N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.
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ABSTRACT:
SUBMITTER: Baggio C
PROVIDER: S-EPMC7568863 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Herein we review contemporary synthetic and protein design strategies to stabilize the alpha-helical motif in short peptides and miniature proteins. Advances in organometallic catalyst design, specifically for the olefin metathesis reaction, enable the use of hydrocarbon bridges to either crosslink side chains of specific residues or mimic intramolecular hydrogen bonds with carbon-carbon bonds. The resulting hydrocarbon-stapled and hydrogen bond surrogate alpha-helices provide unique synthetic ligands for targeting biomolecules. In the protein design realm, several classes of miniature proteins that display stable helical domains have been engineered and manipulated with powerful in vitro selection technologies to yield libraries of sequences that retain their helical folds. Rational re-design of these scaffolds provide distinctive reagents for the modulation of protein-protein interactions.
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Project description:The triple-helical structure of collagen has been accurately reproduced in numerous chemical and recombinant model systems. Triple-helical peptides and proteins have found application for dissecting collagen-stabilizing forces, isolating receptor- and protein-binding sites in collagen, mechanistic examination of collagenolytic proteases, and development of novel biomaterials. Introduction of native-like sequences into triple-helical constructs can reduce the thermal stability of the triple-helix to below that of the physiological environment. In turn, incorporation of nonnative amino acids and/or templates can enhance triple-helix stability. We presently describe approaches by which triple-helical structure can be modulated for use under physiological or near-physiological conditions.
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Project description:Aromatic interactions such as ?-? interaction and cation-? interaction are present in membrane proteins and play important roles in both structure and function. To systematically investigate the effect of aromatic residues on the structural stability and ion permeability of peptide-formed ion channels, we designed several peptides with one or two tryptophan (Trp) residues incorporated at different positions in amphipathic ?-helical peptides. Circular dichroism (CD) studies revealed the preferable position of Trp residues for self-association in these designed peptides. Systematically designed di-substituted peptides with two Trps at each helix termini demonstrated intermolecular Trp-Trp interactions caused by aggregation. In the presence of liposomes, Trp on the hydrophilic face of the peptide enhanced interaction with the lipid membrane to increase the amphipathic ?-helical contents. Appropriate incorporation and positioning of Trp enabled peptides to form more stable channels and had notable effects with Trp di-substituted peptides. The ion channel forming capability of a series of these peptides showed that the cation-? interactions between Trp and Lys residues in adjacent transmembrane helices contribute to remarkable stabilization of the channel structure.
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Project description:Recent work has shown that the incorporation of an all-hydrocarbon "staple" into peptides can greatly increase their alpha-helix propensity, leading to an improvement in pharmaceutical properties such as proteolytic stability, receptor affinity, and cell permeability. Stapled peptides thus show promise as a new class of drugs capable of accessing intractable targets such as those that engage in intracellular protein-protein interactions. The extent of alpha-helix stabilization provided by stapling has proven to be substantially context dependent, requiring cumbersome screening to identify the optimal site for staple incorporation. In certain cases, a staple encompassing one turn of the helix (attached at residues i and i+4) furnishes greater helix stabilization than one encompassing two turns (i,i+7 staple), which runs counter to expectation based on polymer theory. These findings highlight the need for a more thorough understanding of the forces that underlie helix stabilization by hydrocarbon staples. Here we report all-atom Monte Carlo folding simulations comparing unmodified peptides derived from RNase A and BID BH3 with various i,i+4 and i,i+7 stapled versions thereof. The results of these simulations were found to be in quantitative agreement with experimentally determined helix propensities. We also discovered that staples can stabilize quasi-stable decoy conformations, and that the removal of these states plays a major role in determining the helix stability of stapled peptides. Finally, we critically investigate why our method works, exposing the underlying physical forces that stabilize stapled peptides.
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Project description:Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical ?-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.
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Project description:The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.
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Project description:Despite continuing advances in the development of novel cellular-, antibody-, and chemotherapeutic-based strategies to enhance immune reactivity, the presence of regulatory T cells (Tregs) remains a complicating factor to their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg deletional strategies, we investigated the ability of hydrocarbon stapled alpha-helical (SAH) peptides to target FOXP3, the master transcription factor regulator of Treg development, maintenance, and suppressive function. Using the crystal structure of the FOXP3 homodimer as a guide, we developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain (SAH-FOXP3) to block this key FOXP3 protein-protein interaction (PPI) through molecular mimicry. We show that lead SAH-FOXP3s bind FOXP3, are cell permeable, non-toxic to T cells, induce dose-dependent transcript and protein level alterations of FOXP3 target genes, impede Treg function, and lead to Treg gene expression changes in vivo consistent with Foxp3 dysfunction.
2022-09-29 | GSE201116 | GEO
Project description:BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with ?-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.
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Project description:We demonstrate covalent bond formation between an RNA aptamer containing a cysteamine-tethered nucleobase and helix-threading peptides (HTPs) containing α-bromoacetamide N-termini. The reaction is high yielding and inhibited by a DNA strand Watson-Crick complementary to the aptamer sequence indicating covalent reaction is dependent on the high affinity HTP-binding site present in the folded aptamer. These results are important for future structural studies of HTP-RNA complexes and methods for the discovery of new high affinity analogs via covalent tethering strategies.
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Project description:Side-chain-to-side-chain cyclization is frequently used to stabilize the α-helical conformation of short peptides. In a previous study, we incorporated a lactam bridge between the side chains of Lys-i and Asp-i+4 in the nonapeptide 1Y, cyclo-(2,6)-(Ac-VKRLQDLQY-NH2 ), an artificial ligand of the inhibitor of DNA binding and cell differentiation (ID) protein with antiproliferative activity on cancer cells. Herein, we show that only the cyclized five-residue segment adopts a helical turn whereas the C-terminal residues remain flexible. Moreover, we present nine 1Y analogs arising from different combinations of hydrophobic residues (leucine, isoleucine, norleucine, valine, and tyrosine) at positions 1, 4, 7, and 9. All cyclopeptides except one build a lactam-bridged helical turn; however, residue-4 reveals less helix character than the neighboring Arg-3 and Gln-5, especially with residue-4 being isoleucine, valine, and tyrosine. Surprisingly, only two cyclopeptides exhibit helix propagation until the C-terminus, whereas the others share a remarkable outward tilting of the backbone carbonyl of the lactam-bridged Asp-6 (>40° deviation from the orientation parallel to the helix axis), which prevents the formation of the H-bond between Arg-3 CO and residue-7 NH: As a result, the propagation of the helix beyond the lactam-bridged sequence becomes unfavorable. We conclude that, depending on the amino-acid sequence, the lactam bridge between Lys-i and Asp-i+4 can stabilize a helical turn but deviations from the ideal helix geometry are possible: Indeed, besides the outward tilting of the backbone carbonyls, the residues per turn increased from 3.6 (typical of a regular α-helix) to 4.2, suggesting a partial helix unwinding.
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