms by which amino acids and similar building blocks could have been produced and elaborated into proto-peptides. Next, non-covalent interactions of peptides with other peptides as well as with nucleic acids, lipids, carbohydrates, metal ions, and aromatic molecules are discussed in relation to the possible roles of such interactions in chemical evolution of structure and function. Finally, we describe research involving structural alternatives to peptides and covalent adducts between amino acids/peptides and other classes of molecules. We propose that ample future breakthroughs in origin-of-life chemistry will stem from investigations of interconnected chemical systems in which synergistic interactions between different classes of molecules emerge.

Selective incorporation of proteinaceous over nonproteinaceous cationic amino acids in model prebiotic oligomerization reactions.

Numerous long-standing questions in origins-of-life research center on the history of biopolymers. For example, how and why did nature select the polypeptide backbone and proteinaceous side chains? Depsipeptides, containing both ester and amide linkages, have been proposed as ancestors of polypeptides. In this paper, we investigate cationic depsipeptides that form under mild dry-down reactions. We compare the oligomerization of various cationic amino acids, including the cationic proteinaceous amino acids (lysine, Lys; arginine, Arg; and histidine, His), along with nonproteinaceous analogs of Lys harboring fewer methylene groups in their side chains. These analogs, which have been discussed as potential prebiotic alternatives to Lys, are ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid (Orn, Dab, and Dpr). We observe that the proteinaceous amino acids condense more extensively than these nonproteinaceous amino acids. Orn and Dab readily cyclize into lactams, while Dab and Dpr condense less efficiently. Furthermore, the proteinaceous amino acids exhibit more selective oligomerization through their α-amines relative to their side-chain groups. This selectivity results in predominantly linear depsipeptides in which the amino acids are α-amine-linked, analogous to today's proteins. These results suggest a chemical basis for the selection of Lys, Arg, and His over other cationic amino acids for incorporation into proto-proteins on the early Earth. Given that electrostatics are key elements of protein-RNA and protein-DNA interactions in extant life, we hypothesize that cationic side chains incorporated into proto-peptides, as reported in this study, served in a variety of functions with ancestral nucleic acid polymers in the early stages of life.

Design of a DNA-Programmed Plasminogen Activator.

Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.

Templated Self-Assembly of Dynamic Peptide Nucleic Acids.

Template-directed macromolecule synthesis is a hallmark of living systems. Inspired by this natural process, several fundamentally novel mechanisms for template-directed assembly of nucleic acid analogues have been developed. Although these approaches have broad significance, including potential applications in biotechnology and implications for the origins of life, there are unresolved challenges in how to characterize in detail the complex assembly equilibria associated with dynamic templated reactions. Here we describe mechanistic studies of template-directed dynamic assembly for thioester peptide nucleic acid (tPNA), an informational polymer that responds to selection pressures under enzyme-free conditions. To overcome some of the inherent challenges of mechanistic studies of dynamic oligomers, we designed, synthesized, and implemented tPNA-DNA conjugates. The DNA primer region affords a high level of control over the location and register of the tPNA backbone in relation to the template strand. We characterized the degree and kinetics of dynamic nucleobase mismatch correction at defined backbone positions. Furthermore, we report the fidelity of dynamic assembly in tPNA as a function of position along the peptide backbone. Finally, we present theoretical studies that explore the level of fidelity that can be expected for an oligomer having a given hybridization affinity in dynamic templated reactions and provide guidance for the future development of sequence self-editing polymers and materials. As our results demonstrate, the use of molecular conjugates of constitutionally static and dynamic polymers establishes a new methodology for expediting the characterization of the complex chemical equilibria that underlie the assembly of dynamic informational polymers.

Self-Assembling Cyclic d,l-α-Peptides as Modulators of Plasma HDL Function. A Supramolecular Approach toward Antiatherosclerotic Agents.

There is great interest in developing new modes of therapy for atherosclerosis to treat coronary heart disease and stroke, particularly ones that involve modulation of high-density lipoproteins (HDLs). Here, we describe a new supramolecular chemotype for altering HDL morphology and function. Guided by rational design and SAR-driven peptide sequence enumerations, we have synthesized and determined the HDL remodeling activities of over 80 cyclic d,l-α-peptides. We have identified a few distinct sequence motifs that are effective in vitro in remodeling human and mouse plasma HDLs to increase the concentration of lipid-poor pre-beta HDLs, which are key initial acceptors of cholesterol in the reverse cholesterol transport (RCT) process, and concomitantly promote cholesterol efflux from macrophage cells. Functional assays with various control peptides, such as scrambled sequences, linear and enantiomeric cyclic peptide variants, and backbone-modified structures that limit peptide self-assembly, provide strong support for the supramolecular mode of action. Importantly, when the lead cyclic peptide c[wLwReQeR] was administered to mice (ip), it also promoted the formation of small, lipid-poor HDLs in vivo, displayed good plasma half-life (∼6 h), did not appear to have adverse side effects, and exerted potent anti-inflammatory effects in an acute in vivo inflammation assay. Given that previously reported HDL remodeling peptides have been based on α-helical apoA-I mimetic architectures, the present study, involving a new structural class, represents a promising step toward new potential therapeutics to combat atherosclerosis.

Cyclic tetrapeptide HDAC inhibitors as potential therapeutics for spinal muscular atrophy: Screening with iPSC-derived neuronal cells.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is caused by inactivating mutations in the Survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein expression. Humans possess a paralog gene, SMN2, which contains a splicing defect in exon 7 leading to diminished expression of full-length, fully functional SMN protein. Increasing SMN2 expression has been a focus of therapeutic development for SMA. Multiple studies have reported the efficacy of histone deacetylase inhibitors (HDACi) in this regard. However, clinical trials involving HDACi have been unsatisfactory, possibly because previous efforts to identify HDACi to treat SMA have employed non-neuronal cells as the screening platform. To address this issue, we generated an SMA-patient specific, induced pluripotent stem cell (iPSC) derived neuronal cell line that contains homogenous Tuj1+neurons. We screened a small library of cyclic tetrapeptide HDACi using this SMA neuronal platform and discovered compounds that elevate SMN2 expression by an impressive twofold or higher. These candidates are also capable of forming gems intranuclearly in SMA neurons, demonstrating biological activity. Our study identifies new potential HDACi therapeutics for SMA screened using a disease-relevant SMA neuronal cellular model.

Natural and Synthetic Macrocyclic Inhibitors of the Histone Deacetylase Enzymes.

Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration in the US, and several are currently in clinical trials. However, none of these compounds possesses particularly good isozyme selectivity, which would be a highly desirable feature in a tool compound. Whether selective inhibition of individual HDAC isozymes will provide improved drug candidates remains to be seen. Nevertheless, it has been speculated that using macrocyclic compounds to target HDAC enzymes might hold an advantage over the use of traditional hydroxamic-acid-containing inhibitors, which rely on chelation to the conserved active-site zinc ion. Here we review the literature on macrocyclic HDAC inhibitors obtained from natural sources and on structure-activity relationship studies inspired by these molecules, as well as on efforts aimed at fully synthetic macrocyclic HDAC inhibitors.

Peptide Bond Formation in Water Mediated by Carbon Disulfide.

Demonstrating plausible nonenzymatic polymerization mechanisms for prebiotic monomers represents a fundamental goal in prebiotic chemistry. While a great deal is now known about the potentially prebiotic synthesis of amino acids, our understanding of abiogenic polymerization processes to form polypeptides is less well developed. Here, we show that carbon disulfide (CS2), a component of volcanic emission and sulfide mineral weathering, and a widely used synthetic reagent and solvent, promotes peptide bond formation in modest yields (up to ∼20%) from α-amino acids under mild aqueous conditions. Exposure of a variety of α-amino acids to CS2 initially yields aminoacyl dithiocarbamates, which in turn generate reactive 2-thiono-5-oxazolidone intermediates, the thio analogues of N-carboxyanhydrides. Along with peptides, thiourea and thiohydantoin species are produced. Amino acid stereochemistry was preserved in the formation of peptides. Our findings reveal that CS2 could contribute to peptide bond formation, and possibly other condensation reactions, in abiogenic settings.

In vivo efficacy of HDL-like nanolipid particles containing multivalent peptide mimetics of apolipoprotein A-I.

We have observed that molecular constructs based on multiple apoA-I mimetic peptides attached to a branched scaffold display promising anti-atherosclerosis functions in vitro. Building on these promising results, we now describe chronic in vivo studies to assess anti-atherosclerotic efficacy of HDL-like nanoparticles assembled from a trimeric construct, administered over 10 weeks either ip or orally to LDL receptor-null mice. When dosed ip, the trimer-based nanolipids markedly reduced plasma LDL-cholesterol levels by 40%, unlike many other apoA-I mimetic peptides, and were substantially atheroprotective. Surprisingly, these nanoparticles were also effective when administered orally at a dose of 75 mg/kg, despite the peptide construct being composed of l-amino acids and being undetectable in the plasma. The orally administered nanoparticles reduced whole aorta lesion areas by 55% and aortic sinus lesion volumes by 71%. Reductions in plasma cholesterol were due to the loss of non-HDL lipoproteins, while plasma HDL-cholesterol levels were increased. At a 10-fold lower oral dose, the nanoparticles were marginally effective in reducing atherosclerotic lesions. Intriguingly, analogous results were obtained with nanolipids of the corresponding monomeric peptide. These nanolipid formulations provide an avenue for developing orally efficacious therapeutic agents to manage atherosclerosis.

Molecules that mimic apolipoprotein A-I: potential agents for treating atherosclerosis.

Certain amphipathic α-helical peptides can functionally mimic many of the properties of full-length apolipoproteins, thereby offering an approach to modulate high-density lipoprotein (HDL) for combating atherosclerosis. In this Perspective, we summarize the key findings and advances over the past 25 years in the development of peptides that mimic apolipoproteins, especially apolipoprotein A-I (apoA-I). This assemblage of information provides a reasonably clear picture of the state of the art in the apolipoprotein mimetic field, an appreciation of the potential for such agents in pharmacotherapy, and a sense of the opportunities for optimizing the functional properties of HDL.

Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.

We describe an approach for engineering peptide-lipid nanoparticles that function similarly to high-density lipoprotein (HDL). Branched, multivalent constructs, bearing multiple 23- or 16-amino-acid peptides, were designed, synthesized, and combined with phospholipids to produce nanometer-scale discoidal HDL-like particles. A variety of biophysical techniques were employed to characterize the constructs, including size exclusion chromatography, analytical ultracentrifuge sedimentation, circular dichroism, transmission electron microscopy, and fluorescence spectroscopy. The nanoparticles functioned in vitro (human and mouse plasma) and in vivo (mice) to rapidly remodel large native HDLs into small lipid-poor HDL particles, which are key acceptors of cholesterol in reverse cholesterol transport. Fluorescent labeling studies showed that the constituents of the nanoparticles readily distributed into native HDLs, such that the peptide constructs coexisted with apolipoprotein A-I (apoA-I), the main structural protein in HDLs. Importantly, nanolipid particles containing multivalent peptides promoted efficient cellular cholesterol efflux and were functionally superior to those derived from monomeric apoA-I mimetic peptides. The multivalent peptide-lipid nanoparticles were also remarkably stable toward enzymatic digestion in vitro and displayed long half-lives and desirable pharmacokinetic profiles in mice, providing a real practical advantage over previously studied linear or tandem helical peptides. Encouragingly, a two-week exploratory efficacy study in a widely used animal model for atherosclerosis research (LDLr-null mice) using nanoparticles constructed from a trimeric peptide demonstrated an exceptional 50% reduction in the plasma total cholesterol levels compared to the control group. Altogether, the studies reported here point to an attractive avenue for designing synthetic, HDL-like nanoparticles, with potential for treating atherosclerosis.

Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3ß-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3ß-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

Macrocyclic peptoid-Peptide hybrids as inhibitors of class I histone deacetylases.

We report the design, synthesis, and biological evaluation of the first macrocyclic peptoid-containing histone deacetylase (HDAC) inhibitors. The compounds selectively inhibit human class I HDAC isoforms in vitro, with no inhibition of the tubulin deacetylase activity associated with class IIb HDAC6 in cultured Jurkat cells. Compared to the natural product apicidin (1), one inhibitor (compound 10) showed equivalent potency against K-562 cells, but was more cytoselective across a panel of cancer cell lines.