Toxicology lecture

2. EXPANDING THE LIGANDABLE PROTEOME
A new method for finding drug candidates that bind to specific proteins. It’s important because of its widespread application to many
proteins at once, even to the ones within their native cellular environments.
The new technique involved finding ligands for many proteins previously thought to bind poorly to small molecules—> determine the
functions of their protein targets and serve as starting compounds for drug development.
Data —> widely increased targetability of the human proteome by way of small molecules
Finding out which proteins in our cells are ligandable is currently a major theoretical step in drug development. Only about 3% of
human proteins have been targeted successfully with current drugs on the market. The new method is empirical, instead of
theoretical, to show the fraction of the human proteome that is ligandable (ie small-molecule-friendly targets).
The new method is FBLD-inspired (fragment-based ligand discovery). FBLD uses candidate ligand molecules that are half the size
of small molecules in pill-based drugs. These are fragment molecules. Tiny fragment molecules are more effective than larger,
drug-sized ones to find possible ligands because they require smaller libraries of compounds for a complete search.

3. New method:
Candidate fragment molecules are attached to a class of other molecules; when they get close enough to the cysteine amino acids
on proteins, they react strongly with the cysteine —> ligands are locked to the proteins via strong covalent bonding.
The covalent bonding = large potency boost.
Then...
The creation of a screening system to apply the covalent-bonding fragment molecules, one by one, to whole collections of human
proteins (even on proteins that are still living in petri dishes). What this does: researchers can detect and identify the small molecule
fragments that are covalently-bound to proteins, which shows then which sites on the protein are responsible for binding.
Next step:
Testing a small library of the cysteine-reactive fragments to the proteins in 2 kinds of cancer cells. —> the small-molecule fragments
liganded >750 different cysteines on more than 600 individual proteins (ie 20% of the proteins assayed in the samples)
Some of those liganded proteins were transcription factors (previously considered ~unligandable, so undruggable). 85% of the
liganded proteins they found to be ligandable weren’t listed in a standard database of proteins with known small-molecule ligands.

4. Quantification of cysteine reactivity via:
isotopic tandem orthogonal proteolysis‒activity- based protein profiling (isoTOP-ABPP)—to perform covalent FBLD in native
biological systems. → the new method
isoTOP-ABPP steps:
1. Lysate or intact cells are pre-treated with dimethylsulfoxide (DMSO) or an electrophilic small-molecule fragment then
exposed to a broad-spectrum cysteine-reactive probe, iodoacetamide (IA)-alkyne 1.
2. Proteins harboring cysteine residues from DMSO- and fragment-treated samples are conjugated by click chemistry
(azide-alkyne cycloaddition) to isotopically differentiated azide-biotin tags (heavy and light), combined, enriched by
streptavidin, and digested proteolytically to yield isotopic peptide pairs that are analyzed via liquid chromatography-mass
spectrometry (LC-MS).
3. Quantification of MS1 chromatographic peak ratios for peptide pairs identifies fragment-competed Cys residues as those
displaying high competition ratios, or R values, in DMSO/ fragment comparisons.

5. The small library of cysteine reactive fragments, fig 1:
BIG PICTURE: A fragment library (with mostly chloroacetamide or acrylamide
electrophiles (Fig. 1b and Extended Data Fig. 1)—well-characterized cysteine-reactive
groups). These electrophiles were attached to structurally diverse small-molecule
recognition (or binding) elements to create library members. The researchers wanted to
probe the ligandability of cysteines in the human proteome, so they screened the
electrophile library at a high concentration (500μM) similar to the compound
concentrations used in FBLD experiments.
1a) General protocol for competitive isoTOP-ABPP. Competition ratios or R values, are measured
by dividing the MS1 ion peaks for IA-alkyne (1)-labelled peptides in DMSO-treated (heavy, blue)
versus fragment-treated (light, red) samples.
1b) General structure of electrophilic fragment library, in which the reactive (electrophilic) and
binding groups are coloured green and black.
1c) Competitive isoTOP-ABPP analysis of the MDA-MB-231 cell proteome pre-treated with the
electrophilic 3 and 14 fragments, along with the non-electrophilic 17. Proteomic reactivity values, or
liganded cysteine rates, for fragments were calculated as the percentage of total cysteines with R
values ≥ 4 in DMSO/fragment (heavy/light) comparisons.
1d) Representative MS1 peptide ion chromatograms from competitive isoTOP-ABPP experiments
marking liganded cysteines selectively targeted by one of three fragments, 3, 14 and 23.

6. Extended Data Figure 1 | Composition of fragment electrophile library and structures of
additional tool compounds, click probes and fragments .

12. Researchers looked at 2 enzymes:
-PRMT1
-MAP3 kinase MLTK
Found that fragment electrophiles
inhibited the enzymes

16. ●

17. ● Competitive isoTOP-ABPP experiments identified distinct subsets of
ligands that targeted a conserved cysteine in isocitrate dehydrogenase 1
(IDH1) and 2 (IDH2) (C269 and C308, respectively).
● IDH1 and IDH2 are mutated in a number of human cancers to produce
enzyme variants with neomorphic catalytic activity that converts isocitrate
to the oncometabolite 2-hydroxyglutarate (2-HG)
● Reversible inhibitors selective for mutant forms of IDH1 and IDH2 have
been developed and are under clinical investigation for cancer treatment.

20. ● The fragment ligands inhibited the activity of wild-type but not a C269S
mutant of IDH1
● Blocked the R132H oncogenic mutant of IDH1 both in vitro and in cells.
● The differences could reflect the impact of transport and/or metabolic
pathways on the cellular concentrations of fragment electrophiles.

21. 7a) Representative MS1 chromatograms for CASP8 tryptic peptide containing the catalytic
cysteine C360 quantified by isoTOP-ABPP in cell lysates or cells treated with fragment 4 (250
μM, in vitro; 100 μM, in situ) and control fragment 21 (500 μM, in vitro; 200 μM, in situ).
BIG PICTURE: Several fragments targeted the catalytic cysteine nucleophile C360 of the
protease caspase-8 (CASP8) in isoTOP-ABPP experiments performed in vitro and in situ
(Extended Data Fig. 7a and Extended Data Table 1).
7bc) BIG PICTURE: Curiously, however, these fragments exhibited little to no inhibition of active
CASP8 using either substrate or activity-based probe (Rho-DEVD-AOMK probe) assays
(Extended Data Fig. 7b, c). more specifically, b and c showcase fragment reactivity with casp-8:
7b) Neither 7 nor control fragment 62 (100 μM each) inhibited recombinant. purified-active
CASP3 and CASP8 were assayed using DEVD-AMC and IETD-AFC fluorogenic substrates,
respectively. DEVD-CHO (20 μM) inhibited both caspases.
7c) Fragment reactivity with recombinant: purified-active CASP8 added to cell lysates, where
reactivity was detected in competition assays using the caspase activity probe
Rho-DEVD-AOMK probe.

22. BIG PICTURE: This crazy outcome— that the fragments showed
little to no inhibition of active CASP8 using either substrate or
activity-based probe—was explained when they found out that the
electrophilic fragments selectively labelled the inactive zymogen
(pro-), but not active form of CASP8.
7d) Western blot of proteomes from MDA-MB-231, Jurkat, and CASP8-null Jurkat
proteomes showing that CASP8 was only found in the pro-enzyme form in these
cells.
7e) Fragment reactivity with recombinant: purified pro-CASP8 added to cell lysates
to a final concentration of 1 μM protein, where reactivity was detected in competition
assays with the IA-rhodamine probe (2 μM). Reactivity in 21, 3, 14, 31 fragment.
7f) Inactive control fragment 62 did not compete IA-rhodamine labelling of C360 of
pro-CASP8.
7g) Apparent IC50 curve for blockade of IA-rhodamine labelling of pro-CASP8
(C409S) by 7. As compound concentration increased, casp8 labeling decreased
7h) 7 (50 μM) fully competed IA-alkyne labelling of C360 of endogenous CASP8 in
cell lysates as measured by isoTOP-ABPP. Representative MS1 chromatograms
are shown for the C360-containing peptide of CASP8.

23. BIG PICTURE: All of these essentially show that the
electrophilic fragments selectively labelled the inactive
zymogen (pro-), but not active form of CASP8. Which is why
the fragments showed little to no inhibition of active CASP8
using the substrate or activity-based probes
-They created a clickable analogue of the most potent
pro-CASP8 ligand 7 and found that this probe (25 μM)
strongly labelled pro-CASP8, but not a pro-CASP8 C360S
mutant (Fig. 7i), and it directly modified C360 of CASP8 in
Jurkat cell lysates.
- Compound 7 blocked labelling of pro-CASP8 by 61, but did
not inhibit labelling of active CASP8 or other caspases by
the Rho-DEVD-AOMK probe (Fig. 7k, l). Conversely, the
general caspase inhibitor Ac-DEVD-CHO blocked
Rho-DEVD-AOMK labelling of active CASP8 and other
caspases, but not 61 labelling of pro-CASP8 (Fig. 7k, l).
7i) Concentration-dependent reactivity of
click probe 61, with recombinant, purified
pro-CASP8 (D374A, D384A) added to cell
lysates to a final concentration of 1 μM
protein.
7j) Blocked IA-alkyne labelling of C360 of
pro-CASP8, but not active CASP8, as
measured by isoTOP-ABPP. Recombinant
pro- and active CASP8 were added to
Ramos lysates at 1 μM and then treated
with 7 (30 μM) followed by isoTOP-ABPP.
7k) Fragments 7 and 62 did not block
labelling by Rho-DEVD-AOMK of
recombinant; purified active CASP8 and
active CASP3 added to MDA-MB-231 cell
lysates .
7l) 7 does not inhibit active caspases.
Recombinant, active caspases were added
to MDA-MD-231 lysate, treated with
(z-VAD-FMK), followed by labelling with
the Rho-DEVD-AOMK probe.

24. BIG PICTURE: They confirmed that 7, but not a
structurally related inactive probe (62 - the
control) blocked Fas ligand (FasL)-, but not
staurosporine (STS)-induced apoptosis in Jurkat
cells (Extended Data Fig. 7n–p).
7 blocks extrinsic apoptosis. Control 62 fragment
doesn’t. This, and studying the fragments’ effects
on these pathways helps to improve our collective
understanding of the functions of Casp-8 and
Casp-10, which are currently poorly understood
with respect to apoptosis.
7m) Representative MS1 chromatograms
for tryptic peptides containing the catalytic
cysteines of CASP8 (C360), CASP2
(C320), and CASP7 (C186) quantified by
isoTOP-ABPP in Jurkat cell lysates
treated with 7 or 62.
7n) 7, but not control fragment 62, blocked
extrinsic, but not intrinsic apoptosis. Jurkat
cells (1.5 million cells per ml) were
incubated with 7 or 62 (30 μM) for 30 min
before addition of staurosporine or
SuperFasLigand (100 ng ml−1).
7o) For cells treated as described in n,
cleavage of PARP (96 kDa), CASP8
(p43/p41, p18), and CASP3 (p17) was
visualized by western blot.
7p) 7 protects Jurkat cells from extrinsic,
but not intrinsic apoptosis. Cleavage of
PARP, CASP8 and CASP3 detected by
western blotting as shown in o was
quantified for three (STS) or two (FasL)
independent experiments.

25. BIG PICTURE: fragment 63, a more potent version of fragment 7,
gets separated into 63R and 63S. 63R is CASP8-selective;
Fragment 7 is dual CASP8–CASP10. Now, tthey can investigate the
biological functions of these proteases to better our understanding
of the caspases’ role in apoptosis (which are not that understood
partly because of lack of selective inhibitors for these enzymes,
which the researchers successfully made).
8a) Chemical proteomic experiments revealed that 7 fully inhibited
CASP8, as well as the related initiator caspase CASP10
8b-e) Confirms that 7 blocked labelling of pro-CASP10 by 61 (Fig.
8b–d), but did not inhibit active CASP10 or a substrate assay (Fig.
8e).
8c,d,f) fragment 63 was separated into 63-R and 63-S, with 63-R
showing improved activity against CASP8 compared to compound 7
and negligible cross-reactivity with CASP10.
8d,g,h) 63-S was much less active against CASP8 (apparent IC50
value of 15 μM, 8gh) and also inactive against CASP10 (8d).
a, Representative MS1 peptide signals
showing R values for caspases detected by
quantitative proteomics using probe 61.
b, 7 blocked 61 labelling of pro-CASP8 and
CASP10, whereas 63-R selectively blocked
probe labelling of pro-CASP8.
c, 7, but not 63-R blocked probe labelling of
pro-CASP10. Recombinant pro-CASP10 was
added to MDA-MB-231 lysates to a final
concentration of 300 nM, treated with the
indicated compounds, labeled with probe 61.
d, Apparent IC50 curve for blockade of 61
labelling of pro-CASP10 by 7, 63-R or 63-S.
e, Neither 7 nor 63-R (25 μM each) inhibited
the activity of recombinant, purified active
CASP10, which was assayed after addition of
the protein to MDA-MB-231 lysate using
fluorometric (AEVD-AMC) substrate.
DEVD-CHO (20 μM) inhibited the activity of
CASP10.
f, IC50 curve for blockade of 61 labelling of
pro-CASP8 and pro-CASP10 by 63-R.
g, 63-R shows increased potency against
pro-CASP8. Recombinant pro-CASP8 was
added to MDA-MB-231 lysates to a final
concentration of 300 nM, treated with the
indicated compounds, labelled with probe 61.
h, Apparent IC50 curve for blockade of 61
labelling of pro-CASP8 by 63-R compared
with 63-S. The structure of 63-S is shown.
i, CASP10 is more highly expressed in
primary human T cells compared to Jurkat
cells. Western blot analysis of full-length
CASP10, CASP8 and GAPDH expression
levels in Jurkat and T-cell lysates.

26. BIG PICTURE: CASP10 is involved in intrinsic apoptosis in primary
human T cells.
63-R fully blocked FasL-induced apoptosis in Jurkat cells and did so with
greater potency than 7 (Fig. 8j)
Similar results in HeLa cells—express CASP8, but not CASP10 (Fig. 8l).
But FasL-induced apoptosis in primary human T cells showed substantial
resistance to 63-R and instead was completely inhibited by the dual
CASP8/10 ligand 7.
Chemical proteomics — 7 blocked CASP8 and CASP10. 63-R inhibited
CASP8, but not CASP10, in primary human T cells and Jurkat cells.
7, but not 63-R, prevented proteolytic processing of CASP8 and CASP10
in primary human T cells (Fig. 8m).
These data, taken together, support substantive functions for both CASP8
and CASP10 in primary human T cells & confirm that deleterious mutations
in either CASP8 or CASP10 can lead to autoimmune syndromes in
humans.
j, Jurkat cells were incubated with 7
or 63-R at the indicated
concentrations for 30 min before
addition of staurosporine (2 μM) or
SuperFasLigand (100 ng ml−1).
Cells were incubated for 4 h and
viability was quantified with
CellTiter-Glo (CTG).
k, Jurkat cells treated as in j, but
with 63-R or 63-S.
l, HeLa cells were seeded and 24 h
later treated with the indicated
compounds for 30 min before the
addition of SuperFasLigand and
cycloheximide. Cells were
incubated for 6 h and viability was
quantified.
m, T cells treated as in Fig. 4d,
then cleavage of CASP10, CASP8,
CASP3 and RIPK was visualized by
western blotting.

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