Designing helical peptide inhibitors of protein–protein interactions

Short helical peptides combine characteristics of small molecules and large proteins and provide an exciting area of opportunity in protein design. A growing number of studies report novel helical peptide inhibitors of protein-protein interactions. New techniques have been developed for peptide design and for chemically stabilizing peptides in a helical conformation, which frequently improves protease resistance and cell permeability. We summarize advances in peptide crosslinking chemistry and give examples of peptide design studies targeting coiled-coil transcription factors, Bcl-2 family proteins, MDM2/MDMX, and HIV gp41, among other targets.National Institute of General Medical Sciences (U.S.) (Award GM067681)National Institute of General Medical Sciences (U.S.) (Award GM110048)National Institute of General Medical Sciences (U.S.) (Award GM084181

Rapid Optimization of Mcl-1 Inhibitors using Stapled Peptide Libraries Including Non-Natural Side Chains

Alpha helices form a critical part of the binding interface for many protein-protein interactions, and chemically stabilized synthetic helical peptides can be effective inhibitors of such helix-mediated complexes. In particular, hydrocarbon stapling of peptides to generate constrained helices can improve binding affinity and other peptide properties, but determining the best stapled peptide variant often requires laborious trial and error. Here, we describe the rapid discovery and optimization of a stapled-helix peptide that binds to Mcl-1, an antiapoptotic protein that is overexpressed in many chemoresistant cancers. To accelerate discovery, we developed a peptide library synthesis and screening scheme capable of identifying subtle affinity differences among Mcl-1-binding stapled peptides. We used our method to sample combinations of non-natural amino-acid substitutions that we introduced into Mcl-1 inhibitors in the context of a fixed helix-stabilizing hydrocarbon staple that increased peptide helical content and reduced proteolysis. Peptides discovered in our screen contained surprising substitutions at sites that are conserved in natural binding partners. Library-identified peptide M3d is the most potent molecule yet tested for selectively triggering mitochondrial permeabilization in Mcl-1 dependent cell lines. Our library approach for optimizing helical peptide inhibitors can be readily applied to the study of other biomedically important targets.National Institute of General Medical Sciences (U.S.) (Award R01GM110048

The protofilament architecture of a de novo designed coiled coil-based amyloidogenic peptide

International audienceAmyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson's and Alzheimer's diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements

α-Helikale "Coiled Coil"-Nachahmung durch alternierende β- und γ-Aminosäuresequenzen

The goal of this work was to develop helical conformations by using synthetic foldamers that could be functionally modulated to selectively disrupt unfavourable helix-helix interactions. Adhering to the principle of “equal backbone atoms”, the alternating βγ sequence appears to be well-suited to mimic an α-helical conformation. As a case study, the backbone modification is applied to a natural helical protein folding motif, the α-helical coiled coil. First an extended sequence of and amino acids was substituted in a coiled coil forming sequence. The helical structure is induced through oligomerization while the individual βγ segment is mostly unstructured. The natural side chains were preserved to more accurately imitating natural packing of αβγ chimera. The self- and hetero-assembly of a series of αβγ-chimeric sequences is investigated in model peptide systems as well as the biologically-derived GCN4pLI sequence by means of a variety of theoretical and experimental methods and assays, such as MD simulations, CD, SEC, AU, TEM, as well as FRET, disulfide exchange and template-directed native chemical ligation assays. The next task was to optimize the interactions between the artificial sequence and the native partner by means of two medium-throughput methods. Spot synthesis/analysis and phage display techniques revealed the key side chain properties that are required for coiled coil assembly. The phage display technique selected α-sequences with primary structures that would not have been considered in a rational design approach, but were observed to be better binding partners for the αβγ-chimera. Further, the function of the artificial folding motif was studied in the context of its catalytic activity in the native chemical ligation. In order to bring the essential functional groups together and present a well-formed catalytic site, αβγ-chimera had to be and were shown to mimic the natural α-helical coiled coil structure. The structural consequences of the ααα→βγ isosteric backbone substitution, such as disruption in local packing or conformational degrees of freedom due to further loss of H-bond are other interesting aspects which were studied in detail. As determined by disulfide exchange assays, the pairing of αβγ- chimeric sequences with the native GCN4pLI sequence is thermodynamically allowed only in the case of an ideal arrangement of β- and γ-residues. This indicates a similarity in the local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Altogether, the observations are consistent with the theoretical studies and show that the stability of a αβγ-peptide bundle can be tuned by controlling the extent of the side chain interactions at the interhelical recognition domains.Die vorliegende Arbeit beschäftigt sich mit synthetischen β,γ-Foldameren, die dazu designed worden, die α-helikale Konformation zu imitieren. Im Rahmen der Wirkstoffentwicklung könnte es mit Hilfe derartiger Strukturen ermöglicht werden, Einfluss auf unerwünschte z.B. im Rahmen bestimmter Krankheiten auftretenden Protein-Protein-Interaktionen zu nehmen. Basierend auf dem Prinzip, die Anzahl der Atome im Peptidrückgrat konstant zu halten, sollten alternierende Sequenzen aus β- und γ- Aminosäuren eine der α-helikalen Konformation nahe kommende Struktur einnehmen. Als Modell, um diese Hypothese zu überprüfen, wird im Rahmen dieser Studie das α-helikale Coiled-Coil- Faltungsmotiv verwendet. Hierzu wurde ein Segment einer Coiled-Coil bildenden α-Sequenz durch β- und γ-Aminosäuren ersetzt, um somit ein α,β,γ-Chimär zu generieren. Das Design der α,β,γ-Sequenz wurde dabei so gewählt, dass die natürlichen Seitenketten beibehalten wurden, um dadurch das native Packungsmuster zu gewährleisten. Die Helixbildung in diesem System wird durch Oligomerisierung induziert, während die isolierte β,γ-Sequenz größtenteils unstrukturiert vorliegt. Im Folgenden wurden verschiedene α,β,γ-Chimäre hinsichtlich ihrer Oligomerisierung sowohl an dem Modell als auch im Rahmen einer natürlich vorkommenden Coiled-Coil-Sequenz, GCN4pLI, untersucht. Dabei kam eine von Vielzahl von theoretischen und experimentellen Methoden und Assays wie Molekulardynamic-Simulationen, CD-Spektroskopie, Größenausschluss- Chromatografie, analytische Ultrazentrifugation, Transmissionselektronen- Spektroskopie sowie auch Fluoreszenz-Resonanz-Energietransfer, Disulfid- Austauschexperimente und „Native Chemische Ligation“ zur Anwendung. Im Anschluss an diese Untersuchungen wurden mit Hilfe zweier Medium-Throughput- Methoden (Spot-Synthese und Phage-Display) optimale Interaktionspartner für die artifizielle α,β,γ-Sequenz identifiziert. Während der rationale Ansatz bei der Spot-Synthese von vorn herein bestimmte Sequenzen ausschloss, schöpfte die Phage-Display-Technik aus dem gesamten Pool codierter Aminosäuren, sodass hiermit dem Wildtyp zwar strukturell wenig ähnelnde jedoch strukturell stabilere Bindungspartner gefunden werden konnten. Die Funktion des künstlichen Faltungsmotivs wurde im Kontext der templat-gesteuerten „Nativen Chemischen Ligation“ untersucht. Um die reaktiven funktionellen Gruppen in räumliche Nähe zu bringen, muss die α,β,γ-Sequenz die natürliche α-helikale Struktur imitieren können. Diese Eigenschaft konnte im Rahmen dieser Untersuchungen bestätigt werden. Weitere Auswirkungen der strukturellen Veränderungen durch den isosteren α,α,α→β,γ-Austausch, wie z.B. Verlust lokaler Packung und Gewinn an konformationeller Flexibilität durch die Eliminierung von Wasserstoffbrücken im Rückgrat wurden ebenfalls untersucht. In Disulfid-Austausch-Experimenten konnte gezeigt werden, dass die Paarung der chimären Sequenz mit der natürlichen GCN4pLI-Sequenz thermodynamisch nur erlaubt ist, wenn die β- und γ-Reste einer bestimmten Sequenz (βγβγ) folgen. Dies weist darauf hin, dass nur in diesem Fall die lokale Packung der Reste dem natürlichen Packungsmuster hinreichend ähnlich ist. Diese Beobachtungen stimmen mit den theoretischen Studien überein und zeigen, dass die Stabilität der Interaktionen mit α,β,γ-chimären Sequenzen durch das Ausmaß an Wechselwirkungen zwischen den Seitenketten in der helikalen Interaktionsdomäne kontrolliert werden kann

Investigation of the network of preferred interactions in an artificial coiled-coil association using the peptide array technique

We screened a randomized library and identified natural peptides that bound selectively to a chimeric peptide containing α-, β- and γ-amino acids. The SPOT arrays provide a means for the systematic study of the possible interaction space accessible to the αβγ-chimera. The mutational analysis reveals the dependence of the binding affinities of α-peptides to the αβγ-chimera, on the hydrophobicity and bulkiness of the side chains at the corresponding hydrophobic interface. The stability of the resulting heteroassemblies was further confirmed in solution by CD and thermal denaturation

An Unusual Interstrand H‑Bond Stabilizes the Heteroassembly of Helical αβγ-Chimeras with Natural Peptides

The substitution of α-amino acids by homologated amino acids has a strong impact on the overall structure and topology of peptides, usually leading to a loss in thermal stability. Here, we report on the identification of an ideal core packing between an α-helical peptide and an αβγ-chimera via phage display. Selected peptides assemble with the chimeric sequence with thermal stabilities that are comparable to that of the parent bundle consisting purely of α-amino acids. With the help of MD simulations and mutational analysis this stability could be explained by the formation of an interhelical H-bond between the selected cysteine and a backbone carbonyl of the β/γ-segment. Gained results can be directly applied in the design of biologically relevant peptides containing β- and γ-amino acids

Iterative optimization yields Mcl-1–targeting stapled peptides with selective cytotoxicity to Mcl-1–dependent cancer cells

Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1–binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1–dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or “staple” that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation work-flow applicable to a host of challenging biomedical targets. Keywords: stapled peptide; Mcl-1; apoptosis; BH3 mimetic; inhibitorUnited States. Department of Energy (Contract DE-AC02-06CH11357)National Institutes of Health (U.S.) (Grant R01 GM110048