Huy N. Hoang

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency

Recombinant proteins are important therapeutics due to potent, highly specific, and nontoxic actions in vivo. However, they are expensive medicines to manufacture, chemically unstable, and difficult to administer with low patient uptake and compliance. Small molecule drugs are cheaper and more bioavailable, but less target-specific in vivo and often have associated side effects. Here we combine some advantages of proteins and small molecules by taking short amino acid sequences that confer potency and selectivity to proteins, and fixing them as small constrained molecules that are chemically and structurally stable and easy to make. Proteins often use short α-helices of just 1–4 helical turns (4–15 amino acids) to interact with biological targets, but peptides this short usually have negligible α-helicity in water. Here we show that short peptides, corresponding to helical epitopes from viral, bacterial, or human proteins, can be strategically fixed in highly α-helical structures in water. These helix-constrained compounds have similar biological potencies as proteins that bear the same helical sequences. Examples are (i) a picomolar inhibitor of Respiratory Syncytial Virus F protein mediated fusion with host cells, (ii) a nanomolar inhibitor of RNA binding to the transporter protein HIV-Rev, (iii) a submicromolar inhibitor of Streptococcus pneumoniae growth induced by quorum sensing pheromone Competence Stimulating Peptide, and (iv) a picomolar agonist of the GPCR pain receptor opioid receptor like receptor ORL-1. This approach can be generally applicable to downsizing helical regions of proteins with broad applications to biology and medicine.

Comparative α-Helicity of Cyclic Pentapeptides in Water†

Helix-constrained polypeptides have attracted great interest for modulating protein-protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1-X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water. In this very stringent test of helix induction, a Lys1→Asp5 lactam linker conferred greatest α-helicity, hydrocarbon and triazole linkers induced a mix of α- and 3₁₀-helicity, while thio- and dithioether linkers produced less helicity. The lactam-linked cyclic pentapeptide was also the most effective α-helix nucleator attached to a 13-residue model peptide.

Improving on Nature: Making a Cyclic Heptapeptide Orally Bioavailable†

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.

Metal clips that induce unstructured pentapeptides to be α-helical in water

Short peptides corresponding to protein helices do not form thermodynamically stable helical structures in water, a solvent that strongly competes for hydrogen-bonding amides of the peptide backbone. Metalloproteins often feature metal ions coordinated to amino acids within hydrogen-bonded helical regions of protein structure, so there is a prospect of metals stabilizing or inducing helical structures in short peptides. However, this has only previously been observed in nonaqueous solvents or under strongly helix-favoring conditions in water. Here cis-[Ru(NH(3))(4)(solvent)(2)](2+) and [Pd(en)(solvent)(2)](2+) are compared in water for their capacity as metal clips to induce alpha-helicity in completely unstructured peptides as short as five residues, Ac-HARAH-NH(2) and Ac-MARAM-NH(2). More alpha-helicity was observed for the latter pentapeptide and, when chelated to ruthenium, it showed the greatest alpha-helicity yet reported for a short metallopeptide in water (approximately 80%). Helicity was clearly induced rather than stabilized, and the two methionines were 10(13)-fold more effective than two histidines in stabilizing the lower oxidation state Ru(II) over Ru(III). The study identifies key factors that influence stability of an alpha-helical turn in water, suggests metal ions as tools for peptide folding, and raises an intriguing possibility of transiently coordinated metal ions playing important roles in native folding of polypeptides in water.

Total synthesis, structure, and oral absorption of a thiazole cyclic peptide, sanguinamide A.

The first total synthesis and three-dimensional solution structure are reported for sanguinamide A, a thiazole-containing cyclic peptide from the sea slug H. sanguineus. Solution phase fragment synthesis, solid phase fragment assembly, and solution macrocyclization were combined to give (1) in 10% yield. Spectral properties were identical for the natural product, requiring revision of its structure from (2) to (1). Intramolecular transannular hydrogen bonds help to bury polar atoms, which enables oral absorption from the gut.

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor

Human anaphylatoxin C3a, formed through cleavage of complement protein C3, is a potent effector of innate immunity via activation of its G protein coupled receptor, human C3aR. Previously reported short peptide ligands for this receptor either have low potency or lack receptor selectivity. Here we report the first small peptide agonists that are both potent and selective for human C3aR, derived from structure-activity relationships of peptides based on the C-terminus of C3a. Affinity for C3aR was examined by competitive binding with (125)I-labeled C3a to human PBMCs [corrected], agonist versus antagonist activity measured using fluorescence detection of intracellular calcium, and general selectivity monitored by C3a-induced receptor desensitization. An NMR structure for an agonist in DMSO showed a beta-turn motif that may be important for C3aR binding and activation. Derivatization produced a noncompetitive and insurmountable antagonist of C3aR. Small molecule C3a agonists and antagonists may be valuable probes of immunity and inflammatory diseases.

Novel Helix-Constrained Nociceptin Derivatives Are Potent Agonists and Antagonists of ERK Phosphorylation and Thermal Analgesia in Mice

The nociceptin opioid peptide receptor (NOP, NOR, ORL-1) is a GPCR that recognizes nociceptin, a 17-residue peptide hormone. Nociceptin regulates pain transmission, learning, memory, anxiety, locomotion, cardiovascular and respiratory stress, food intake, and immunity. Nociceptin was constrained using an optimized helix-inducing cyclization strategy to produce the most potent NOP agonist (EC50 = 40 pM) and antagonist (IC50 = 7.5 nM) known. Alpha helical structures were measured in water by CD and 2D (1)H NMR spectroscopy. Agonist and antagonist potencies, evaluated by ERK phosphorylation in mouse neuroblastoma cells natively expressing NOR, increased 20-fold and 5-fold, respectively, over nociceptin. Helix-constrained peptides with key amino acid substitutions had much higher in vitro activity, serum stability, and thermal analgesic activity in mice, without cytotoxicity. The most potent agonist increased hot plate contact time from seconds up to 60 min; the antagonist prevented this effect. Such helix-constrained peptides may be valuable physiological probes and therapeutics for treating some forms of pain.

Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases.