Selektive Detektion kooperativ bindender Fragmente in einem Hochdurchsatz‐Ligationsassay zur Entwicklung eines pikomolaren Caspase‐3‐Inhibitors

Abstract

Die Fragment-basierte Entwicklung von Leitstrukturen wurde in den vergangenen Jahren populär, da sie eine effizientere Abtastung des chemischen Raums ermöglicht als das Hochdurchsatz-Screening und daher schneller zu Treffern ("Hits") führt.[1] Seitdem das Konzept der Fragment-basierten Leitstrukturentwicklung erstmals vorgeschlagen wurde, [2] sind diverse Detektionsmethoden verwendet worden, um die Bindung von Fragmenten an Proteintemplate nachzuweisen. Dazu gehörten HPLC, [3] NMR-Spektroskopie, [4] Röntgen-Kristallographie [5] und Massenspektrometrie. [6] Kürzlich wurde das dynamische Ligationsscreening eingeführt, um reversibel verknüpfte Fragmente in einem biochemischen Assay durch ihre Kompetition mit einem fluorogenen Enzymsubstrat nachzuweisen. [7] Die bislang publizierten

Dynamic Substrate Enhancement for the Identification of Specific, Second‐Site‐Binding Fragments Targeting a Set of Protein Tyrosine Phosphatases

“…After a fixed time, the enzymatic reaction was terminated, and the released phosphate (P i ) was quantified as the green ammonium phosphomolybdate complex of Malachite Green at 630 nm ( Figure 1). [14] In agreement with our recent work on caspase-3, [12] three outcomes of this experiment are conceivable. A) The quantity of P i is decreased; this would suggest that the tested fragment acts as an inhibitor of the PTP.…”

“…DLS has been used to identify fragments in a high-throughput assay either through enhancing the inhibition of a fluorogenic substrate, [11] or enhancing binding as detected in an anisotropy (fluorescence-polarization) assay. [12] In addition, dynamic substrate enhancement, as demonstrated here, supports the recent finding of Ellman's group that screening substrate libraries can be useful for the development of improved enzyme inhibitors. [13] Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets.…”

Multivalent Design of Apoptosis‐Inducing Bid‐BH3 Peptide–Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

“…After this initial demonstration of the activity of the conjugate, we investigated the impact of multivalency on the induction of apoptosis using conjugates with different numbers of BH3 peptides per polymers. For a reliable quantification of apoptosis induction, the activity of the protease caspase‐3 was measured 25. This specific enzyme was selected as a biomarker, as in every apoptotic process, caspase‐3 is the central effector protease, which is activated by a pro‐apoptotic signal (such as the presence of BH3 peptides) and then leads to the proteolytic degradation of the cell.…”

“…For a reliable quantification of apoptosis induction, the activity of the protease caspase-3 was measured. [25] This specific enzyme was selected as a biomarker, as in every apoptotic process, caspase-3 is the central effector protease, which is activated by a pro-apoptotic signal (such as the presence of BH3 peptides) and then leads to the proteolytic degradation of the cell. Notably, caspase-3 is activated only during apoptosis, but not in other processes leading to cell death such as necrosis.…”

Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes

“…The situation changed when caspase‐1 served as a cysteine protease target not related to ubiquitin‐derived substrates. For this enzyme the tetrapeptidyl aldehyde was known as a low‐nanomolar inhibitor;9, 10 however, the propargyl amide of this short peptide was inactive in the inhibition experiments. Elongation of the progargyl amide to 16 amino acids was required to regain potent inhibition of the enzyme.…”