Gold-Based Pharmacophore Inhibits Intracellular MYC Protein

Article abstract of DOI:10.1002/chem.202004962

Direct targeting of intrinsically disordered proteins, including MYC, by small molecules for biomedical applications would resolve a longstanding issue in chemical biology and medicine. Thus, we developed gold-based small-molecule MYC reagents that engage MYC inside cells and modulate MYC transcriptional activity. Lead compounds comprise an affinity ligand and a gold(I) or gold(III) warhead capable of protein chemical modification. Cell-based MYC target engagement studies via CETSA and co-immunoprecipitation reveal specific interaction of compounds with MYC in cells. The lead gold(I) reagent, 1, demonstrates superior cell-killing potential (up to 35-fold) in a MYC-dependent manner when compared to 10058-F4 in cells including the TNBC, MDA-MB-231. Subsequently, 1 suppresses MYC transcription factor activity via functional colorimetric assays, and gene-profiling using whole-cell transcriptomics reveals significant modulation of MYC target genes by 1. These findings point to metal-mediated ligand affinity chemistry (MLAC) based on gold as a promising strategy to develop chemical probes and anticancer therapeutics targeting MYC.

Full text of DOI:10.1002/chem.202004962

Accepted Article
Accepted Article
Title: Gold-Based Pharmacophore Inhibits Intracellular MYC Protein
Authors: Samuel G Awuah, Samuel Ofori, and Sailajah Gukathasan
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202004962
Link to VoR: https://doi.org/10.1002/chem.202004962
01/2020

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
Gold-Based Pharmacophore Inhibits Intracellular MYC Protein
Samuel Ofori,[a] Sailajah Gukathasan,[a]and Samuel G. Awuah*[a][b]
[*a]
S. Ofori, S. Gukathasan, Prof. S.G. Awuah
Department of Chemistry
University of Kentucky
505 Rose Street, Lexington Kentucky, 40506[b] Center for Pharmaceutical and Research Innovation
College of Pharmacy, Department of Pharmaceutical Sciences
University of Kentucky, Lexington Kentucky, 40536
E-mail: awuah@uky.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: Direct targeting of intrinsically disordered proteins,
including MYC by small-molecules for biomedical applications will
resolve a longstanding issue in chemical biology and medicine. Thus,
we developed gold-based small-molecule MYC reagents that engage
MYC inside cells and modulate MYC transcriptional activity. Lead
compounds comprise an affinity ligand and a gold(I) or gold(III)
warhead capable of protein chemical modification. Cell-based MYC
target engagement studies via CETSA and co-immunoprecipitation
reveal specific interaction of compounds with MYC in cells. The lead
gold(I) reagent, 1, demonstrates superior cell-killing potential (up to
35-fold) in a MYC-dependent manner when compared to 10058-F4 in
cells including, the TNBC, MDA-MB-231. Subsequently, 1 suppresses
MYC transcription factor activity via functional colorimetric assays,
and gene-profiling using whole-cell transcriptomics reveals significant
modulation of MYC target genes by 1. These findings point to metal-
mediated ligand affinity chemistry (MLAC) based on gold as a
promising strategy to develop chemical probes and anticancer
therapeutics targeting MYC.
Fig. 1: Schematic illustration of protein labeling via biorthogonal
chemistry, noncovalent binding, covalent binding and our metal-
mediated ligand affinity chemistry for endogenous covalent
modification of MYC.
Most of these known organic warheads modify specific, but
Introduction
8b, 9]
few amino acid types.[6,
Therefore, the expansion of
potent covalent drugs with high selectivity for their target will
benefit from chemoselective warheads that react with several
Targeting proteins via chemical modification is a new powerful
methodology to elucidate protein function.[1] This has led to
the identification of biological targets, antibody-drug
conjugates, and small-molecule covalent drugs.[2] In the past
two decades, bioorthogonal methods have advanced to
include chemically selective reactions of fluorescent agents,[3]
drugs,[4], or affinity tags with proteins carrying functionalized
amino acids,[5] and are now valuable strategies for selective
protein labeling[6] (Fig.1a). Bioorthogonal methods, however,
involve two steps[7]: i) incorporation of the bioorthogonal
handle (e.g. non-canonical amino acid, enzyme domain,
peptide sequences) and ii) the binding of functional molecules
such as drugs, affinity tags, and fluorophores. This two-step
protocol often involves genetic manipulation, which makes it
challenging to chemically modify endogenous proteins in
living systems. Thus, we propose the development of a
transition metal-based chemical strategy that is ligand-
directed to the endogenous protein of interest in a single step,
which we refer to as metal-mediated ligand affinity chemistry
(MLAC). (Fig. 1c). Covalent drugs, composed mainly of
organic small-molecules, exemplify ligand affinity chemistries
in which ligands bind canonical amino acids of desired
proteins in cells or animals via electrophilic warheads.[3, 8]
nucleophilic amino acid side chains[8b, 10] including, (Lys[1b, 11]
His[12], Met[13], Arg[14], Tyr[15], Ser, Glu).
,
Transition-metal complexes provide new opportunities for
bioconjugation[16] of peptides[17] and proteins.[18] Important
examples of direct metalation or ligation of amino acids have
been reported using Au[19], Ru[20], Pd[21], Rh[22], and Ni[23]
.
Gold complexes have been reported to react with cysteines[24]
,
and other amino acid residues. Lysine residues are ~5.9%
abundant within the human proteome and is an attractive
target for site selective modification. A significant number of
lysine residues are within the binding region of most PPIs.[25]
Hence, significant progress has been made in targeted
covalent modification of these amino acids in peptides or
proteins. Chemoselective cysteine arylation methods using
Au and Pd have been demonstrated by Wong et al.,[26]
Buchwald et al.[27] and Spokoyny et al.[28] Equally, regio- and
chemoselective Cu[29] and Pd[30] mediated arylation of lysine
have also been reported. Despite the success, extension of
these ideas to the much more complex intracellular matrix of
living systems is underexplored.[19c] Finally, incorporating
transition-metal based warheads in therapeutics as covalent
1
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
carried out using standard coupling reactions of the affinity ligand,
10058-F4 with Au-F4Phos or Au-Oxime (Fig. 2).
drugs could offer new opportunities to target proteins deemed
undruggable due to unconventional binding modes of
transition-metal complexes.
Intracellular targeting of PPIs such as MYC proteins, which
are implicated in ~70% of all human cancers through gene
amplification, translocation, and aberrant mRNA regulation, is
crucial for chemical probe development and as anticancer
therapeutics.[31] The MYC protein heterodimerizes with MAX
and binds to a conserved consensus DNA E-box sequence[32]
,
which regulates downstream target genes involved in
proliferation, cell cycle progression[33], and differentiation.[31c-
Fig. 2. Metal-mediated ligand affinity chemistry reagents under
investigation in this report.
e]
Whereas MYC is a validated cancer target, the lack of defined
pocket domains and related toxicity to normal tissues render
them undruggable[34]. Moreover, no small molecule inhibitor of
MYC exists in the clinic.[35] Innovative strategies including,
covalent modification of MYC remain an untapped
All of the final compounds were well characterized by NMR
spectroscopy, high-resolution mass spectrometry and the
purity was ascertained by HPLC, >97% (Fig S1 – S20). We
examined the solution stability of 1 and 2 in RPMI, DMEM, or
BSA (Fig. S21). The UV-vis absorption profile of compound 1
was minimally altered in the presence of all three solutions
over a 24 h period, whereas 2 showed major changes in UV
profile, particularly in BSA. Further, we used HPLC to analyze
related decomposition of the lead compound, 1 in RPMI,
DMEM, or BSA (Fig. S22-S24) and found that 1 remains intact
over 24 h with only 4-8% loss in concentration based on area
under the curve. Together, 1 has better solution stability than
2. Conceivably, the high redox potential of gold(III)
compounds facilitate thiol-induced reduction and this may be
the reason behind the altered UV-vis profile of 2.
Subsequently, we investigated the reactivity of the gold
reagents with lysine. The Au+3 cyclometalated warhead reacts
with nucleophiles via a ligand associative mechanism and, at
appreciable energy, proceed to arylated products via
reductive elimination,[40] whereas the ability for Au+1 to
mediate transfer arylation is not well understood. Therefore,
we reacted 1 with a model lysine in aqueous media at
physiological conditions and found that the ε amino group was
arylated with the affinity ligand as confirmed by LC-MS (Fig.
3).
methodology but could yield transformational outcomes.[31c,
31e]
Herein we describe ligand affinity chemistry using gold
complexes as the electrophilic reactive group. Importantly, we
employ this strategy to inhibit c-MYC protein and its
transcriptional activity in cells. The excellent bioorthogonal
kinetics permit selective chemical modification of proteins
even in their native cellular environment.[36] We demonstrate
via mass spectrometry, cellular target engagement,
comparative cellular assays, functional transcriptional assays,
and whole cell transcriptomics that gold warheads are a
promising warhead for irreversible inhibition of expressed
MYC proteins; and they suppress MYC transcriptional
program in living cancer cells.
Results and Discussion
Stimulated by post-translational modification at lysine such as
succinylation,[37] which involves the addition of succinyl group
to a lysine residue of a protein molecule, we hypothesized that
gold-based reagents could mediate transfer arylation at lysine
like succinylation or acetylation events in biology.[38] To test
this hypothesis, we developed gold-based warheads for
chemical modification of proteins based on reactivity studies
of gold(I) and gold(III) complexes with model amino acid,
lysine. Gold (I) reagents are known to bind several amino
acids in protein structures[39] via direct coordination. Thus, we
designed metal-mediated ligand affinity chemistry (MLAC)
reagents that take advantage of the electrophilic gold center
to promote arylation at lysine. Our design consists of the c-
MYC affinity ligand 10058-F4 (Fig. 2) conjugated to gold
warheads. These gold warheads, namely, Au-F4Phos and
Au-Oxime (Fig. 2) are Au+1 and Au+3 complexes respectively.
As there exist several lysine residues in the binding pocket of
10058-F4, MYC (394-416), we envisioned our MLACs will
inhibit MYC by covalently modifying the lysine residues within
this binding region.
Fig 3. N-acetyllysine covalent modification with Au(I) MLAC reagent,
1. (a) Scheme for the arylation reaction between N-acetyllysine and 1.
General reaction conditions: N-acetyllysine was reacted with N-
acetyllysine was reacted with 1 (1 mol equiv.) in 0.01 % HCl in ACN :
H2O (v/v = 20: 80, pH = 8.0) at 37 °C for 2 h. (b) ESI-MS analysis of
Guided by preliminary molecular modelling, we designed gold
warheads that possess aromatic rings for sufficient π-π
interactions with key amino acid residues within the 10058-F4
binding region of MYC. The synthesis of MLAC reagents 1 and 2
along with control compounds N-Boc-Phos and N-Boc-Oxime were
2
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
reaction mixture of N-acetyllysine reacted with 1 in (a). Insert shows
isotopic distribution of respective products formed.
Fig. 4: Covalent modification of c-MYC peptide on lysine (residue 412) via Au1+ reagent ,1 a) Proposed reaction for labeling c-MYC peptide
on lysine via complex 1. b) LC-MS/MS of complex 1 labeled c-MYC peptide (res. 394-416) confirmed the covalent modification on lys-412 by
the addition of +215.04 Da. c) Proposed mechanism for Au1+ mediated lysine modification using complex 1.
Further, we studied the ability for 1 to covalently modify lysine
residues on a MYC peptide. We used a 23 amino acid peptide
that encompasses the binding sites of 10058-F4. Compound
1 labels the peptide covalently in aqueous solution under
physiological condition. Next, we subjected the product to
enzymatic (tryptic) digestion and analyzed the resulting
peptides by LC-MS/MS. We found that the modification
occurred in the peptide VILKKATAYILSVQAEEQKLISE
(modified residue underlined and italicized− total of 91%
sequence coverage) as observed in the MS/MS spectrum,
which corresponds to lysine 412 (Fig. 4). Lysine 412 was the
only residue modified by MLAC reagent 1, suggestive of
regioselectivity for the reaction.
The proposed mechanism, as shown in Fig. 4c, illustrates the
ability of the soft Lewis acid, the gold(I) center to coordinate
polarizable thiocarbonyl sulphur atom of 1. This highly
deactivates the thiocarbonyl carbon of the thiozalidinone core
of 1; in turn, making it relatively susceptible to the nucleophilic
attack by the ε-amino of lysine within the c-MYC peptide,
ultimately resulting in the modified peptide adduct.
by the nucleus, within a short time. Thus, P493-6 cells were
treated with 1 and 2, for 1 h, respectively. The nuclear
fractions were subsequently isolated, and the amount of gold
in the various nuclear extracts was quantified by graphite
furnace – atomic absorbance spectroscopy (GF-AAS) (Fig
S25). We found that 1 and 2, at 1 h, were taken-up and
distributed in cellular nucleus at 0.582 and 1.507 pmol per
million cells. This is an indication that the MLAC reagents can
reach their target within the nucleus.
We proceeded to study the intracellular effects of the MLAC
reagents. The use of cellular thermal stabilization assay
(CETSA) assesses drug-protein interaction in the native
environment of the cell based on temperature-dependent
ligand induced changes.[41] We employed the inducible c-MYC
expression system in P493-6 Burkitt’s lymphoma cells for our
CETSA studies.[42] The c-MYC OFF (+Tetracycline, 10 ng/ml),
c-MYC low (+Tetracycline, 10 ng/ml + β-estradiol), and c-MYC
ON (-Tetracycline) were confirmed by cell scattering using
flow cytometry and Western blot (Fig. 4A and 4B) respectively.
Treatment of c-MYC ON P493-6 cells with 1 or 2 (10 μM) for
1 h prior to CETSA protocols led to significant thermal
stabilization of the MYC protein, indicative of a direct, specific,
and strong binding of 1 or 2 to MYC (Fig. 4C and 4D).
Next, we performed uptake studies to determine the nuclear
uptake of our compounds. The localization of c-MYC in the
nucleus requires that compounds targeting MYC be taken up
3
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
Quantification of Western blot band intensities from the
CETSA experiment revealed that the onset of MYC melting for
control samples was ~42 ºC, 44 ºC for compound 2 treated
cells, and 47 ºC for compound 1 treated cells. This suggest
that 1 induces high thermal stability of MYC in cells. The
rational design of reagents to target intrinsically disordered
protein structures, such as c-MYC, as demonstrated with the
correlational CETSA put forth, is promising. Importantly,
thermal stabilization or destabilization of MYC by reported
MYC ligands such as 10058-F4, 10074-G5, 361 or JKY-2-169
is dictated by the binding site of the ligand to MYC, or
potentially the cell type used.[34b, 43] It is conceivable that the
use of the gold warheads in our MLAC strategy lead to
covalent modification of the MYC protein, which gave rise to
the observed thermal stabilization by 1 and 2.
Fig. 5: Inducible c-MYC expression in P493-6 Burkitt’s lymphoma cells via treatment with 0.1 µg/ml tetracycline (c-MYC-OFF), both 0.1 µg/ml
tetracycline and 1 µM β-estradiol (c-MYC LOW), or untreated (c-MYC-HIGH) for 72 hours, and then analyzed by (a) Overlay of histograms of
P493-6 cell sizes after the treatment and (b) related immunoblots c, d) Immunoblots and related CETSA assay melt curves of MYC protein,
normalized to β-Actin, in P493-6 cells treated with 10 µM of 1, 2 or DMSO. The graph shows the quantification of MYC protein versus
temperature points based on western blot analyses. Each data point is presented as mean ± SD obtained from three independent biological
experiments. Full blots have been included in the SI.
To determine the effect of 1 or 2 on MYC protein in P493-6
cells, we used Western blot to assess the levels of MYC
protein following exposure to the respective MLAC
compounds 1 or 2 (10 μM) in a time-dependent manner (0, 6,
12, 24 h) (Fig. 6A). The results suggest depletion of MYC
protein expression induced by 1 or 2. When 10058-F4 was
used, a high concentration of 60 μM was required to achieve
a similar outcome to 1 or 2. This is suggestive of direct
interaction of 1 or 2 with MYC protein. At high concentrations,
10058-F4 is known to inhibit Myc transcriptional activity and
decrease cell growth in myc-transformed fibroblasts. Thus, the
ability for MLAC reagents 1 or 2 to reduce MYC expression at
sub-micromolar concentrations has significant implications on
Myc transcriptional program and growth inhibition.
Furthermore, the effect of intracellular MYC-MAX interaction
was examined by co-immunoprecipitation (co-IP) (Fig. 6B).
Treatment of P493-6 cells with 1 at 10 μM for 3 h led to MYC
depletion, implying a MYC-MAX disruption. Overall, these
studies establish that gold-based MYC ligand affinity
chemistry exemplified in 1 binds MYC and possibly disrupts
the MYC-MAX complex.
Next, we sought to determine whether 1 or 2 could inhibit
MYC-dependent cancer cell proliferation. Using
a
comparative profiling approach, we assessed the comparative
Myc inhibiton of 1, 2, or control compounds in a panel of MYC-
dependent and MYC-independent cell lines (Fig. 6 C-D, Fig.
S26- S35). Both 1 and 2 inhibited cell proliferation of MYC -
dependent cancer cells including, breast cancer (MCF7, MDA-
MB-231, MDA-MB-468, HCC1937), lymphoma (P493-6) with
IC50s in the low-micromolar range, but with minimal effect on
RPE-NEO, which is a MYC-independent cell line.
4
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
Comparative Myc inhibition was examined in isogenic cell
lines including, P493-6 with MYC in the ON or OFF states.
We found that MYC OFF P493-6 cells were more resistant to
1 or 2 than MYC ON P493-6 cells (Fig 6C-D, Fig. S26-27).
Additionally, using normal retinal pigment epithelial (RPE)
cells and Myc-transformed RPEs, 1 and 2 demonstrate
significant Myc Inhibition (Fig. S31-S32). We examined the
impact of the gold warheads, Au-F4Phos and Au-Oxime
alone and found that they do not exhibit potent Myc inhibition
in the isogenic P493-6 or RPE cells used (Fig. S34-S35).
Also, the non-affinity compounds (N-Boc-Phos and N-Boc-
Oxime) were used as controls to ascertain whether the MYC
selectivity observed for 1 and 2 were due to the hybrid-
scaffold itself. These control compounds do not inhibit MYC
in high MYC P493-6 cells, and are not active in the c-Myc
TF activity assay (Fig. 7. C). Overall, the MLAC reagents are
potent inhibitors of c-MYC.
Fig. 6: Cellular effects of MLAC reagents. (a) Immunoblots showing the levels of MYC after P493-6 cells were treated with compounds, 1, 2,
10058-F4 or DMSO at the specified concentration, and time points. (b) Immunoblots showing the levels of MYC co-immunoprecipitated with
MAX in P493-6 cells with or without treatment of 1. (c) Cytotoxicity profile of (c) 1 (d) 2 in c-MYC-OFF, and c-Myc-HIGH P493 cells. e)
Comparative cytotoxicity profiles of 1 and 10058-F4 in MDA-MB-231 cell lines. Each data point is presented as mean ± SD obtained from three
independent biological experiments
Table 1: Cell viability of 1, 2, 10058-F4 across various cell lines
MLAC reagents show low-micromolar IC50 values with high
selectivity (Table 1). For example, as seen in Fig 6E,
compound 1 exhibits high potency (~10-fold) in MYC-
dependent MDA-MB-231, in comparison to 10058-F4.
Together, these results suggest improved growth inhibition in
a MYC-dependent manner consistent with direct MYC protein
binding by 1 or 2. We tested the hypothesis that MYC MLAC
binders, 1 and 2, modulate Myc-driven transcription through a
transcription factor assay of c-MYC in P493 cells and MDA-
MB-231 nuclear extracts. A colorimetric assay that can detect
the activation of transcription factors was used. In this assay,
a double-stranded oligonucleotide containing the c-Myc E-box
consensus sequence (5-CACGTG-3‘) is coated on the bottom
of 96-well plate. Active c-MYC within nuclear extracts can then
be captured after a short incubation, which can be detected
following c-MYC primary antibody and HRP-conjugated
secondary antibody using spectrophotometric measurements at
450 nm (Fig. S36A). We treated P493-6 or MDA-MB-231 cells
with compound 1 (10 μM) for 12 h, nuclear extracts were
obtained and used in the transcription factor assay. Low levels
of active MYC were detected for nuclear extracts from treated
cells compared to vehicle treated cells (Fig. 7A-B). We found
that the effect of 1 on c-MYC P493-6 transcription was more
pronounced in comparison to MDA-MB-231.This is because
P493-6 B-cells possess a high dependence on Myc for
Cell Lines
IC50 (µM)
10058-F4
1
2
P493-6
(HIGH MYC)
-
1.14
0.05
9.27 0.45
6.03 0.71
22.90 3.20
3.23 0.14
1.59 0.81
P493-6 (NO
MYC)
-
10.23 0.08
11.74 0.79
18.19 1.11
5.75 0.31
RPE-MYC
79.43 3.87
87.09 1.9
RPE-NEO
MDA-MB-231 106.0 ± 4.10
MDA-MB-468
63.09 9. 6
4.12
0.18
6.32 0.92
4.67 0.17
13.48 0.26
HCC1937
69.19 3.33
5.37 1.3
MCF-7
95.50 5.10
9.26 1.19
The MYC ligand, 10058-F4, exhibit IC50s in the range of 60 –
90 μM across the panel of cells used in this study, while our
proliferation
whereas
MDA-MB-231
has
moderate
dependence.
5
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
These results demonstrate the ability of MLAC reagents to
modulate c-Myc transcriptional activity with high potency.
Control compounds, N-Boc Phos and N-Boc Oxime, did not
have any significant effect on Myc-driven transcription (Fig
7C). This implies that the transcriptional activity results
obtained for our MLACs are not just due to the reactivity of the
gold warheads alone. But by a combined effect of the gold
warhead and the MYC-inhibiting 10058-F4 core.
Fig. 7: Regulation of Myc-transcriptional activity by MLAC reagents. (a) Immunoblots showing the levels of MYC after P493-6 cells were treated
with compounds, 1, 2, 10058-F4 or DMSO at the specified concentration, and time points. (b) Immunoblots showing the levels of MYC co-
immunoprecipitated with MAX in P493-6 cells with or without treatment of 1. (c) Cytotoxicity profile of (c) 1 (d) 2 in c-MYC-OFF, and c-Myc-
HIGH P493 cells. e) Comparative cytotoxicity profiles of 1 and 10058-F4 in MDA-MB-231 cell lines. Each data point is presented as mean ±
SD obtained from three independent biological experiments.
For subsequent transcriptional effects imposed by MLAC
reagents 1 and 2, we conducted RNA-sequencing (RNA-seq)
following compound exposure in MDA-MB-231 cells, Fig. 7 (D
and E). In the heat map of differentially expressed genes,
compound 1 downregulates key Myc related genes (Fig. 7E).
The downstream targets of Myc include genes which
modulate cell growth, cell cycle arrest, cell adhesion, cell-cell
communication, and other related biological processes.[44] Of
these, compound 1 effect the downregulation of many direct
c-Myc target genes as presented in the heat map of the
and, cell adhesion and migration processes gene[49] FN1 (fig.
7E).
The MYC oncogene promotes cell cycle phase transitions through
50]
varied mechanisms.[46b,
Per the gene ontology (GO)
enrichment plots (Fig. 7.D), compound 1 perturbs the cell
cycle G1/S phase transition, which is not different from 10058-
F4, and causes cell cycle arrest in the G1/S phase[51] of most
tumor cell lines. Other enriched categories in the GO plots
include mitotic cell cycle phase transition, cell-cycle G1/S
phase transition and DNA-dependent DNA replication.
differentially
expressed
genes
(Fig.
7E).
These
Also, the upregulation of MYC is linked to aneuploidy in
tumors; this it does by reorganizing mitotic gene expression,
which results in mitotic spindle defects[52] and chromosomal
instability in tumor cells used. Because the GO plots shows
enhanced enrichment for biological processes related to
kinetochore, mitosis and chromosome, our compound, 1,
downregulated direct c-Myc genes, effected by compound 1
in P493-6 cells, include cell division control gene[45] CDCA7;
cyclins[46]: CCNE2, CCNA2; transcription factor gene[47] E2F8;
G-protein coupled receptor signaling pathway gene[48] VIPR1;
6
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
directly perturbs these processes. Complex 1 could be
preventing the induction of chromosomal instability via
microtubule nucleation at the centrosome. Hence, these GO
findings corroborate the preceding observation that 1 directly
targets MYC and modulates key Myc genes and related
biological processes.
We are grateful to the University of Kentucky for start-up
funding. The authors acknowledge support of the Center for
Pharmaceutical Research and Innovation (NIH P20
GM130456) and UK Igniting Research Collaboration (IRC).
Notes
The Authors declare no competing financial interest.
Conclusion
Keywords: MYC • gold complexes • affinity • ligand •
In summary, we have developed a gold-based ligand affinity
chemistry to modulate MYC protein activity in cells. Our gold
complexes modulate intracellular MYC. Disrupting protein-
protein interactions such as the MYC-MAX heterodimer
remains a challenge in chemical biology. Thus, innovative
strategies capable of chemical modification of MYC can
overcome this barrier. Our data suggest that the use of gold
warheads can mediate the modification of amino acids, such
as lysine and by tethering these gold warheads to affinity
ligands, desired proteins in their native live environments can
be targeted, as demonstrated with MYC. This chemical
modification strategy induced irreversible inhibition of MYC
protein expression in a time-dependent manner as well as
Myc transcriptional activity. In this regard, our report is among
the first direct covalent inhibitors of MYC based on transition
metal warheads. The reactivity of gold complexes including
those reported in this study with cysteine amino acid residues
poses a challenge to lysine chemo-selectivity. Future studies
to advance this proof-of-concept study will focus on structure
activity relationship studies that limit reactivity of gold-
complexes with cysteine-containing proteins or biological
targets for reduced off-target effect. This study provides
opportunities to target PPIs and expands the development of
metal-based probes/therapeutics by chemical modification.
chemical modification
[1] a) C. D. Spicer, B. G. Davis, Nature Communications 2014, 5,
4740; b) O. Boutureira, G. J. L. Bernardes, Chemical Reviews
2015, 115, 2174-2195.
[2] M. Schenone, V. Dančík, B. K. Wagner, P. A. Clemons, Nature
chemical biology 2013, 9, 232-240.
[3] B. Yu, Z. Qin, G. T. Wijewickrama, P. Edirisinghe, J. L. Bolton,
G. R. J. Thatcher, Bioconjug Chem 2009, 20, 728-741.
[4] a) X. Ji, Z. Pan, B. Yu, L. K. De La Cruz, Y. Zheng, B. Ke, B.
Wang, Chemical Society Reviews 2019, 48, 1077-1094; b) Z.
Shao, W. Liu, H. Tao, F. Liu, R. Zeng, P. A. Champagne, Y.
Cao, K. N. Houk, Y. Liang, Chemical communications
(Cambridge, England) 2018, 54, 14089-14092.
[5] I. Segal, D. Nachmias, A. Konig, A. Alon, E. Arbely, N. Elia,
BMC Biology 2020, 18, 5.
[6] T. Tamura, I. Hamachi, Journal of the American Chemical
Society 2019, 141, 2782-2799.
[7] R. K. V. Lim, Q. Lin, Science China. Chemistry 2010, 53, 61-70.
[8] a) P. Agarwal, J. van der Weijden, E. M. Sletten, D. Rabuka, C.
R. Bertozzi, Proceedings of the National Academy of Sciences
2013, 110, 46; b) J. N. deGruyter, L. R. Malins, P. S. Baran,
Biochemistry 2017, 56, 3863-3873.
[9] K. K. Hallenbeck, D. M. Turner, A. R. Renslo, M. R. Arkin,
Current topics in medicinal chemistry 2017, 17, 4-15.
[10] a) B. Đ. Glišić, U. Rychlewska, M. I. Djuran, Dalton
Transactions 2012, 41, 6887-6901; b) L. Messori, M. A. Cinellu,
A. Merlino, ACS Medicinal Chemistry Letters 2014, 5, 1110-
1113; c) S. M. Hacker, K. M. Backus, M. R. Lazear, S. Forli, B.
E. Correia, B. F. Cravatt, Nature Chemistry 2017, 9, 1181-
1190; d) M. J. Matos, B. L. Oliveira, N. Martínez-Sáez, A.
Guerreiro, P. M. S. D. Cal, J. Bertoldo, M. Maneiro, E. Perkins,
J. Howard, M. J. Deery, J. M. Chalker, F. Corzana, G. Jiménez-
Osés, G. J. L. Bernardes, Journal of the American Chemical
Society 2018, 140, 4004-4017.
[11] L. Cesaro, L. A. Pinna, M. Salvi, Curr Genomics 2015, 16, 128-
138.
[12] S. Jia, D. He, C. J. Chang, Journal of the American Chemical
Society 2019, 141, 7294-7301.
[13] S. Lin, X. Yang, S. Jia, A. M. Weeks, M. Hornsby, P. S. Lee, R.
V. Nichiporuk, A. T. Iavarone, J. A. Wells, F. D. Toste, C. J.
Chang, Science 2017, 355, 597-602.
[14] V. M. Makhnyr, Chemistry of Natural Compounds 1985, 21,
553-566.
[15] S. Sato, M. Matsumura, T. Kadonosono, S. Abe, T. Ueno, H.
Ueda, H. Nakamura, Bioconjug Chem 2020, 31, 1417-1424.
[16] a) E. Meggers, Chemical Communications 2009, 1001-1010; b)
J. Ohata, S. C. Martin, Z. T. Ball, Angewandte Chemie
International Edition 2019, 58, 6176-6199; c) A. Casini, C.
Gabbiani, E. Michelucci, G. Pieraccini, G. Moneti, P. J. Dyson,
L. Messori, JBIC Journal of Biological Inorganic Chemistry
2009, 14, 761-770; d) A. Casini, C. Hartinger, C. Gabbiani, E.
Mini, P. J. Dyson, B. K. Keppler, L. Messori, Journal of
Inorganic Biochemistry 2008, 102, 564-575; e) A. Casini, C.
Gabbiani, G. Mastrobuoni, L. Messori, G. Moneti, G. Pieraccini,
ChemMedChem 2006, 1, 413-417.
Acknowledgements
We are thankful to all, at the University of Kentucky, who
provided support for completion of the experiments detailed in
this manuscript. The UK NMR Center supported by NSF
(CHE-997738). For the flow cytometry experiments we would
like to thank Greg Bauman, Ph.D. (UK Flow Cytometry and
Immune Function core supported by the Office of the Vice
President of Research, the Markey Cancer Center, and NCI
Center Core Support Grant (P30 CA177558). Special thanks
to Dr. Jong Hyun Kim for the Graphite Furnace Atomic
Absorption Spectrometric measurements and analysis. Also,
we are grateful for the use of Dr. Steven Van Lanen’s
laboratory (UK College of Pharmacy) for the use of their LC-
MS. Many thanks to the staff of the Mass Spectrometry facility,
College of Arts and Sciences, University of Colorado Boulder
for running the LC-MS/MS of the complex with the peptide.
Keywords: MYC • gold complexes • affinity • ligand •
chemical modification
[17] D. G. Rivera, G. M. Ojeda-Carralero, L. Reguera, E. V. Van der
Eycken, Chemical Society Reviews 2020, 49, 2039-2059.
Funding Sources
7
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
[18] M. Salmain, N. Fischer-Durand, B. Rudolf, European Journal of
Inorganic Chemistry 2020, 2020, 21-35.
F. Anker, B. Lysy, V. Sagar, Y. Luan, Z. R. Chalmers, K. Unno,
H. Mok, R. Vatapalli, Y. A. Yoo, Y. Rodriguez, I. Kandela, J. B.
Parker, D. Chakravarti, R. K. Mishra, G. E. Schiltz, S. A.
Abdulkadir, Cancer Cell 2019, 36, 483-497.e415; c) J. R.
Whitfield, M.-E. Beaulieu, L. Soucek, Frontiers in cell and
developmental biology 2017, 5, 10-10.
[19] a) A. On-Yee Chan, J. Lui-Lui Tsai, V. Kar-Yan Lo, G.-L. Li, M.-
K. Wong, C.-M. Che, Chemical Communications 2013, 49,
1428-1430; b) K. K.-Y. Kung, H.-M. Ko, J.-F. Cui, H.-C. Chong,
Y.-C. Leung, M.-K. Wong, Chemical Communications 2014, 50,
11899-11902; c) K. Tsubokura, K. K. H. Vong, A. R. Pradipta,
A. Ogura, S. Urano, T. Tahara, S. Nozaki, H. Onoe, Y. Nakao,
R. Sibgatullina, A. Kurbangalieva, Y. Watanabe, K. Tanaka,
Angewandte Chemie International Edition 2017, 56, 3579-3584;
d) M. N. Wenzel, R. Bonsignore, S. R. Thomas, D. Bourissou,
G. Barone, A. Casini, Chemistry – A European Journal 2019,
25, 7628-7634.
[36] N. Krall, F. P. da Cruz, O. Boutureira, G. J. L. Bernardes,
Nature Chemistry 2016, 8, 103-113.
[37] Z. Zhang, M. Tan, Z. Xie, L. Dai, Y. Chen, Y. Zhao, Nat Chem
Biol 2011, 7, 58-63.
[38] a) H. Xu, X. Chen, X. Xu, R. Shi, S. Suo, K. Cheng, Z. Zheng,
M. Wang, L. Wang, Y. Zhao, B. Tian, Y. Hua, Scientific Reports
2016, 6, 30212; b) C. Gaviard, I. Broutin, P. Cosette, E. Dé, T.
Jouenne, J. Hardouin, Journal of Proteome Research 2018, 17,
2449-2459.
[20] A. Schischko, H. Ren, N. Kaplaneris, L. Ackermann,
Angewandte Chemie International Edition 2017, 56, 1576-1580.
[21] E. V. Vinogradova, C. Zhang, A. M. Spokoyny, B. L. Pentelute,
S. L. Buchwald, Nature 2015, 526, 687-691.
[39] G. Ferraro, C. Gabbiani, A. Merlino, Bioconjug Chem 2016, 27,
1584-1587.
[22] J. M. Antos, M. B. Francis, Journal of the American Chemical
Society 2004, 126, 10256-10257.
[40] J. H. Kim, R. T. Mertens, A. Agarwal, S. Parkin, G. Berger, S.
G. Awuah, Dalton Trans 2019, 48, 6273-6282.
[23] K. Hanaya, J. Ohata, M. K. Miller, A. E. Mangubat-Medina, M.
J. Swierczynski, D. C. Yang, R. M. Rosenthal, B. V. Popp, Z. T.
Ball, Chemical Communications 2019, 55, 2841-2844.
[24] A. Gutiérrez, I. Marzo, C. Cativiela, A. Laguna, M. C. Gimeno,
Chemistry – A European Journal 2015, 21, 11088-11095.
[25] a) J. Gavenonis, B. A. Sheneman, T. R. Siegert, M. R.
Eshelman, J. A. Kritzer, Nat Chem Biol 2014, 10, 716-722; b)
A. Cuesta, J. Taunton, Annu Rev Biochem 2019, 88, 365-381;
c) M. Gehringer, S. A. Laufer, Journal of Medicinal Chemistry
2019, 62, 5673-5724.
[26] H.-M. Ko, J.-R. Deng, J.-F. Cui, K. K.-Y. Kung, Y.-C. Leung, M.-
K. Wong, Bioorg Med Chem 2020, 28, 115375.
[27] A. J. Rojas, C. Zhang, E. V. Vinogradova, N. H. Buchwald, J.
Reilly, B. L. Pentelute, S. L. Buchwald, Chemical Science 2017,
8, 4257-4263.
[41] R. Jafari, H. Almqvist, H. Axelsson, M. Ignatushchenko, T.
Lundbäck, P. Nordlund, D. M. Molina, Nature Protocols 2014,
9, 2100-2122.
[42] M. Schuhmacher, F. Kohlhuber, M. Hölzel, C. Kaiser, H.
Burtscher, M. Jarsch, G. W. Bornkamm, G. Laux, A. Polack, U.
H. Weidle, D. Eick, Nucleic acids research 2001, 29, 397-406.
[43] H. Axelsson, H. Almqvist, M. Otrocka, M. Vallin, S. Lundqvist,
P. Hansson, U. Karlsson, T. Lundbäck, B. Seashore-Ludlow,
ACS Chemical Biology 2018, 13, 942-950.
[44] M. Zhang, H.-Y. Fan, S.-C. Li, Biomedicine & Pharmacotherapy
2015, 73, 123-128.
[45] a) M. Eilers, R. N. Eisenman, Genes Dev 2008, 22, 2755-2766;
b) R. M. Gill, T. V. Gabor, A. L. Couzens, M. P. Scheid, Mol
Cell Biol 2013, 33, 498-513.
[46] a) T. Ryl, E. E. Kuchen, E. Bell, C. Shao, A. F. Flórez, G.
Mönke, S. Gogolin, M. Friedrich, F. Lamprecht, F. Westermann,
T. Höfer, Cell Syst 2017, 5, 237-250.e238; b) L. García-
Gutiérrez, M. D. Delgado, J. León, Genes (Basel) 2019, 10,
244.
[47] G. Leone, R. Sears, E. Huang, R. Rempel, F. Nuckolls, C.-H.
Park, P. Giangrande, L. Wu, H. I. Saavedra, S. J. Field, M. A.
Thompson, H. Yang, Y. Fujiwara, M. E. Greenberg, S. Orkin, C.
Smith, J. R. Nevins, Molecular Cell 2001, 8, 105-113.
[48] H. Y. Lee, J. Cha, S. K. Kim, J. H. Park, K. H. Song, P. Kim, M.-
Y. Kim, Molecular Cancer Research 2019, 17, 544.
[49] H. A. Coller, C. Grandori, P. Tamayo, T. Colbert, E. S. Lander,
R. N. Eisenman, T. R. Golub, Proceedings of the National
Academy of Sciences 2000, 97, 3260.
[28] M. S. Messina, J. M. Stauber, M. A. Waddington, A. L.
Rheingold, H. D. Maynard, A. M. Spokoyny, Journal of the
American Chemical Society 2018, 140, 7065-7069.
[29] M. K. Miller, H. Wang, K. Hanaya, O. Zhang, A. Berlaga, Z. T.
Ball, Chemical Science 2020, 11, 10501-10505.
[30] H. G. Lee, G. Lautrette, B. L. Pentelute, S. L. Buchwald,
Angewandte Chemie International Edition 2017, 56, 3177-3181.
[31] a) A. A. Ivanov, F. R. Khuri, H. Fu, Trends Pharmacol Sci 2013,
34, 393-400; b) M. Kalkat, J. De Melo, K. A. Hickman, C.
Lourenco, C. Redel, D. Resetca, A. Tamachi, W. B. Tu, L. Z.
Penn, Genes 2017, 8, 151; c) C. Grandori, S. M. Cowley, L. P.
James, R. N. Eisenman, Annual Review of Cell and
Developmental Biology 2000, 16, 653-699; d) M. Conacci-
Sorrell, L. McFerrin, R. N. Eisenman, Cold Spring Harbor
Perspectives in Medicine 2014, 4; e) P. Dong, M. V. Maddali, J.
K. Srimani, F. Thélot, J. R. Nevins, B. Mathey-Prevot, L. You,
Nature Communications 2014, 5, 4750.
[32] B. Lüscher, L.-G. Larsson, Oncogene 1999, 18, 2955-2966.
[33] E. V. Schmidt, Oncogene 1999, 18, 2988-2996.
[34] a) H. Chen, H. Liu, G. Qing, Signal Transduction and Targeted
Therapy 2018, 3, 5; b) L. A. Carabet, P. S. Rennie, A.
Cherkasov, International journal of molecular sciences 2018,
20, 120.
[50] G. Bretones, M. D. Delgado, J. León, Biochim Biophys Acta
2015, 1849, 506-516.
[51] J. Wang, X. Ma, H. M. Jones, L. L.-Y. Chan, F. Song, W.
Zhang, V. L. Bae-Jump, C. Zhou, J Transl Med 2014, 12, 226-
226.
[52] A. Goga, J. Rohrberg, A. Corella, M. Taileb, S. Kilinc, M. L.
Jokisch, R. Camarda, A. Zhou, S. Balakrishnan, A. N. Chang,
H. Klein-Connolly, Cancer Research 2019, 79, P3-09-01.
[35] a) B. L. Allen-Petersen, R. C. Sears, BioDrugs 2019, 33, 539-
553; b) H. Han, A. D. Jain, M. I. Truica, J. Izquierdo-Ferrer, J.
Entry for the Table of Content
8
This article is protected by copyright. All rights reserved.

10.1002/chem.202004962
Chemistry - A European Journal
RESEARCH ARTICLE
Gold-based warheads capable of protein binding and ligand-
affinity chemistry enable modification of the MYC protein and
inhibition of MYC activity in cells. This is a proof-of-concept study
to modify intracellular protein-protein interaction such as
MYC/MAX using metal-mediated ligand affinity chemistry (MLAC)
based on gold.
9
This article is protected by copyright. All rights reserved.