Gloria Ruiz-Gómez

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.

Update 1 of: Over One Hundred Peptide-Activated G Protein-Coupled Receptors Recognize Ligands with Turn Structure

1. GPCRs: Importance and Occurrence G protein-coupled receptors (GPCRs) are seven transmembrane helical bundle proteins (Figure 1) found on the surface of all cells.1–3 They mediate cellular responses to a diverse range of extracellular stimuli, including both endogenous chemical signals and exogenous environmental agents (e.g., light, amino acids, peptides, proteins, small organic molecules such as amines and lipids, nucleosides, nucleotides, metal ions, and pharmaceuticals). Once activated by an extracellular signal, GPCRs activate heterotrimeric G proteins that interact promiscuously with multiple receptors....

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.

Complement component C2, inhibiting a latent serine protease in the classical pathway of complement activation.

The innate immune response to infection or injury involves an antigen-antibody triggered classical pathway (CP) of complement activation, in which soluble and cell surface plasma proteins cooperatively effect elimination of foreign organisms and damaged host cells. However, protracted or dysfunctional complement activation can lead to inflammatory diseases. Complement component 2 bound to C4b is cleaved by classical (C1s) or lectin (MASP2) proteases to produce C4bC2a, a very short-lived C3 convertase (t(1/2) 2 min) that in turn cleaves C3 to C3a and C3b, leading ultimately to formation of Membrane Attack Complex (MAC) and lysis of bacteria and damaged cells. C2 has the same serine protease domain as C4bC2a but in an inactive zymogen-like conformation, requiring cofactor-induced conformational change for activity. Here, we show that C2 has catalytic protease activity in its own right above pH 7, in the absence of cofactor, processing C3 and C3-derived chromogenic peptide fragments. In contrast to the instability of C3 convertase (t(1/2) 2 min, pH 7), the C2 enzyme is indefinitely stable under alkaline conditions, facilitating studies of its catalytic properties and development of small molecule inhibitors. We characterize the catalytic activity of C2 against C3 and short paranitroanilide peptide substrates, and identify potent small molecule inhibitors of C2 that also inhibit classical pathway C3 convertase, MAC formation, and hemolysis of sensitized sheep erythrocytes. These results provide a new avenue and valuable new insights to inhibiting CP complement activation relevant to inflammatory diseases.

Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency

Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103-127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8-14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.

Helix-constrained nociceptin peptides are potent agonists and antagonists of ORL-1 and nociception

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.