compatibility with native protein structures and effects on protein–protein interactions

Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids—aliphatic, aromatic, charged and polar as well as proline—is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references)

Systematic Evaluation of Fluorination as Modification for Peptide‐Based Fusion Inhibitors against HIV‐1 Infection

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics

Untersuchung der Interaktionsprofile nichtnatürlicher Aminosäuren zur Vorhersage ihrer spezifischen Anwendung an α-helikalen Grenzflächen

1 Introduction 1 2 Theoretical Background and Scientific Context 3 2.1 The α-Helical Coiled-Coil Folding Motif 3 2.2 Modifying Helical Structures with Nonnatural Amino Acids 12 2.3 Phage Display as an Example for Protein Evolution 29 3 Aim 35 4 Concept and Previous Studies 37 4.1 Applied Screening System 37 4.2 Previous Phage-Display Experiments with VPE/VPK 40 5 Results and Conclusions 43 5.1 Accommodating Fluorinated Amino Acids in Parallel Coiled- Coil Dimers 43 5.2 Stabilizing a Coiled Coil that Contains a Set of Alternating β -and γ-Amino Acids 53 5.3 Towards Protease Stable Fluorinated HIV Entry Inhibitors 63 6 Summary and Outlook 75 7 Experimental Procedures and Analytical Methods 79 7.1 Peptide Synthesis and Characterization 79 7.2 Structural Analysis 89 7.3 Phage Display 95 9 Supplementary Data 107 9.1 Phage Display with Fluorinated Amino Acids 107 9.2 Phage Display with βγ-Foldamers 114 10 Literature 123Nonnatural amino acids are frequently incorporated into peptide-based pharmaceuticals to improve bioavailability and specificity. Systematic studies of how such residues interact with natural amino acids in the context of native protein folds may yield important information that can facilitate the prediction of the properties of novel nonnatural peptide therapeutics or biomaterials. To this end, the current study made use of phage display to screen large numbers of helical microenvironments to identify, based on binding affinity, natural peptide sequences that preferably interact with sequences containing either fluoroalkyl substituted amino acids or alternating sets of β- and γ-amino acids. The use of peptides that contain different analogues of (S)-2-aminobutyric acid with different numbers of fluorine atoms, and thus different side-chain volumes, showed that the incorporation of these amino acids into the hydrophobic core of a parallel heterodimeric coiled coil leads to similar pairing characteristics. Despite their differences in hydrophobicity and size, all investigated amino acids prefer to interact with the aliphatic amino acids leucine and isoleucine. However, the selection led to an optimized side chain packing which is expressed in thermal stability enhancements. It was verified that coiled coils readily accommodate diverse fluorinated aliphatic amino acids as nonnatural building blocks within their hydrophobic cores. In contrast, diverse α-amino acid patterns were found with recognition specificity for βγ-foldameric sequences, at the interface of parallel or antiparallel helical assemblies. Here, the sequences selected by phage display show that the incorporation of a polar H-bond donor functionality can significantly improve helical interactions involving backbone extended amino acids, because these donors are able to engage a free backbone carbonyl of αβγ-chimeras in an interstrand H-bond. A mutation leading to an increase of the surface area of the hydrophobic core had a similar effect on the thermal stability. Finally, initial studies were carried out towards the rational design of a protease resistant peptide-based inhibitor of HIV’s gp41 envelope protein subunit. Gp41 is thought to play a key role in infection by facilitating host cell entry via the assembly of a bundle of six α-helices composed of three N-terminal heptad repeat (NHR) segments and three C-terminal heptad repeat (CHR) segments. Several short C-peptide analogues of differing length were intrahelically crosslinked to increase α-helicity, and tested for binding affinity to NHR segments. A CHR derived peptide sequence was generated that shows affinity for a NHR segment, and may therefore inhibit bundle formation. The highly conserved Trp-Trp-Ile motif, which is crucial for tight helix alignment, constitutes the center of this CHR peptide and will serve as a starting point for systematic substitution studies with fluorinated aliphatic amino acid analogues.Nichtnatürliche Aminosäuren werden häufig in peptidbasierte Pharmazeutika eingebaut, um deren Bioverfügbarkeit und Spezifität zu erhöhen. Die systematische Untersuchung der Interaktionsprofile solcher Aminosäuren mit ihren natürlichen Analoga in nativen Proteinumgebungen liefert wertvolle Erkenntnisse, die Vorhersagen über die Eigenschaften neuartiger nichtnatürlicher peptidbasierter Therapeutika und Biomaterialen ermöglichen. In dieser Arbeit sollten mithilfe von phage display Wechselwirkungspartner identifiziert werden, die bevorzugt mit fluoralkylsubstituierten Aminosäuren oder mit Motiven alternierender β- und γ-Aminosäuren, im Kontakt helikaler Strukturen interagieren. Die Untersuchung von Peptiden mit unterschiedlichen fluorierten Analoga von (S)-2-Aminobuttersäure zeigte, dass der Einbau solcher Aminosäuren in den hydrophoben Kern eines parallelen, heterodimeren coiled coil-Faltungsmotivs ähnliche Wechselwirkungspartner hervorbringt. Trotz deutlicher Unterschiede in Größe und Hydrophobie der fluorierten Seitenketten, bevorzugen alle hier untersuchten Aminobuttersäureanaloga die aliphatischen Aminosäuren Leucin oder Isoleucin als ihre natürlichen Wechselwirkungspartner. Die Selektion führte zu einer optimierten Packung um die fluorierten Seitenketten, was sich in einer thermischen Stabilitätserhöhung äußerte. Diese Studie verifiziert, dass coiled coil-Strukturen bereitwillig verschiedene fluoralkylsubstituierte Aminosäuren in ihrem hydro-phoben Kern als nichtnatürliche Bausteine akzeptieren. Im Gegensatz dazu wurden mittels phage display diverse heptad repeat-Muster selektiert, die Erkennungsspezifitäten für βγ-Foldamere an der Grenzfläche von parallelen und antiparallelen α-helikalen Anordnungen aufweisen. Mit den selektierten Sequenzen konnte gezeigt werden, dass durch die Einführung von ausgewählten, polaren Wasserstoff-brückendonoren die Stabilität von Interaktionen zwischen rückgraterweiternden β- und γ-Aminosäuren und α-peptidischen Motiven erhöht wird. Diese Donoren sind in der Lage Wasserstoffbrücken mit freien Rückgratcarbonylgruppen von αβγ-Chimären auszubilden. Zusätzlich können auch Mutationen, die die Oberfläche des hydrophoben Kerns vergrößern zu einer thermischen Stabilisierung beitragen. Schließlich wurden einleitende Studien zur rationalen Entwicklung von proteasestabilen, peptidbasierten Inhibitoren der gp41 Hüllproteinuntereinheit von HIV durchgeführt. Es wird angenommen, dass gp41 während der Infektion eine Schlüsselrolle einnimmt, indem es ein Bündel aus sechs Helices ausbildet, das sich aus drei N-terminalen Heptad- Repeat (NHR) Segmenten und drei C-terminalen Heptad-Repeat (CHR) Segmenten zusammensetzt. Mehrere C-Peptid Analoga unterschiedlicher Länge wurden intrahelikal verbrückt, um ihre α-Helizität zu erhöhen. Die Auswirkungen dieser Modifikation auf die Bindungsaffinität zu NHR Segmenten wurden untersucht. Außerdem wurde eine CHR Sequenz generiert, die bindungsaffin zu einem NHR Segment ist und somit möglicherweise die Ausbildung des hexameren Bündels inhibieren kann. Das hochkonservierte Trp-Trp-Ile Motiv, das für die Bündelausbildung ausschlaggebend ist, befindet sich in der Mitte dieser Sequenz und wird in künftigen Untersuchungen als Ausgangspunkt für Substitutionsstudien mit fluorierten Aminosäuren fungieren

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics

Synthesis of enantiomerically pure (2S,3S)-5,5,5-trifluoroisoleucine and (2R,3S)-5,5,5-trifluoro-allo-isoleucine

A practical route for the stereoselective synthesis of (2S,3S)-5,5,5-trifluoroisoleucine (L-5-F3Ile) and (2R,3S)-5,5,5-trifluoro-allo-isoleucine (D-5-F3-allo-Ile) was developed. The hydrophobicity of L-5-F3Ile was examined and it was incorporated into a model peptide via solid phase peptide synthesis to determine its α-helix propensity. The α-helix propensity of 5-F3Ile is significantly lower than Ile, but surprisingly high when compared with 4’-F3Ile

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

Coiled-Coils in Phage Display Screening: Insight into Exceptional Selectivity Provided by Molecular Dynamics

Involved in numerous key biological functions, protein helix–helix interactions follow a well-defined intermolecular recognition pattern. The characteristic structure of the α-helical coiled-coil allows for the specific randomization of clearly defined interaction partners within heteromeric systems. In this work, a rationally designed heterodimeric coiled-coil was used to investigate potential factors influencing the sequence selectivity in interhelical interactions

Human monoclonal antibodies against chikungunya virus target multiple distinct epitopes in the E1 and E2 glycoproteins.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the β-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention