ROSETTA-informed design of structurally stabilized cyclic anti-amyloid peptides

Abstract

β-amyloid oligomers are thought to be the most toxic species formed en route to fibril deposition in Alzheimer’s disease. Transthyretin is a natural sequestering agent of β-amyloid oligomers: the binding site to β-amyloid has been traced to strands G/H of the inner β-sheet of transthyretin. A linear peptide, with the same primary sequence as the β-amyloid binding domain on transthyretin, was moderately effective at inhibiting β-amyloid fibril growth. Insertion of a β-turn template and cyclization greatly increased stability against proteolysis and improved efficacy as an amyloid inhibitor. However, the cyclic peptide still contained a significant amount of disorder. Using the Simple Cyclic Peptide Application within ROSETTA as an in silico predictor of cyclic peptide conformation and stability, we investigated putative structural enhancements, including stabilization by disulfide linkages and insertion of a second β-turn template. Several candidates were synthesized and tested for secondary structure and ability to inhibit β-amyloid aggregation. The results demonstrate that cyclization, β-sheet structure and conformational homogeneity are all preferable design features, whereas disulfide bond formation across the two β-strands is not preferable.

Unlocking novel therapies: cyclic peptide design for amyloidogenic targets through synergies of experiments, simulations, and machine learning

“…154 Cyclization of CG8 via a disulfide bond using the simple cyclic peptide application (SCPA) 155 within ROSETTA and the addition of a second d -proline (TKVVTpPRYTIAKLSSpPSYSQ) lead to increased peptide stability, enhanced conformational uniformity, and a higher β-sheet content. 156 These findings highlight the added value of of cyclization and conformational homogeneity as design strategies.…”

The emerging role of computational design in peptide macrocycle drug discovery

“…Est et al were recently able to show improvements to the amyloid inhibitory activity of a transthyretin-derived cyclic peptide using mutations and disulfide placements screened virtually with the simple_cycpep_predict application. They found experimentally that conformational rigidity in a slightly twisted active β-sheet conformation was a key determinant of antiamyloid activity, and were able to predict experimental success of peptide designs using the application [22]. Figure 3A shows the typical drug discovery process.…”

“…Peptide stapling [16,17], cross-linking [18,19], and macrocyclization [20,21] approaches can limit the conformational flexibility of larger molecules, allowing presentation of more chemical groups for favorable interactions with a target without creating an unduly flexible molecule with a prohibitive entropic penalty to binding. However, covalent linkages alone do not guarantee rigidity, let alone rigidity in an active conformation [21,22]. Recently, computational tools have been developed both for rationally designing peptide macrocycles and for reliably stabilizing a peptide in a desired conformation [23][24][25].…”

Automated Design of Macrocycles for Therapeutic Applications: From Small Molecules to Peptides and Proteins

“…Implementation of reliable in silico approaches for an accurate design are therefore needed to guide experimental efforts towards the most promising compounds. 82,[93][94][95][96] To perform rational design, a reliable description of macrocycles' conformational landscape is essential.…”

“…Considering each residue in a peptide as potential cyclization site and taking into account the diversity of chemical strategies for cyclization might lead to a vast number of molecules that could be synthesized. Implementation of reliable in silico approaches for an accurate design is therefore needed to guide experimental efforts toward the most promising compounds. ,− To perform rational design, a reliable description of macrocycles’ conformational landscape is essential. Unfortunately, the rugged conformational landscape limits the effectiveness of standard approaches for small molecules to reliably predict macrocycle conformations. ,, Consequently, significant work has been made into enhanced sampling algorithms toward macrocycle conformation prediction. − Additional work has been put into docking or otherwise calculating macrocycle binding free energies. − More recently, a structure-based design of peptide macrocycles targeting the interaction site of human adaptor protein 14-3-3 using free-energy perturbation (FEP) calculations was proposed .…”