6 research outputs found
A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides
Thiopeptides make up a group of structurally complex
peptidic natural
products holding promise in bioengineering applications. The previously
established thiopeptide/mRNA display platform enables de novo discovery
of natural product-like thiopeptides with designed bioactivities.
However, in contrast to natural thiopeptides, the discovered structures
are composed predominantly of proteinogenic amino acids, which results
in low metabolic stability in many cases. Here, we redevelop the platform
and demonstrate that the utilization of compact reprogrammed genetic
codes in mRNA display libraries can lead to the discovery of thiopeptides
predominantly composed of nonproteinogenic structural elements. We
demonstrate the feasibility of our designs by conducting affinity
selections against Traf2- and NCK-interacting kinase (TNIK). The experiment
identified a series of thiopeptides with high affinity to the target
protein (the best KD = 2.1 nM) and kinase
inhibitory activity (the best IC50 = 0.15 ÎŒM). The
discovered compounds, which bore as many as 15 nonproteinogenic amino
acids in an 18-residue macrocycle, demonstrated high metabolic stability
in human serum with a half-life of up to 99 h. An X-ray cocrystal
structure of TNIK in complex with a discovered thiopeptide revealed
how nonproteinogenic building blocks facilitate the target engagement
and orchestrate the folding of the thiopeptide into a noncanonical
conformation. Altogether, the established platform takes a step toward
the discovery of thiopeptides with high metabolic stability for early
drug discovery applications
Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries
A methodology to
achieve high-throughput de novo sequencing of
synthetic peptide mixtures is reported. The approach leverages shotgun
nanoliquid chromatography coupled with tandem mass spectrometry-based
de novo sequencing of library mixtures (up to 2000 peptides) as well
as automated data analysis protocols to filter away incorrect assignments,
noise, and synthetic side-products. For increasing the confidence
in the sequencing results, mass spectrometry-friendly library designs
were developed that enabled unambiguous decoding of up to 600 peptide
sequences per hour while maintaining greater than 85% sequence identification
rates in most cases. The reliability of the reported decoding strategy
was additionally confirmed by matching fragmentation spectra for select
authentic peptides identified from library sequencing samples. The
methods reported here are directly applicable to screening techniques
that yield mixtures of active compounds, including particle sorting
of one-bead one-compound libraries and affinity enrichment of synthetic
library mixtures performed in solution
A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides
Thiopeptides make up a group of structurally complex
peptidic natural
products holding promise in bioengineering applications. The previously
established thiopeptide/mRNA display platform enables de novo discovery
of natural product-like thiopeptides with designed bioactivities.
However, in contrast to natural thiopeptides, the discovered structures
are composed predominantly of proteinogenic amino acids, which results
in low metabolic stability in many cases. Here, we redevelop the platform
and demonstrate that the utilization of compact reprogrammed genetic
codes in mRNA display libraries can lead to the discovery of thiopeptides
predominantly composed of nonproteinogenic structural elements. We
demonstrate the feasibility of our designs by conducting affinity
selections against Traf2- and NCK-interacting kinase (TNIK). The experiment
identified a series of thiopeptides with high affinity to the target
protein (the best KD = 2.1 nM) and kinase
inhibitory activity (the best IC50 = 0.15 ÎŒM). The
discovered compounds, which bore as many as 15 nonproteinogenic amino
acids in an 18-residue macrocycle, demonstrated high metabolic stability
in human serum with a half-life of up to 99 h. An X-ray cocrystal
structure of TNIK in complex with a discovered thiopeptide revealed
how nonproteinogenic building blocks facilitate the target engagement
and orchestrate the folding of the thiopeptide into a noncanonical
conformation. Altogether, the established platform takes a step toward
the discovery of thiopeptides with high metabolic stability for early
drug discovery applications
Di- and Triphenylacetates of Lanthanum and Neodymium. Synthesis, Structural Diversity, and Application in Diene Polymerization
A new
family of lanthanide complexes with di- and triphenylacetate
ligands was prepared from LnCl<sub>3</sub>. Dimeric diphenylacetates
were the only isolated products of the reaction between LnCl<sub>3</sub> (Ln = La, Nd) and Ph<sub>2</sub>CHCOONa. The exchange reaction between
LnCl<sub>3</sub> and Ph<sub>3</sub>CCOONa led to the formation of
three structurally different types of neodymium and lanthanum complexes
under different reaction conditions. Various coordination modes of
the carboxylate ligands were revealed, including Îș<sup>2</sup>- and Îș<sup>1</sup>-terminal coordination, ÎŒ<sub>2</sub>-Îș<sup>1</sup>:Îș<sup>1</sup>-bridging, and ÎŒ<sub>2</sub>-Îș<sup>1</sup>:Îș<sup>2</sup>-semibridging. The
treatment of La and Nd tris-triphenylacetates with excess AlEt<sub>3</sub> produces the dimeric tetraethylaluminates [Ln<sub>2</sub>((ÎŒ<sub>2</sub>-Îș<sup>1</sup>:Îș<sup>1</sup>-Ph<sub>3</sub>CCOO)<sub>4</sub>(AlEt<sub>4</sub>)<sub>2</sub>]. The structures
of the key La and Nd triphenylacetates that were obtained from these
reactions were verified by X-ray diffraction, and the complex [(THF)<sub>4</sub>Nd<sub>2</sub>(Ph<sub>2</sub>CHCOO)<sub>2</sub>(ÎŒ<sub>2</sub>-Îș<sup>1</sup>:Îș<sup>2</sup>-Ph<sub>2</sub>CHCOO)<sub>2</sub>(ÎŒ<sub>2</sub>- Îș<sup>1</sup>:Îș<sup>1</sup>-Ph<sub>2</sub>CHCOO)<sub>2</sub>] was studied by high-resolution
X-ray diffraction. Neodymium di- and triphenylacetate complexes can
serve as precursors of catalysts for butadiene and isoprene stereospecific
1,4-cis polymerization
dâAmino Acid Scan of Two Small Proteins
A âD-scanâ
of two small proteins, the disulfide-rich <i>Ecballium elaterium</i> trypsin inhibitor II (EETI-II) and a
minimized Z domain of protein A (Z33), is reported. For each protein,
the stereochemistry of one amino acid at a time was inverted to generate
a series of diastereomers. In much the same way an alanine scan determines
necessary residues for protein function, the D-scan elucidated the
critical stereocenters of the 30-residue EETI-II and the 33-residue
Z33. The folding properties and activity of each variant were investigated.
A total of 24 out of 30 EETI-II D-scan analogues folded to give a
three-disulfide product. Of the 24 variants that folded, half were
high-affinity trypsin inhibitors, and three were as active as the
wild type (WT). Of these 12 active variants, most were substantially
less stable to reduction than WT EETI-II (WT first reduction potential
â270.0 ± 1.5 mV, WT second reduction potential â307.2
± 1.1 mV). Similarly, ten Z33 analogues retained high binding
affinity to IgG (<i>K</i><sub>D</sub> < 250 nM, WT: 24
± 1 nM) and 12 additional analogues had reduced but appreciable
IgG binding affinity (<i>K</i><sub>D</sub> between 250 nM
and 2.5 ÎŒM). As with EETI-II, most Z33 analogues were substantially
less stable than the WT (Î<i>G</i>(H<sub>2</sub>O,
263 K) = 2.4 ± 1.2 kcal/mol). Collectively, our findings show
that the D-scan is powerful new strategy for studying how the stereochemistry
of amino acids affects the structure and function of proteins
Polyphenylcyclopentadienyl Ligands as an Effective Light-Harvesting ÏâBonded Antenna for Lanthanide +3 Ions
A new approach to
design âantenna-ligandsâ to enhance the photoluminescence
of lanthanide coordination compounds has been developed based on a
Ï-type ligandîžthe polyphenyl-substituted cyclopentadienyl.
The complexes of di-, tri-, and tetraphenyl cyclopentadienyl ligands
with Tb and Gd have been synthesized and all the possible structural
types from mononuclear to di- and tetranuclear complexes, as well
as a coordination polymer were obtained. All types of the complexes
have been studied by single-crystal X-ray diffraction and optical
spectroscopy. All terbium complexes are luminescent at ambient temperature
and two of them have relatively high quantum yields (50 and 60%).
Analysis of energy transfer process has been performed and supported
by quantum chemical calculations. The role of a low-lying intraligand
charge transfer state formed by extra coordination with K<sup>+</sup> in the Tb<sup>3+</sup> ion luminescence sensitization is discussed.
New aspects for design of lanthanide complexes containing Ï-type
ligands with desired luminescence properties have been proposed