Sensitized Detection of Inhibitory Fragments and Iterative Development of Non‐Peptidic Protease Inhibitors by Dynamic Ligation Screening

Abstract

Dedicated to Professor Günther Jung on the occasion of his 70th birthdayThe conventional approach to identify biologically active, druglike small molecules is based on high-throughput screening (HTS) of chemical libraries. However, the composition of large chemical libraries and their screening are time-consuming and expensive endeavors; the success relies heavily on the quality of the available libraries, and even the largest library can span only a minute section of the virtual chemical space. Therefore, over the past decade several strategies have been proposed to facilitate the development process by using the protein target as a template for ligand assembly.[1-3] The binding of low-molecular-weight fragments has been detected "directly" by NMR spectroscopy [2a, b] or X-ray crystallography. [2c, d] These biophysical methods have been demonstrated to provide low-affinity ligands as rational starting points for the iterative development of potent protein binders. Alternatively, protein-binding molecules have been identified from mixtures of compounds formed in dynamic equilibria. In the presence of a protein the equilibrium was shifted, and the best binding products were concentrated in the mixture and could be detected by chromatography, mass spectrometry, or NMR spectroscopy.[3a, b] The reported fragment-based methods have in common that they detect binding, not biological activity. Moreover, all these methods require large amounts of protein and test compounds and suffer from the difficult, timeconsuming, and expensive detection of active compounds.We envisioned that the detection of bioactive ligands should be sensitized considerably if reversibly formed ligation products compete in dynamic equilibrium with a fluorogenic reporter substrate for an enzyme (Figure 1). This approach would combine dynamic, target-assisted formation of inhibitory species and detection by a fluorescence-based screening methodology; thus, we designated it dynamic ligation screening (DLS). In DLS, the application of chemically reactive inhibitors as directing probes should enable the testing of inhibitory fragments for a defined binding site on the protein surface. Using an enzymatic reaction for fragment detection amplifies the signals and thus reduces the required amount of protein drastically. Finally, enzymatic detection with a fluorescent reporter molecule should enable high-throughput screening (HTS) in microtiter plates (MTPs); thus, for the first time conventional HTS methodology could be employed in fragment-based dynamic ligand development.The SARS coronavirus main protease (SARS-CoV M pro ; SARS = severe acute respiratory syndrome) was selected as the protein target to demonstrate the DLS approach. SARSCoV M pro is a cysteine protease that is essential for replication of the virus inside the infected host cell. Thus, it has been proposed as a drug target for SARS and-owing to the reported high homology among coronaviral main proteasesalso for other coronaviral infections.[5] Several irreversible (covalent) peptide-based...

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept

“…However, the binding of small fragments is weak and needs highly sensitive methods such as X-ray crystallography [7], NMR spectroscopy [8] or the amplification of binding, e.g. by dynamic ligation screening [9].…”

Catalytic activation of pre-substrates via dynamic fragment assembly on protein templates

“…Dynamic ligation screening (DLS) has been introduced as an approach for the sensitive, site-selective detection of proteinbinding fragments 17 . In this method, a chemically reactive protein ligand serves as a directing probe for the screening of fragment libraries at a spatially defined protein site 18 .…”

Recent Advances in Targeting Viral Proteases for the Discovery of Novel Antivirals

“…(11)), and, if performed subsequently in "reverse mode", it paves the way for the development of non-peptidic inhibitors (27, Fig. (11)) [113].…”

“…This property has been made use of for screening a small library of non-peptidic amines that would replace the cysteine from the aldehyde to form an imine with the latter, in the presence of the enzyme. This novel approach has been coined "Dynamic Ligation Screening" [113]. In this context, the proteolytic cleavage of a fluorogenic substrate is perturbed by the competing directing inhibitory aldehyde, but inhibition of the cleavage reaction will be observed to larger extent, if the aldehyde undergoes imine formation with fragment-type components of a screening library, thereby forming a product with enhanced affinity compared to the directing probe.…”