Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small-molecule/protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ϵ-amino group in lysine, this strategy generates a number of specific challenges. Herein, we review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.
Demonstrating
intracellular protein target engagement is an essential step in the
development and progression of new chemical probes and potential small
molecule therapeutics. However, this can be particularly challenging
for poorly studied and noncatalytic proteins, as robust proximal biomarkers
are rarely known. To confirm that our recently discovered chemical
probe 1 (CCT251236) binds the putative transcription
factor regulator pirin in living cells, we developed a heterobifunctional
protein degradation probe. Focusing on linker design and physicochemical
properties, we generated a highly active probe 16 (CCT367766)
in only three iterations, validating our efficient strategy for degradation
probe design against nonvalidated protein targets.
Phenotypic screens, which focus on
measuring and quantifying discrete
cellular changes rather than affinity for individual recombinant proteins,
have recently attracted renewed interest as an efficient strategy
for drug discovery. In this article, we describe the discovery of
a new chemical probe, bisamide (CCT251236), identified using an unbiased
phenotypic screen to detect inhibitors of the HSF1 stress pathway.
The chemical probe is orally bioavailable and displays efficacy in
a human ovarian carcinoma xenograft model. By developing cell-based
SAR and using chemical proteomics, we identified pirin as a high affinity
molecular target, which was confirmed by SPR and crystallography.
The stress‐inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine‐targeted irreversible inhibitor. Using rational design, we adapted a validated 8‐N‐benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine‐56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.
A general strategy for the production of pyrrolizidine alkaloids is described, starting from intermediate (+)-9. The key features are diastereoselective dihydroxylation, inversion at the ring junction by hydroboration of an enamine, and ring closure to form the bicyclo ring system. This route is attractive because of its brevity and versatility; four natural products were prepared with differing stereochemistry and substitution patterns. Finally, this work allowed assignment of the absolute stereochemistry of 2,3,7-triepiaustraline and hyacinthacine A 7.
HSP70 is a molecular
chaperone and a key component of the heat-shock
response. Because of its proposed importance in oncology, this protein
has become a popular target for drug discovery, efforts which have
as yet brought little success. This study demonstrates that adenosine-derived
HSP70 inhibitors potentially bind to the protein with a novel mechanism
of action, the stabilization by desolvation of an intramolecular salt-bridge
which induces a conformational change in the protein, leading to high
affinity ligands. We also demonstrate that through the application
of this mechanism, adenosine-derived HSP70 inhibitors can be optimized
in a rational manner.
The covalent inhibition mechanism of action, which overcomes
competition
with high-affinity, high-abundance substrates of challenging protein
targets, can deliver effective chemical probes and drugs. The success
of this strategy has centered on exposed cysteine residues as nucleophiles
but the low abundance of cysteine in the proteome has limited its
application. We have recently reported our discovery that lysine-56
in the difficult-to-drug target HSP72 could form a covalent bond with
a small-molecule inhibitor. We now disclose the optimization of these
targeted covalent inhibitors using rational design. Essential to our
optimization was the development of a new covalent fluorescence polarization
assay, which allows for the direct measurement of the key kinetic
parameter in covalent inhibitor design, k
inact
/K
I
, extrapolation of
the underlying parameters, k
inact and K
i
, and direct comparison to
reversible analogues. Using our approach, we demonstrate a >100-fold
enhancement in covalent efficiency and key learnings in lysine-selective
electrophile optimization.
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