5 research outputs found
Structure–Activity Relationship Study of Opiorphin, a Human Dual Ectopeptidase Inhibitor with Antinociceptive Properties
Toward developing new potential analgesics, this first
structure–activity
relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human
peptide inhibiting enkephalin degradation, was performed. A systematic
Ala scanning proved that Phe<sup>3</sup> is a key residue for neprilysin
and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series
of Phe<sup>3</sup>-halogenated analogues revealed that halogen bonding
based optimization strategies are not applicable to this residue.
Additional substituted Phe<sup>3</sup> derivatives showed that replacing l-Phe<sup>3</sup> for d-Phe<sup>3</sup> increased the
AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular
mechanics calculations indicated that the improved potency may be
due to CH−π stacking interactions between the aromatic
ring of d-Phe<sup>3</sup> and the Hγ protons of Arg<sup>2</sup>. This structural motif is not possible for the native opiorphin
and may be useful for the design of further potent and metabolically
stable analogues
Interactions of Bacterial Cell Division Protein FtsZ with C8-Substituted Guanine Nucleotide Inhibitors. A Combined NMR, Biochemical and Molecular Modeling Perspective
FtsZ
is the key protein of bacterial cell-division and target for
new antibiotics. Selective inhibition of FtsZ polymerization without
impairing the assembly of the eukaryotic homologue tubulin was demonstrated
with C8-substituted guanine nucleotides. By combining NMR techniques
with biochemical and molecular modeling procedures, we have investigated
the molecular recognition of C8-substituted-nucleotides by FtsZ from <i>Methanococcus jannaschii</i> (Mj-FtsZ) and <i>Bacillus
subtilis</i> (Bs-FtsZ). STD epitope mapping and trNOESY bioactive
conformation analysis of each nucleotide were employed to deduce differences
in their recognition mode by each FtsZ species. GMP binds in the same
anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn
conformation. However, the anti conformation of 8-morpholino-GMP is
selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries.
The inhibitory potencies of the C8-modified-nucleotides on the assembly
of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities.
Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces
formation of Mj-FtsZ curved filaments, resembling polymers formed
by the inactive forms of this protein. NMR data, combined with molecular
modeling protocols, permit explanation of the mechanism of FtsZ assembly
impairment by C8-substituted GTP analogs. The presence of the C8-substituent
induces electrostatic remodeling and small structural displacements
at the association interface between FtsZ monomers to form filaments,
leading to complete assembly inhibition or to formation of abnormal
FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be
simply explained by steric clashes of the C8-GTP-analogs with the
incoming FtsZ monomer. This information may facilitate the design
of antibacterial FtsZ inhibitors replacing GTP
The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR
The identification of MUC1 tumor-associated
Tn antigen (αGalpNAc1-<i>O</i>-Ser/Thr) has boosted
the development of anticancer vaccines.
Combining microarrays and saturation transfer difference NMR, we have
characterized the fine-epitope mapping of a MUC1 chemical library
(naked and Tn-glycosylated) toward two families of cancer-related
monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs
clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and
bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn
mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind
naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference
for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr
analogue, stressing the role of the underlying amino acid (serine
or threonine) in the binding process. The reported strategy can be
employed, in general, to unveil the key minimal structural features
that modulate antigen–antibody recognition, with particular
relevance for the development of Tn-MUC1-based anticancer vaccines
Detection of Tumor-Associated Glycopeptides by Lectins: The Peptide Context Modulates Carbohydrate Recognition
Tn
antigen (α-<i>O</i>-GalNAc-Ser/Thr) is a convenient
cancer biomarker that is recognized by antibodies and lectins. This
work yields remarkable results for two plant lectins in terms of epitope
recognition and reveals that these receptors show higher affinity
for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR)
peptide region of mucin MUC1. In contrast, a significant affinity
loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala
(AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our
data indicate that the charged residues, Arg and Asp, present in the
PDTR sequence establish noteworthy fundamental interactions with the
lectin surface as well as fix the conformation of the peptide backbone,
favoring the presentation of the sugar moiety toward the lectin. These
results may help to better understand glycopeptide–lectin interactions
and may contribute to engineer new binding sites, allowing novel glycosensors
for Tn antigen detection to be designed
Exploiting the Therapeutic Potential of 8‑β‑d‑Glucopyranosylgenistein: Synthesis, Antidiabetic Activity, and Molecular Interaction with Islet Amyloid Polypeptide and Amyloid β‑Peptide (1–42)
8-β-d-Glucopyranosylgenistein
(<b>1</b>),
the major component of <i>Genista tenera</i>, was synthesized
and showed an extensive therapeutical impact in the treatment of STZ-induced
diabetic rats, producing normalization of fasting hyperglycemia and
amelioration of excessive postprandial glucose excursions and and
increasing β-cell sensitivity, insulin secretion, and circulating
insulin within 7 days at a dose of 4 (mg/kg bw)/day. Suppression of
islet amyloid polypeptide (IAPP) fibril formation by compound <b>1</b> was demonstrated by thioflavin T fluorescence and atomic
force microscopy. Molecular recognition studies with IAPP and Aβ<sub>1–42</sub> employing saturation transfer difference (STD)
confirmed the same binding mode for both amyloid peptides as suggested
by their deduced epitope. Insights into the preferred conformation
in the bound state and conformers’ geometry resulting from
interaction with Aβ<sub>1–42</sub> were also given by
STD, trNOESY, and MM calculations. These studies strongly support
8-β-d-glucopyranosylgenistein as a promising molecular
entity for intervention in amyloid events of both diabetes and the
frequently associated Alzheimer’s disease