3 research outputs found
Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical Synthesis and High-Resolution X‑ray Structure of ShK Toxin by Racemic Protein Crystallography
We
have re-examined the utility of native chemical ligation at
-Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the
improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native
chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites
without side reactions. After optimization, ligation at a -Glu-Cys-
site could also be used as a ligation site, with minimal levels of
byproduct formation. However, -Asp-Cys- is not appropriate for use
as a site for native chemical ligation because of formation of significant
amounts of β-linked byproduct. The feasibility of native chemical
ligation at -Gln-Cys- enabled a convergent total chemical synthesis
of the enantiomeric forms of the ShK toxin protein molecule. The d-ShK protein molecule was ∼50,000-fold less active in
blocking the Kv1.3 channel than the l-ShK protein molecule.
Racemic protein crystallography was used to obtain high-resolution
X-ray diffraction data for ShK toxin. The structure was solved by
direct methods and showed significant differences from the previously
reported NMR structures in some regions of the ShK protein molecule
Fully Convergent Chemical Synthesis of Ester Insulin: Determination of the High Resolution X‑ray Structure by Racemic Protein Crystallography
Efficient total synthesis of insulin is important to
enable the
application of medicinal chemistry to the optimization of the properties
of this important protein molecule. Recently we described “ester
insulin”a novel form of insulin in which the function
of the 35 residue C-peptide of proinsulin is replaced by a single
covalent bondas a key intermediate for the efficient total
synthesis of insulin. Here we describe a fully convergent synthetic
route to the ester insulin molecule from three unprotected peptide
segments of approximately equal size. The synthetic ester insulin
polypeptide chain folded much more rapidly than proinsulin, and at
physiological pH. Both the d-protein and l-protein
enantiomers of monomeric DKP ester insulin (i.e., [Asp<sup>B10</sup>, Lys<sup>B28</sup>, Pro<sup>B29</sup>]ester insulin) were prepared
by total chemical synthesis. The atomic structure of the synthetic
ester insulin molecule was determined by racemic protein X-ray crystallography
to a resolution of 1.6 Å. Diffraction quality crystals were readily
obtained from the racemic mixture of {d-DKP ester insulin
+ l-DKP ester insulin}, whereas crystals were not obtained
from the l-ester insulin alone even after extensive trials.
Both the d-protein and l-protein enantiomers of
monomeric DKP ester insulin were assayed for receptor binding and
in diabetic rats, before and after conversion by saponification to
the corresponding DKP insulin enantiomers. l-DKP ester insulin
bound weakly to the insulin receptor, while synthetic l-DKP
insulin derived from the l-DKP ester insulin intermediate
was fully active in binding to the insulin receptor. The d- and l-DKP ester insulins and d-DKP insulin were
inactive in lowering blood glucose in diabetic rats, while synthetic l-DKP insulin was fully active in this biological assay. The
structural basis of the lack of biological activity of ester insulin
is discussed
A Potent d‑Protein Antagonist of VEGF‑A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating <i>in Vivo</i>
Polypeptides
composed entirely of d-amino acids and the
achiral amino acid glycine (d-proteins) inherently have <i>in vivo</i> properties that are proposed to be near-optimal
for a large molecule therapeutic agent. Specifically, d-proteins
are resistant to degradation by proteases and are anticipated to be
nonimmunogenic. Furthermore, d-proteins are manufactured
chemically and can be engineered to have other desirable properties,
such as improved stability, affinity, and pharmacokinetics. Thus,
a well-designed d-protein therapeutic would likely have significant
advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth
factor A (VEGF-A) that inhibited binding to its receptor. However,
RFX001.D is unstable at physiological temperatures (<i>T</i><sub>m</sub> = 33 °C). Here, we describe RFX037.D, a variant
of RFX001.D with extreme thermal stability (<i>T</i><sub>m</sub> > 95 °C), high affinity for VEGF-A (<i>K</i><sub>d</sub> = 6 nM), and improved receptor blocking. Comparison
of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has
a longer half-life <i>in vivo</i> in mice. Significantly,
RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer
generated a strong immune response. These results confirm the potential
utility of synthetic d-proteins as alternatives to therapeutic
antibodies