Designing Short Peptides with High Affinity for Organic Molecules: A Combined Docking, Molecular Dynamics, And Monte Carlo Approach

We present a method for designing artificial receptors capable of binding with high affinity to a chosen target organic molecule. The primary sequence of the peptide is optimized to maximize its binding affinity. Our algorithm builds on a combination of molecular dynamics, semiflexible docking, and replica exchange Monte Carlo and performs simultaneous sampling in sequence and conformational spaces carefully selecting the degree of flexibility in the mutated peptides. The approach is used to design a decapeptide able to bind efavirenz. The calculated binding energy of the designed peptide (approximately −12 kcal/mol) was confirmed experimentally by fluorescence measurements. NMR spectroscopy confirmed the interactions between the peptide and the efavirenz molecule predicted by the algorithm

Structure of the human serum albumin HSA-100 fragment.

<p>(<b>a</b>) The full-length protein and HSA-100 fragment are shown as white and red ribbons, respectively. The structure was obtained from the Protein Data Bank (1BKE). (<b>b</b>) Designed construct bearing the polypeptide GST-HSA100. (<b>c</b>) SDS-PAGE and anti-FLAG tag Western blot of purified GST-HSA100 protein. (<b>d</b>) Tryptic map of GST-HSA100. Tryptic peptides obtained by GST-HSA100 digestion were analyzed by LC-MS/MS. Only peptides covering the GST-HSA100 sequence are shown. Their identities were assigned on the basis of molecular mass (red bars) or peptide sequence (blue bars).</p

Aldolase activity of GST-HSA100.

<p>Michaelis–Menten plot of the initial velocities for the aldol addition of acetone to aldehyde <b>5</b> catalyzed by HSA (•) and GST-HSA100 (○).</p

Control of reactivity with GST-HSA100.

<p>(<b>a</b>) Diastereoselective reduction of 1,3-diketones. (<b>b</b>) Albumin-catalyzed aldol addition.</p

Binding parameters for warfarin and efavirenz.

[a]<p>Stern-Volmer quenching association constant (10⁻³Lmol⁻¹).</p>[b]<p>Bimolecular quenching kinetic constant (10⁻¹²Lmol⁻¹s⁻¹) assuming τ₀ = 5 ns for the tryptophan fluorescence decay.</p>[c]<p>Dissociation constant (10⁻⁴molL⁻¹) for the protein-ligand complexes from Hill analysis of the quenching data.</p>[d]<p>Number of binding sites per molecule of protein from Hill analysis of the quenching data.</p>[e]<p>SPR steady state association equilibrium constant (10⁻³ Lmol⁻¹).</p

Control of reactivity.

[a]<p>observed pseudo first order value (10⁶ min⁻¹).</p>[b]<p>apparent value (10⁶ min⁻¹) in 10% aqueous acetone.</p>[c]<p>apparent value (10³molL⁻¹). [d] Lmol⁻¹.</p

Circular dichroism of GST-HSA100.

<p>(<b>a</b>) Far-UV CD spectra. Solid line, 25°C; dashed line, 60°C; dotted line, 75°C. The spectra were recorded at 0.2 µM protein in 10 mM phosphate buffer, pH 7.4. (<b>b</b>) Near-UV CD spectrum of GST-HSA100.</p

GST-HSA100 small ligand binding.

<p>(<b>a</b>) Reference albumin IIa site ligands warfarin (1), efavirenz (2a) and efavirenz with amine linker (2b). (<b>b</b>) Synchronous (Δ = 60 nm) fluorescence spectra of GST-HSA100 1 µM in 10 mM phosphate buffer, pH 7.4. 1, no quencher added; 2, 10 µM efavirenz; 3, 100 µM efavirenz; 4, 200 µM efavirenz; and 1–4 (dotted line), difference between spectrum 1 and 4. (<b>c</b>) Titration of fluorescence emission of HSA, GST-HSA100 and GST by warfarin and efavirenz. •, warfarin-HSA; ▴, warfarin-HSA100; ▪, warfarin-GST; ○, efavirenz-HSA; Δ, efavirenz-HSA100; □, efavirenz-GST.</p

Stable and Long-Lasting, Novel Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release <i>in vitro</i>, and <i>in vivo</i> efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema

Stable and Long-Lasting, Novel Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release <i>in vitro</i>, and <i>in vivo</i> efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema