Fully Functionalized Small-Molecule Probes for Integrated Phenotypic Screening and Target Identification

Phenotypic screening offers a powerful approach to identify
small
molecules that perturb complex biological processes in cells and organisms.
The tendency of small molecules, however, to interact with multiple
protein targets, often with moderate to weak affinities, along with
the lack of straightforward technologies to characterize these interactions
in living systems, has hindered efforts to understand the mechanistic
basis for pharmacological activity. Here we address this challenge
by creating a fully functionalized small-molecule library whose membership
is endowed with: (1) one or more diversity elements to promote interactions
with different protein targets in cells, (2) a photoreactive group
for UV light-induced covalent cross-linking to interacting proteins,
and (3) an alkyne handle for reporter tag conjugation to visualize
and identify cross-linked proteins. A library member was found to
inhibit cancer cell proliferation selectively under nutrient-limiting
(low glucose) conditions. Quantitative chemoproteomics identified
MT-ND1, an integral membrane subunit of the ∼1 MDa NADH:ubiquinone
oxidoreductase (complex 1) involved in oxidative phosphorylation,
as a specific target of the active probe. We further demonstrated
that the active probe inhibits complex 1 activity in vitro (IC50 = 720 nM), an effect that is known to induce cell death
in low-glucose conditions. Based on this proof of principle study,
we anticipate that the generation and integration of fully functionalized
compound libraries into phenotypic screening programs should facilitate
the discovery of bioactive probes that are amenable to accelerated
target identification and mechanistic characterization using advanced
chemoproteomic technologies.