14 research outputs found
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
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
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
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