Full text of DOI:10.1039/d0md00310g

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RESEARCH ARTICLE
Discovery, affinity maturation and multimerization
of small molecule ligands against human
Cite this: DOI: 10.1039/d0md00310g
tyrosinase and tyrosinase-related protein 1†
a
a
ab
cd
Marco Catalano, ‡ Gabriele Bassi,‡ Giulia Rotondi, Lyna Khettabi,
a
a
a
Maria Dichiara, Patrizia Murer, Jörg Scheuermann,
Montserrat Soler-Lopez and Dario Neri*
c
a
Human tyrosinase (hTYR) and tyrosinase-related protein 1 (hTYRP1) are closely-related enzymes involved in
the synthesis of melanin, which are selectively expressed in melanocytes and, in a pathological context, in
melanoma lesions. We used a previously described tyrosinase inhibitor (Thiamidol™) and DNA-encoded
library technology for the discovery of novel hTYR and hTYRP1 ligands, that could be used as vehicles for
melanoma targeting. Performing de novo selections with DNA-encoded libraries, we discovered novel
ligands capable of binding to both hTYR and hTYRP1. More potent ligands were obtained by multimerizing
Thiamidol™ moieties, leading to homotetrameric structures that avidly bound to melanoma cells, as
revealed by flow cytometry. These findings suggest that melanoma lesions may, in the future, be targeted
not only by monoclonal antibody reagents but also by small organic ligands.
Received 10th September 2020,
Accepted 28th October 2020
DOI: 10.1039/d0md00310g
rsc.li/medchem
However, the development of ADC products can be
problematic, due to high costs-of-goods and to off-target
Introduction
9
,10
The majority of conventional cytotoxic agents used for cancer
therapy do not preferentially localize to neoplastic lesions.
toxicity, which can be severe.
It is widely recognized that
1
,2
the therapeutic index of ADCs is typically much higher in
rodent models of cancer compared to the situation in
humans, but the reasons for these discrepancies are still not
This pharmacokinetic limitation contributes to undesired
toxicity and prevents dose escalation to therapeutic regimens,
that would be needed in order to achieve a durable
therapeutic benefit.
One avenue for improving the therapeutic index of
chemotherapeutic agents consists in coupling them to
monoclonal antibodies, serving as selective delivery vehicles.
There has been a substantial interest in the development of
1
1–13
fully understood.
The tumor-homing properties of
anticancer antibodies are often better in mice compared to
patients, which is due (in part) to the fact that highly
vascularized and rapidly growing tumors are typically used in
1
4,15
the experimental setting.
Small organic ligands have been proposed as an
alternative to antibodies for the delivery of cytotoxics to
3
–6
antibody–drug conjugates (ADCs) for the therapy of cancer.
Nine products [Mylotarg™, Adcetris™, Kadcyla™,
1
6–18
tumors.
Small molecule-drug conjugates (SMDCs) can be
Besponsa™, Polivy™, Padcev™, Trodelvy™, Enhertu™,
Blenrep™] have gained marketing authorization for the
treatment of different types of malignancies and various
produced inexpensively and in large quantities. Moreover, a
recent comparative analysis of the two platforms has shown
that SMDCs may accumulate more efficiently and more
homogenously in tumors compared to ADCs (which are
7
,8
other ADCs are currently being tested in clinical trials.
1
9,20
typically confined to the perivascular space),
molecules extravasate more rapidly than antibodies.
since small
2
1,22
a
Department of Chemistry and Applied Biosciences, Swiss Federal Institute of
Interestingly, in a side-by-side comparison, ADCs and SMDCs
exhibited a comparable performance in mouse models of
cancer, even though the tumor uptake of the SMDC products
Technology (ETH Zürich), Vladimir-Prelog-Weg 4, CH-8093 Zürich, Switzerland.
E-mail: dario.neri@pharma.ethz.ch
b
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome,
P.le A. Moro 5, 00185 Rome, Italy
1
9
was clearly superior.
c
Structural Biology Group, European Synchrotron Radiation Facility, 71 Avenue des
Until recently, the development of SMDCs has been
limited by the availability of small organic ligands, specific to
tumor-associated antigens. Folate derivatives, somatostatin
analogues, as well as synthetic ligands to prostate-specific
membrane antigen (PSMA) and carbonic anhydrase IX (CAIX),
Martyrs, 38000 Grenoble, France
d
CNRS, DCM, Université Grenoble Alpes, 38000 Grenoble, France
†
Electronic supplementary information (ESI) available: ESI including material
and methods used in this work. See DOI: 10.1039/d0md00310g
These authors equally contributed to this work.
‡
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ligands specific to hTYR and/or hTYRP1 could be ideally
suited for tumor-targeting applications. TA99 is a monoclonal
antibody specific to hTYRP1, which has been shown to
4
8
selectively target melanoma and melanocytes in vivo. We
assumed that a small organic ligand, specific to hTYR or
hTYRP1, might represent a valid alternative to TA99 for
melanoma targeting applications.
In this article, we explored the possibility to generate
hTYR and hTYRP1 ligands, that could be used for the
selective recognition of melanoma cells. Our work was
inspired by the recent discovery of Thiamidol™ (Beiersdorf
AG) as a tyrosinase inhibitor selective for the human version
of the enzyme, leading to a product which has been approved
49–52
for the treatment of irregular pigmentation of the skin.
We studied whether a chemical modification of Thiamidol™
could be used for the selective delivery of chemical moieties
to melanoma cells. We found that oligomerization of
Thiamidol™ with dendrimeric scaffolds allowed the
generation of novel multivalent ligands, capable of avid
binding to melanoma cells. Moreover, we performed DEL
selections against immobilized hTYRP1 and hTYR, yielding
to novel chemotypes that are selectively enriched against
both enzymes.
Fig.
1 Human tyrosinase (hTYR) and tyrosinase-related protein 1
(
hTYRP1) are melanoma associated antigens that present favorable
Results and discussion
Binding properties of Thiamidol™ derivatives to hTYR and
ligandability properties. a) Tyrosine is converted in eumelanin through
the interaction with metal ions placed within the active site (depicted
in red); b) chromatographic elution profiles of purified intra
melanosomal human hTYR and hTYRP1 proteins. In the insets, SDS-
PAGE gels showing the purity of the eluted fractions in the peak
volume. The first lane contains the MW marker, confirming the
expected size of the proteins (PDB: 5M8P).
hTYRP1
The structure of hTYRP1 has been elucidated by X-ray
5
3
crystallography and has revealed the presence of a catalytic
pocket, which could accommodate certain organic ligands,
bearing structural analogies to tyrosine, the physiological
substrate for melanin biosynthesis [Fig. 1a]. Pure
preparations of recombinant hTYR and hTYRP1 were used in
our studies, as revealed by size-exclusion chromatography
and SDS-PAGE analysis [Fig. 1b].
represent some of the small ligands, which have been shown
to efficiently target tumors both in mouse models of
cancer
2
3–27
28–32
and in patients.
Further developments in the
SMDC field will crucially rely on the availability of improved
discovery technologies for small organic ligands.
We synthesized
a
terminal alkyne derivative of
1
6
Thiamidol™, which could be suitable for Cu-catalyzed azide–
alkyne cycloaddition (CuAAC) coupling to azide-containing
structures [Fig. 2]. The inhibitory potency of Thiamidol™
and Thiamidol™ alkyne was tested against human
tyrosinase, using an enzymatic assay based on the production
DNA-encoded chemical libraries (DELs) are collections of
organic molecules, individually coupled to distinctive DNA
3
3,34
fragments serving as amplifiable identification barcodes.
DELs can be conveniently synthesized by split&pool
techniques, thus allowing the facile creation of libraries
3
5
containing millions of compounds.
Alternatively, the
discovery of small binding peptides has been facilitated by
the display on filamentous phage of chemically-modified
3
6,37
bicyclic peptides
peptides, containing unnatural amino acids.
applications of these technologies for SMDC development
or by ribosome display of cyclic
38
The
3
9–43
have recently been reported.
Human tyrosinase (hTYR) and tyrosinase-related protein 1
hTYRP1) are closely-related enzymes, which are abundantly
(
expressed in melanosomes and on the surface of melanoma
Fig. 2 A known hTYR inhibitor (Thiamidol™) was modified with a
derivatizable handle for later modifications. The new Thiamidol™
alkyne derivative retains the inhibitory capacity of the parent
compound in presence of human hTYR.
44,45
cells.
They are involved in the biosynthesis of melanin
and are detectable in a very limited set of healthy tissues,
4
6,47
such as skin, eyes and hairs.
We hypothesized that
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of L-DOPA and its subsequent conversion into a coloured
use of glutamic acid analogues for the combinatorial display
of two sets of building blocks [Fig. 3a]. The library contained
366′600 encoded compounds. Decoding of the library after
capture on “empty” streptavidin-based supports using high-
5
3,54
adduct.
The experimental IC50 values for the two
compounds were 2.2 μM and 5.6 μM, respectively [Fig. 2].
These values are in keeping with previous measurements,
50
performed on a series of thiazolyl-resorcinol variants.
throughput DNA sequencing [Fig. 3b] revealed
a
homogeneous distribution of compounds, without striking
5
5
enrichments of individual molecules.
By contrast,
Discovery of novel hTYR and hTYRP1 binders from DEL libraries
selections performed on biotinylated hTYR or hTYRP1 on
streptavidin-coated magnetic beads revealed a characteristic
enrichment of “lines” of compounds, which in DEL
In order to perform DEL selections against hTYR and
hTYRP1, the two enzymes were chemically biotinylated and
used in affinity capture procedures on streptavidin-coated
magnetic beads. We screened
described by our laboratory (termed GB-DEL), featuring the
a DEL library recently
5
5
Fig. 3 Discovery of novel binding fragments for hTYR and hTYRP1
through DNA-encoded library (GB-DEL). a) A single-pharmacophore DEL,
based on a racemic glutamic acid scaffold, was screened in presence of
immobilized hTYR and hTYRP1 proteins; b–d) bi- and tri-dimensional plots
of the resulting selections fingerprints after incubation with streptavidin
coated beads, hTYRP1 and hTYR are reported. Selections against hTYR and
hTYRP1 displayed A26/B389 as the most enriched combination of building
blocks; e) the chemical structures of the enriched fragments are reported,
whose binding affinities were evaluated through fluorescence polarization
measurements in presence of hTYRP1 [Fig. S1†].
Fig. 4 ESAC-based affinity maturation of Thiamidol™ ligand. a) The
single-stranded GB-DEL library was paired with a complementary DNA-
fragment displaying a Thiamidol™ derivative and screened in presence of
hTYR and hTYRP1; b and c) decoding plots of the selection experiment
delineate fragments particularly enriched upon incubation with hTYRP1
and hTYR; d) DNA-linked fragments were paired to fluoresceinated LNA-
scaffolds presenting a Thiamidol™ derivative. Binding affinities towards
hTYRP1 were measured through fluorescence polarization.
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technology is typically indicative of preferential binding by
the corresponding building blocks [Fig. 3c and d]. In
particular, we observed an enrichment for building blocks
A19, A26 and B389, whose structures are depicted in Fig. 3e.
They all include tri-alkylated amine moieties in the chemical
structure, resembling chemical features showed by previously
combination of Thiamidol™ with A221/B147 and A221/B443
did not substantially improve binding performance in
fluorescence polarization studies [Fig. 4d].
Targeting melanoma cells with oligomeric derivatives of
Thiamidol™
5
6,57
described melanoma-targeting agents.
The simultaneous
selection of these building blocks on both hTYR and hTYRP1
may reflect the conservation of the active site of the two
enzymes, in spite of the fact that total amino acid sequence
Ligands for transmembrane antigens can be constructed on
multimeric scaffolds to achieve better targeting performance,
by means of avidity. Avid interactions can improve the total
affinity of multimeric ligands, yielding to longer residency
time on cancer cells and therefore better anticancer
5
8
homology was only 40%.
The compounds were
resynthesized as fluorescein conjugates and tested for
binding in fluorescence polarization, exhibiting binding
potency in the micromolar concentration range [ESI† Fig. S1].
Scarce binding affinities were also detected upon enzymatic
assay in the presence of hTYR [ESI† Fig. S2 and S3]. We also
performed DEL selections in encoded self-assembling
chemical (ESAC) library format, pairing the GB-DEL library
6
2,63
outcomes.
We conjugated the alkyne derivative of
Thiamidol™ (Thiamidol™ alkyne) on azide-containing
scaffolds, obtaining monomeric, dimeric and tetrameric
structures [Fig. 5]. The peptidic scaffold included a charged
aminoacidic triad (Asp–Arg–Asp) to improve solubility and to
confine the ligand in the extracellular space. The terminal
cysteine was found particularly convenient, enabling the easy
conjugation of fluorescein-5-maleimide reagent.
with
a
complementary DNA strand, equipped with
a
a
Thiamidol™ moiety [Fig. 4a]. Also in this case,
characteristic enrichment of lines of compounds was
observed, that corresponded to building blocks A221, B147
and B443 [Fig. 4b and c]. Bidentate ligands were produced
We measured the affinity in solution of oligomeric
Thiamidol™ based ligands using a recently described ELISA
assay on hTYR and hTYRP1 coated wells. Both dimeric and
tetrameric ligands significantly improved the affinity towards
hTYR and hTYRP1, in comparison to an analogue monomeric
6
4
and tested on
a fluoresceinated DNA scaffold using
5
9–61
previously described procedures.
Surprisingly, the
Fig. 5 Thiamidol™ alkyne was conjugated to multimeric scaffolds in order to achieve ligands with different binding avidities. The peptidic linker
composed by the triad Asp–Arg–Asp was conjugated to fluorescein and to Thiamidol™ derivative through three scaffolds with different valences
(i.e., monomeric, dimeric and tetrameric). Binding affinities of monomer, dimer and tetramer were evaluated through ELISA assay on hTYR and
hTYRP1 coated microtiter wells.
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interaction remains much weaker compared to the one of the
TA99 antibody. Furthermore, the conjugation of second
moieties to Thiamidol™ resulted in an apparent loss of binding
affinity, which may impair further developments of
Thiamidol™ based conjugates. This work represents a starting
point for the development of higher affinity organic ligands of
hTYR and/or hTYRP1 that can eventually become a novel
targeted treatment against melanoma.
Conclusions
Collectively, our study suggests that Thiamidol™ derivatives (or
more in general hTYR and hTYRP1 binders) may be considered
for pharmacodelivery strategies against melanoma. Similar to
antibodies, multivalence plays a role in strengthening binding
interactions against protein targets, expressed at high density
on tumor cells. We anticipate that SMDCs directed against
hTYR and/or hTYRP1 may be particularly suited for the
treatment of melanoma, as the cognate antigens exhibit an
extremely restricted pattern of expression in normal tissues,
Fig. 6 Binding properties of Thiamidol™ based fluorescein-labelled
ligands were evaluated in presence of B16F10 melanoma cells. Cells
were incubated with a fixed concentration of ligands (1 μM) and the
residual binding was detected by flow-cytometry. An anti-fluorescein
antibody labelled with Cy5-dye was employed to enhance signal
intensity and reduce autofluorescence background.
47,66
mainly confined to few melanocyte cells in the skin.
A
radiolabelled derivative of an anti-melanin antibody has
structure [Fig. 5]. The tetrameric structure yielded to the
strongest binding interaction with both proteins, reaching
single-digit micromolar affinity. However, this affinity is yet
ca. 1000-fold worse than the anti-TYRP1 antibody (TA99),
67,68
previously been proposed for melanoma treatment
but, to
the best of our knowledge, anti-melanoma ADC products have
not yet been developed. Intact antibodies to hTYRP1 may be
66,69
6
5
suitable for prevention of metastatic spread,
but are not
which reacts to both human and murine antigen.
sufficiently potent for the eradication of established melanoma
lesions. The discovery of more potent ligands specific to the
human version of tyrosinase and tyrosinase-related protein 1
will be crucially important for the development of efficacious
We further validated ligands' binding properties in
presence of melanoma cells, expressing hTYR and hTYRP1
proteins (i.e., B16F10). Melanoma cells were incubated with
fluorescein-labelled ligands, washed from non-binding excess
and stained with an anti-fluorescein antibody labelled with
Cy5-fluorophore [Fig. 6]. The fluorescence signal was detected
by flow-cytometry, clearly indicating the tetramer as the best
binding ligand on B16F10 cells compared to the analogue
dimer and monomer.
70
SMDC therapeutics.
Contributions
M. C. and D. N. designed the project and the experiments. M.
S. L. and L. K. provided recombinant human hTYR and
hTYRP1. G. B. synthesized GB-DEL library and performed
DEL-screening. M. C. and J. S. evaluated HTS data. M. C. and
G. B. performed on-DNA synthesis and measurements. M. C.,
G. R. and M. D. synthesized off-DNA compounds. M. C. and
G. R. performed binding and activity assays. M. C., G. R. and
P. M. performed FACS analysis. All authors gave approval to
the final version of the manuscript.
Discussion
The development of high affinity small molecule ligands for the
human version of tyrosinase and tyrosinase-related protein 1
may result to novel targeting agents for the treatment of
melanoma. Here we presented the use of DEL-technology for
the discovery of such ligands, yielding to selective enrichment
of fragments in the presence of both hTYR and hTYRP1.
Additionally, the single-stranded version of a DEL-library (GB
DEL) was employed for the affinity maturation of Thiamidol™,
obtaining also in this case promising fingerprints. Surprisingly,
a significant discrepancy between screening results and binding
affinities was detected, highlighting the necessity of DEL-
selection experiments that better recapitulate the strength of
the molecular interaction with sequence enrichment. We also
investigated the use of multimeric Thiamidol™ derivatives to
stabilize the binding interaction between the inhibitor and its
cognate proteins (hTYR and hTYRP1). Although the tetrametric
structure displayed ca. 100-fold affinity enhancement compared
to the analogue monomeric ligand, yet the strength of the
Conflicts of interest
The authors declare the following competing financial
interest(s): D. N. is
Philochem AG.
a cofounder and shareholder of
Acknowledgements
The authors gratefully acknowledge financial support from
ETH Zürich, the Swiss National Science Foundation (Grant
Nr. 310030B_182003/1) and European Research Council (ERC)
under the European Union's Horizon 2020 research and
innovation program (grant agreement 670603). L. K.
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acknowledges funding support from the IDEX University
Grenoble Alpes project (ANR-15-IDEX-02) under grant
agreement C7H-ID17-P22-COSMRH.
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