A False‐Positive Screening Hit in Fragment‐Based Lead Discovery: Watch out for the Red Herring

Abstract

With the rising popularity of fragment-based approaches in drug development, more and more attention has to be devoted to the detection of false-positive screening results. In particular, the small size and low affinity of fragments drives screening techniques to their limit. The pursuit of a false-positive hit can cause significant loss of time and resources. Here, we present an instructive and intriguing investigation into the origin of misleading assay results for a fragment that emerged as the most potent binder for the aspartic protease endothiapepsin (EP) across multiple screening assays. This molecule shows its biological effect mainly after conversion into another entity through a reaction cascade that involves major rearrangements of its heterocyclic scaffold. The formed ligand binds EP through an induced-fit mechanism involving remarkable electrostatic interactions. Structural information in the initial screening proved to be crucial for the identification of this false-positive hit.

K2S2O8-Mediated Synthesis of Highly Functionalized Pyrroles via Oxidative Self-Dimerization of N-Propargylamines

“…The utility of the methodology is extended toward the synthesis of pyrrolo­[3,4- d ]­pyridazine 3e , 3i , and 3j (Scheme ). It may be noted that pyrrolo­[3,4- d ]­pyridazines are considered as high-affinity nonamino acid ligands for α2δ subunit of voltage gated calcium channels, and aspartic protease endothiapepsin (EP) inhibition . Thus, the reaction of (4-benzoyl-5-(4-chlorophenyl)-1,2-diphenyl-1 H -pyrrol-3-yl)­(4-chlorphenyl)­methanone ( 2e ), 2i , and 2j with hydrazine hydrate in ethanol at 80 °C resulted in 1,5-bis­(4-chlorophenyl)-4,6,7-triphenyl-6 H -pyrrolo­[3,4- d ]­pyridazine ( 3e ), 3i , and 3j in 64, 71, and 76% yields, respectively.…”

Application of Fragment-Based Drug Discovery to Versatile Targets

“…It has been noted that the shift of Tm is proportional to the concentration or affinity of fragments in most cases, but it is not straightforward to correlate the shifts in Tms of compounds with their binding affinities. It is always a good strategy to confirm the identified hits through other biophysical methods (Cramer et al, 2017;Hassaan et al, 2020; Figure 2). It has been noted that other factors such as protein dynamics might influence Tm changes induced by ligand binding.…”

A Proof-of-Concept Fragment Screening of a Hit-Validated 96-Compounds Library against Human Carbonic Anhydrase II

“…However, the hydrazinecarboximidamide substructure and the difluorophenyl moiety, which are connected by a C sp2 atom, provide an extended π-system that can be expected to act as binding partner for the S-atom of the Met side chain. Given the comparably close distance of 3.3 Å between the O δ1 atom of the side chain of Asp71 of a symmetry mate and the covalently linked N-atom of 7, an interaction can be inferred, which resembles that between an Asp side chain and the positively charged aromatic scaffold of a false-positive fragment screening hit against endothiapepsin, discovered by Cramer et al [15] It is reasonable to assume that, in fact, a charged interaction is formed, given that the hydrazinecarboximidamide substructure can be considered basic, thus bearing a positive charge that can be distributed over several atoms (Scheme 2), which will furthermore be beneficial for the interaction between 7 and the S-atom of Met1. Furthermore, the side chain of Ser73 of the symmetry mate might interact with the N3 atom of 7 that is involved in the delocalization of a putative positive charge.…”

“…Detected fragment hits are subsequently elaborated into lead compounds by growing, merging or linking, which relies upon the potentiation of binding affinity upon combination of weak-affinity contributors [3,4,8]. Based on the considerable knowledge about fragment-based research that our group has amassed over the years [9][10][11][12][13][14][15], a 96-entry fragment library was assembled, to make the utility of fragments available to the broader public [16]. We tested its versatility in screenings against several established proteins, among these the well-established drug target and model protein human carbonic anhydrase II (hCAII).…”