Structure-activity analysis of truncated albumin-binding domains suggests new lead constructs for potential therapeutic delivery.

Rapid clearance by renal filtration is a major impediment to the translation of small bioactive biologics into drugs. To extend serum t1/2, a commonly used approach is to attach drug leads to the G-related albumin-binding domain (ABD) to bind albumin and evade clearance. Despite the success of this approach in extending half-lives of a wide range of biologics, it is unclear whether the existing constructs are optimized for binding and size; any improvements along these lines could lead to improved drugs. Characterization of the biophysics of binding of an ABD to albumin in solution could shed light on this question. Here, we examine the binding of an ABD to human serum albumin using isothermal titration calorimetry and assess the structural integrity of the ABD using CD, NMR, and molecular dynamics. A structure-activity analysis of truncations of the ABD suggests that downsized variants could replace the full-length domain. Reducing size could have the benefit of reducing potential immunogenicity problems. We further showed that one of these variants could be used to design a bifunctional molecule with affinity for albumin and a serum protein involved in cholesterol metabolism, PCSK9, demonstrating the potential utility of these fragments in the design of cholesterol-lowering drugs. Future work could extend these in vitro binding studies to other ABD variants to develop therapeutics. Our study presents new understanding of the solution structural and binding properties of ABDs, which has implications for the design of next-generation long-lasting therapeutics.

Cyclotides as Tools in Chemical Biology.

Among the various molecules that plants produce for defense against pests and pathogens, cyclotides stand out as exceptionally stable and structurally unique. These ribosomally synthesized peptides are around 30 amino acids in size, and are stabilized by a head-to-tail cyclic peptide backbone and three disulfide bonds that form a cystine knot. They occur in certain plants of the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, with an individual plant producing up to hundreds of different cyclotides. Aside from being exploitable as crop protection agents based on their natural pesticidal activities, cyclotides are amenable to repurposing by chemists for use as drug leads or as tools in chemical biology. Their macrocyclic peptide backbone and knotted arrangement of three disulfide bonds engenders cyclotides with resistance to proteolytic degradation, high temperatures, and chemical chaotropes. Furthermore, their small size makes them accessible to synthesis using solid-phase peptide chemistry and so non-natural cyclotides can be designed and synthesized for a variety of applications. Our focus here is on cyclotides as tools in chemical biology, and there are four main areas of application that have appeared in the literature so far: (i) cyclotides as probes of membrane binding; (ii) cyclotides as probes of biosynthetic pathways for peptide cyclization; (iii) cyclotides as probes of protease specificity and function; and (iv) cyclotides as probes of receptor binding and specificity, with the potential for them to be developed as drug leads. The main methods used in these studies include solid-phase peptide chemistry for synthesis and NMR spectroscopy for structural characterization, as well as a wide range of biochemical and biophysical techniques for probing intermolecular interactions. In addition, cyclotides have been examined in diverse biological assays, ranging from enzyme inhibition to cell penetration, intracellular targeting and cytotoxicity. The main finding to have emerged from studies over the past decade is that cyclotides are exceptionally stable under a variety of conditions (in assay buffers, biological fluids, membranes, and recombinant expression systems). Furthermore, they are structurally very well-defined and amenable to sequence substitutions that can introduce new desired biological activities, generally without compromising their exceptional stability. Both features contribute to their use as peptide-based frameworks in drug design. Finally, they occupy a size niche between traditional small-molecule drugs (<500 Da in molecular weight) and protein-based biologics (>5000 Da) and thus can probe receptors, membranes, and protein-protein interactions in different ways to what is possible with either small molecules or biologics. Overall, cyclotides are an exciting class of peptides that have great potential as ultrastable chemical biology probes in a variety of applications. They have the advantage of specificity (typical of proteins) combined with the synthetic accessibility of small molecules.

Discovery, structure, function, and applications of cyclotides: circular proteins from plants.

Cyclotides are plant-derived cyclic peptides that have a head-to-tail cyclic backbone and three conserved disulphide bonds that form a cyclic cystine knot motif. They occur in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, typically with 10-100 cyclotides in a given plant species, in a wide range of tissues, including flowers, leaves, stems, and roots. Some cyclotides are expressed in large amounts (up to 1g kg(-1) wet plant weight) and their natural function appears to be to protect plants from pests or pathogens. This article provides a brief overview of their discovery, distribution in plants, and applications. In particular, their exceptional stability has led to their use as peptide-based scaffolds in drug design applications. They also have potential as natural 'ecofriendly' insecticides, and as protein engineering frameworks.

Melanocortin 1 Receptor Agonists Based on a Bivalent, Bicyclic Peptide Framework.

We have designed a new class of highly potent bivalent melanocortin receptor ligands based on the nature-derived bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1). Incorporation of melanotropin pharmacophores in each of the two turn regions of SFTI-1 resulted in substantial gains in agonist activity particularly at human melanocortin receptors 1 and 3 (hMC1R/hMC3R) compared to monovalent analogues. In in vitro binding and functional assays, the most potent molecule, compound 6, displayed low picomolar agonist activity at hMC1R (pEC50 > 10.3; EC50 < 50 pM; pKi: 10.16 ± 0.04; Ki: 69 ± 5 pM) and is at least 30-fold more selective for this receptor than for hMC3R, hMC4R, or hMC5R. The results are discussed in the context of structural homology models of hMCRs in complex with the developed bivalent ligands.

Copying Life: Synthesis of an Enzymatically Active Mirror-Image DNA-Ligase Made of D-Amino Acids.

Our objective is the creation of a mirror-image synthetic biology: that is, to mimic, entirely independent of Nature, a biological system and to re-create it from artificial component parts. Utilizing enantiomeric L-nucleotides and D-amino acids rather than the natural components, we use chemical synthesis toward a basic, self-replicating mirror-image biological system. Here, we report the synthesis of a functional DNA-ligase in the D-enantiomeric conformation, which is an exact mirror-image of the natural enzyme, exhibiting DNA ligation activity on chirally inverted nucleic acids in L-conformation, but not acting on natural substrates and with natural co-factors. Starting from the known structure of the Paramecium bursaria chlorella virus 1 DNA-ligase and the homologous but shorter DNA-ligase of Haemophilus influenza, we designed and synthesized chemically peptides, which could then be assembled into a full-length molecule yielding a functional protein. The structure and the activity of the mirror-image ligase were characterized, documenting its enantiospecific functionality.

Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix-Loop-Helix Peptides with Cell Penetrating and Tumor Suppressor Properties.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.

Calcium-Mediated Allostery of the EGF Fold.

The epidermal growth factor (EGF)-like domain is one of the most abundant disulfide-containing domains in nature and is involved in many cellular processes critical to life. Although many EGF-like domains participate in calcium-dependent functions by responding to the local calcium concentration, little is known about how this responsiveness is programmed at the molecular level. Here, we reveal the structural and environmental determinants underpinning the folding of a synthetic analogue of the EGF-A domain (from the low-density lipoprotein receptor). We show that calcium sensitivity is enabled by an allosteric folding pathway, in which calcium binding is connected to the peptide core through local inter-residue interactions. In the absence of calcium, the fold favors disorder because the inherently weak core is insufficient to stabilize the active form, resulting in substantial loss in activity of 2 orders of magnitude. The EGF-A fold, which can freely transition between active and disordered states, is volatile, and we found it to be intolerant of mutations, unlike other disulfide-rich peptides that have been used as stabilizing frameworks. This volatility is beneficial for modularity/plasticity and appears to have evolved for such a purpose, allowing cellular pathways to sense and respond to environmental cues.

Development of Novel Melanocortin Receptor Agonists Based on the Cyclic Peptide Framework of Sunflower Trypsin Inhibitor-1.

Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3-5R. Double N-methylated compound 18 showed a p Ki of 8.73 ± 0.08 ( Ki = 1.92 ± 0.34 nM) and a pEC50 of 9.13 ± 0.04 (EC50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.

Boc-SPPS: Compatible Linker for the Synthesis of Peptide o-Aminoanilides.

A protection strategy is described for the efficient synthesis of peptide o-aminoanilides using in situ neutralization protocols for Boc-SPPS. On-resin protection of Boc-protected aminoacyl o-aminoanilides is achieved with 2-chlorobenzyl chloroformate. Activation through a peptidyl-benzotriazole intermediate allows for facile conversion to peptide-thioesters for use in native chemical ligation. In addition to providing a robust alternative to established thioester resins, as a latent thioester, the peptide o-aminoanilide has broad utility in convergent ligation strategies.

An L-RNA Aptamer that Binds and Inhibits RNase.

L-RNA aptamers were developed that bind to barnase RNase and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ∼100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates. L-RNA aptamers are resistant to degradation by ribonucleases, thus enabling them to function in biological samples, most notably for applications in molecular diagnostics and therapeutics. In addition to the irony of using RNA to inhibit RNase, L-RNA aptamers such as those described here could be used to measure the concentration or inhibit the function of RNase in the laboratory or in biological systems.

Murine polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor.

Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4(+) and CD8(+) cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4(+) and CD8(+) cells with a low induction of anti-VLP antibodies.