High-Throughput Screening by Nuclear Magnetic Resonance (HTS by NMR) for the Identification of PPIs Antagonists

Abstract

In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications.

Potent and Selective EphA4 Agonists for the Treatment of ALS

“…Here, instead of testing by NMR mixtures of possible of fragments and subsequently synthesize bi-dentate agents after pairs of ligands have been discovered (Becattini and Pellecchia, 2006; Chen et al, 2007; Petros et al, 2010; Shuker et al, 1996), we combine the fragments directly into a common scaffold and test by NMR the pre-assembled molecules in mixtures gathered in positional scanning fashion (Wu et al, 2015a; Wu et al, 2013). These studies led to the identification of the initial hit molecule compound 22 (Table 1) that was able to bind selectively to the EphA4-LBD in the mid-micromolar range.…”

HTS by NMR for the Identification of Potent and Selective Inhibitors of Metalloenzymes

“…10−18 As an alternative to these strategies, we have recently proposed a novel approach, termed HTS by NMR, 19 in which the principles of positional scanning combinatorial chemistry 20−23 and fragment-based drug design are combined with protein-NMR spectroscopy 1 to iteratively identify and optimize antagonists from collections of >100,000 peptide mimetics. 19,24−26 The approach seems also particularly effective in the fragment-hit to lead optimization stages, when a positional scanning library is generated from an initial weak binder, perhaps common to a class of protein targets and/or previously identified from a FBDD campaign, 19,24,25 and tested by biophysical methods including not only NMR but also ITC. 27 Indeed, we recently demonstrated that testing a positional scanned library using the HTS by NMR approach revealed qualitatively a ranking that not only confirmed the known binding consensus motif for the BIR3 domain of the X-linked Inhibitor of Apoptosis Protein (XIAP), but also identified compounds that closely resembled a clinical agent (GDC-0152) 25 that targets it.…”

“…19,24−26 The approach seems also particularly effective in the fragment-hit to lead optimization stages, when a positional scanning library is generated from an initial weak binder, perhaps common to a class of protein targets and/or previously identified from a FBDD campaign, 19,24,25 and tested by biophysical methods including not only NMR but also ITC. 27 Indeed, we recently demonstrated that testing a positional scanned library using the HTS by NMR approach revealed qualitatively a ranking that not only confirmed the known binding consensus motif for the BIR3 domain of the X-linked Inhibitor of Apoptosis Protein (XIAP), but also identified compounds that closely resembled a clinical agent (GDC-0152) 25 that targets it. Hence, to assess if this approach could be implemented to target metalloproteinases, we first derived a focused positional scanning (POS) combinatorial library of peptide mimetics (of approximately 100,000 compounds) where each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal ( Figure 1A).…”

(S)Pinning down protein interactions by NMR

“…Instead we refer to the many excellent reviews available on these topics. [12][13][14][15][16] The complex landscape of protein interactions…”