9 research outputs found
Visualization of PK401 with purified NSP4 and all NSP’s.
<p>(A) NSP4 was treated with PK401 in a range from 1 to 2000nM. (B) 100nM of NE, PR3, CatG and NSP4 with or without 100nM of PK401. (A, B) Samples were denatured in SDS sample buffer, run in SDS/PAGE followed by membrane transfer. The blot was developed with fluorescently-tagged streptavidin and imaged by fluorescence scanning (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132818#pone.0132818.s001" target="_blank">S1 Text</a>).</p
Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4
<div><p>Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.</p></div
Scheme of the HyCoSuL P1 Arg library.
<p>The general library structure contains tetrapeptide derivatives with the sequence Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents one of 120 fixed natural or unnatural amino acids and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met) with ACC (7-amino-4-carbamoylmethylcoumarin) as a reporter group.</p
Kinetic analysis of tetrapeptide substrates for NSP4.
<p>Results are shown as an average of a minimum of 2 separate experiments with S.D.</p
Structures of the optimized NSP4 substrates based on natural (PK417 and PK418) and natural/unnatural amino acids (PK421 and PK431).
<p>The activity-based probe (PK401), a diphenyl phosphonate derived from the optimal substrate sequence—PK421—is shown as the last structure.</p
Kinetic parameters/constants for the hydrolysis of Ac-hCha-Phe(guan)-Oic-Arg-ACC substrate by neutrophil serine proteases to three significant digits.
<p>NA–no activity detected.</p><p>Kinetic parameters/constants for the hydrolysis of Ac-hCha-Phe(guan)-Oic-Arg-ACC substrate by neutrophil serine proteases to three significant digits.</p
Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg<sup>P</sup>(OPh)<sub>2</sub> (PK401).
<p>NI–no inhibition observed; K<sub>m</sub> values relate to the substrate used for analysis,</p><p>* K<sub>m</sub> for this substrate was above 100μM, the concentration used in the assay. AMC – 7-amino-4-methylcoumarin.</p><p>Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg<sup>P</sup>(OPh)<sub>2</sub> (PK401).</p
Determination of NSP4 substrate specificity.
<p>Preferences in the P4-P2 positions were determined by screening HyCoSuL, which contains tetramer peptides with the general structures Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents fixed natural or unnatural amino acid and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met). Screening was performed on a SpectraMax Gemini plate reader. Substrate hydrolysis rates were normalized to the most active component (100%) y axis. Natural amino acids are colored grey, unnatural black. Results are shown as an average of 3 experiments with S.D.</p
Expedient Synthesis of Highly Potent Antagonists of Inhibitor of Apoptosis Proteins (IAPs) with Unique Selectivity for ML-IAP
A series of novel, potent antagonists of the inhibitor
of apoptosis
proteins (IAPs) were synthesized in a highly convergent and rapid
fashion (≤6 steps) using the Ugi four-component reaction as
the key step, thus enabling rapid optimization of binding potency.
These IAP antagonists compete with caspases 3, 7, and 9 for inhibition
by X chromosome-linked IAP (XIAP) and bind strongly (nanomolar binding
constants) to several crucial members of the IAP family of cancer
pro-survival proteins to promote apoptosis, with a particularly unique
selectivity for melanoma IAP (ML-IAP). Experiments in cell culture
revealed powerful cancer cell growth inhibitory activity in multiple
(breast, ovarian, and prostate) cell lines with single agent toxicity
at low nanomolar levels against SKOV-3 human ovarian carcinoma cells.
Administration of the compounds to human foreskin fibroblast cells
revealed no general toxicity to normal cells. Furthermore, computational
modeling was performed, revealing key contacts between the IAP proteins
and antagonists, suggesting a structural basis for the observed potency