Download Functional protein micropatterning for drug design

Review
Functional protein micropatterning
for drug design and discovery
Changjiang You & Jacob Piehler†
†
1.
Introduction
2.
Surface chemistries and surface architectures
3.
Spatially resolved protein
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immobilization
4.
Applications for drug discovery
5.
Conclusions
6.
Expert opinion
Department of Biology, Division of Biophysics, University of Osnabrück, Osnabrück, Germany
Introduction: The past decade has witnessed tremendous progress in surface
micropatterning techniques for generating arrays of various types of biomolecules. Multiplexed protein micropatterning has tremendous potential for
drug discovery providing versatile means for high throughput assays required
for target and lead identification as well as diagnostics and functional
screening for personalized medicine. However, ensuring the functional integrity of proteins on surfaces has remained challenging, in particular in the case
of membrane proteins, the most important class of drug targets. Yet, generic
strategies to control functional organization of proteins into micropatterns
are emerging.
Areas covered: This review includes an overview introducing the most common approaches for surface modification and functional protein immobilization. The authors present the key photo and soft lithography techniques with
respect to compatibility with functional protein micropatterning and multiplexing capabilities. In the second part, the authors present the key applications of protein micropatterning techniques in drug discovery with a focus on
membrane protein interactions and cellular signaling.
Expert opinion: With the growing importance of target discovery as well as
protein-based therapeutics and personalized medicine, the application of
protein arrays can play a fundamental role in drug discovery. Yet, important
technical breakthroughs are still required for broad application of these
approaches, which will include in vitro “copying” of proteins from cDNA
arrays into micropatterns, direct protein capturing from single cells as well
as protein microarrays in living cells.
Keywords: cell patterning, membrane protein, protein array, protein immobilization, protein
interaction, surface micropatterning
Expert Opin. Drug Discov. [Early Online]
1.
Introduction
Modern drug discovery faces immense analytical challenges, which range from
identification and validation of targets and biomarkers, selection and optimization
of lead compounds to increasing diagnostic efforts for toxicological evaluation and
personalized medicine. In the past decade, high-throughput assays (HTA) based on
microarray technologies have dramatically reshaped this procedure in an efficient
and sample-economical manner. A key requirement for HTA is to massively
parallelize and miniaturize binding and activity assays in order to limit sample
consumption. For this purpose, solid-phase-based assays are most suitable as they
combine highly sensitive spectroscopic readout with convenient automated handling of minute sample volumes by microfluidic techniques. For generating microarrays for HTA, samples have to be immobilized on the substrate surface in a
spatially resolved manner and thousands of interactions are probed simultaneously.
A broad range of compound classes have been employed to generate microarrays
(biochips) for HTA in modern drug discovery,[1] including small molecules,
10.1517/17460441.2016.1109625 © 2015 Taylor & Francis. ISSN 1746-0441, e-ISSN 1746-045X
All rights reserved: reproduction in whole or in part not permitted
1
You & Piehler
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Article highlights.
● Microarray-based technology is a cornerstone for highthroughput assays in modern drug discovery. Protein
microarray techniques are gaining importance not only
for target and biomarker identification and validation,
but also for developing protein-based pharmaceuticals.
● The key challenge in protein microarray technology is to
maintain the functional integrity of immobilized proteins, which requires the substrate surface to be engineered protein compatible as well as site-specific protein
conjugation under physiological conditions.
● Multiplexed protein immobilization on biocompatible surface is very important for high-content, high-throughput
screening. For this purpose, a variety of bioorthogonal
conjugation methods and multiplexed micropatterning
techniques have been developed.
● More sophisticated surface architectures are required for
fabricating membrane protein microarrays, since the
functions of membrane proteins are inextricably linked
to their native lipid environment.
● Live-cell protein microarray techniques are emerging,
which exploit the tremendous progress in single-cell
and single-molecule analysis and promises ultrasensitive
drug screening to single cell–single molecule level under
native conditions.
This box summarizes key points contained in the article.
oligonucleotides, carbohydrates, peptides and proteins.[2,3]
Due to the diversity of samples to be immobilized on different substrate materials, multifarious surface chemistries and
microfabrication methods have been developed to ensure the
specificity and efficiency of interaction screening. Thus,
highly multiplexed oligonucleotide and peptide microarrays
are routinely produced with very high quality and
reproducibility.
However, more than 80% of the therapeutic targets are
proteins, which include membrane receptors, ion channels
and enzymes.[4,5] Moreover, therapeutic proteins have tremendously gained importance during the past decade, which
often require intensive further engineering efforts before
being suitable for application.[6] Traditional technologies
for generating microarray are often not compatible with
proteins as their functional integrity is not sustained under
the immobilization conditions. The metastable nature of
three-dimensional protein structures, which is inextricably
linked to their function, as well as the highly diverse physicochemical and functional properties of proteins demand
dedicated solutions for engineering protein-compatible surface architectures. This is particularly challenging for membrane proteins which constitute a major fraction of the most
relevant drug targets,[5] yet often require a specific lipid
environment for maintaining their structural and functional
integrity.[7] In this review, we focus on emerging strategies
for spatially resolved surface functionalization suitable for
2
functional organization of proteins on surfaces. We present
chemical and biochemical aspects of functional protein
immobilization on solid support as well as micropatterning
methodologies for spatial controlled functionalization under
physiological conditions. Recent applications of using micropatterned protein arrays for drug discovery and screening are
highlighted. Given the significant advances of live-cell micropatterning and single-cell analysis,[8,9] recent applications of
micropatterned protein surfaces for mapping signaling pathways and protein–protein interaction in live cells are presented and their potential in drug discovery is discussed.
2.
Surface chemistries and surface architectures
A variety of substrate materials have been used for functional
protein micropatterning, including nitrocellulose, polydimethylsiloxane (PDMS), glass, silicon wafer and gold-coated
silicon/glass. Glass is widely used due to its excellent compatibility with optical detection techniques, including highly
sensitive fluorescence imaging. Glass or silicon supports
coated with gold or other metals are also very commonly
used, as they support functionalization via self-assembled
monolayers (SAMs) as well as the application of label-free
detection by surface plasmon resonance. For sensitive assays,
functional interfacing of proteins with these substrate materials is a major prerequisite. Except for very few robust proteins
such as antibodies,[10] direct interaction of proteins with the
bare substrate mostly results in loss of their functions because
the interactions with the substrate surface may block active
sites or even denature the native protein structure. In order to
minimize such nonspecific interactions, functional protein
immobilization requires coating with organic polymers for
rendering the surface protein repellent (FIGURE 1A). In turn,
site-specific attachment of the protein to the surface is needed
in order to ensure their presentation at the surface in a
functionally homogenous manner (FIGURE 1A). During the
past 15 years, substantial progress in “soft” surface engineering has been made, providing a versatile spectrum of surface
chemistries and surface functionalities suitable for functional
protein immobilization (summarized in TABLE 1). Choosing
an appropriate strategy not only depends on the substrate
material, but also requires careful considerations, which will
be elaborated in this section.
2.1
Protein-compatible surface modification
As introduced above, the “protein compatibility” of a surface
is largely equivalent to its minimized nonspecific interaction
affinity toward proteins, and strategies, therefore, highly
depend on the class of target proteins. Thus, soluble proteins
typically require hydrophilic but largely uncharged surfaces.
In contrast, membrane proteins are preferably integrated into
lipid bilayers as the lipid environment often is critical for
their activities. In general, molecular layers of non-charged,
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Functional protein micropatterning
Figure 1. Surface engineering of micropatterned protein arrays. A. Generic surface architecture of functional protein
micropatterning including a coating for rendering the substrate biocompatible (cyan layer) as well as spatially resolved
functionalization (orange grids) for specific conjugation with biological components. Several commonly used materials for
biocompatible layer are depicted. B–E. Surface modification for biocompatibility layers on different substrates. The reactive
sites for introducing further functionalization are denoted by orange circles. B. Molecular scheme of PEGylated surface for
glass (silica) and other metal oxide support. C. Self-assembled monolayer for ultrathin coatings of noble metal surfaces. D.
Surface coating with functionalized poly-(L)-lysine graft poly(ethylene glycol) (PEG) on a negatively charged surface (material
independent). E. Formation of lipid bilayer on surface provides the crucial lipid environment for keeping the activity of
membrane proteins. F. Site-specific protein capturing on streptavidin-functionalized surfaces. A biotinylated antibody was
used for multiplexed protein immobilization. G. Site-specific protein capturing onto tris-NTA-functionalized surfaces. H. Sitespecific protein capturing based on self-immobilizing protein tags. As an example, the HaloTag/HaloTag® ligand (HTL)
reaction is shown.
hydrophilic polymers such as dextran hydrogels [31] and
dense layers of poly(ethylene glycol) (PEG) [32] have been
proven efficient for shielding surfaces against nonspecific
protein binding (FIGURE 1A). While PEGs with a molecular mass of 2000–5000 g/mol are commonly used in many
cases, highly ordered SAMs formed on gold surface by
alkyl thiolates with short PEG chains of 3–6 repeat units
also efficiently shield the substrate surface.[33] Simple, yet
highly efficient surface coating with PEG layers is achieved
by a layer-by-layer assembly, in which a poly-L-lysine-graftPEG (PLL-PEG) copolymer is directly deposited on negatively charged surfaces.[14] Noncovalent assembly of lipids
and lipid-like molecules into solid-supported lipid bilayers
has been successfully applied for rendering surface biocompatible, which is important for functional reconstitution of
membrane proteins on surfaces [16] (FIGURE 1B–E). A
more sophisticated form of this surface architecture suitable for the integration of transmembrane proteins are
polymer-supported membranes, where lipid bilayers are
tethered above a thin polymer cushion to further minimize
nonspecific interactions with the solid substrate.[18]
2.2
Surface biofunctionalization for protein immobilization
On the basis of protein-repellent surfaces, further functionalization with anchoring moieties is essential for efficient
protein immobilization. Amine-reactive coupling via aldehydes or activated carboxyl groups have been used for covalent tethering proteins to surfaces through lysine residues
presented on protein surfaces. However, these approaches
yield heterogeneously orientated attachment, which likely
results in heterogeneous functionality due to blocking of
relevant residues of protein.[34] Therefore, site-specific techniques for tethering proteins to surface are desired. Several
chemical reactions such as Staudinger ligation, Diels–Alder
reaction, azide-alkyne click reaction and thiol-ene reaction
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You & Piehler
Table 1. Surface functionalization techniques for functional protein immobilization.
Surface chemistry
Coating of
biocompatible layer
Self-assembled monolayer Au–thiol interaction
PEG brush
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Poly lysine-graft-PEG
Solid support lipid bilayer
Polymer-supported,
tethered lipid bilayer
*Surface functionality
Biotin or streptavidin
Disadvantages
Ref.
Very efficient and stable surface
coating; ultrathin layer, good
coupling to detection (e.g., LSPR)
Specific for applications on gold
surface;
Synthesis of complex
compounds required
Little control on composition;
relatively undefined surface
architecture
Stability is dependent on pH
and ionic strength of mediums
Fragile coating, very sensitive to
exposure to air
[11]
Covalent coupling
Robust coating for versatile
biofunctionalization; bottom-up
synthesis from simple compounds
Noncovalent, multiple
One-step coating for obtaining
charge–charge interaction biocompatible surface
Noncovalent self-assembly One-step coating for obtaining
on bare substrate
biocompatible surface used for
membrane proteins; self-healing
Noncovalent self-assembly Functional reconstitution of
on a polymer cushion
transmembrane proteins is possible;
lipid phase separation can be
controlled
Enzymatic (suicide)
substrates
Protein attachment
Affinity capturing via
biotin–streptavidin
interaction
Affinity capturing via Histag and Ni–NTA
interaction
Affinity capturing by
enzymatic reaction
GFP-trap (anti-GFP
nanobody)
Affinity capturing via GFP
fusion
Ni-NTA
Advantages
Advantages
Very rapid, specific and strong
protein attachment; site-specific
binding is possible
Small and very common affinity tag;
reversibility for repetitive write–erase
processes
Site-specific, covalent protein
attachment, feasible for fabricating
multiplexed arrays
Very rapid, specific and relatively
stable; simple visualization/
quantification
[12,13]
[14,15]
[16,17]
Surface modification is rather
error-prone; membrane
assembly is not spontaneous
[18–
20]
Disadvantages
Proteins have to be biotinylated;
blocking of binding sites by
endogenous biotin
Reversible binding sensitive to
nucleophiles, chelators, bivalent
metal ions, reducing agents
Relatively slow, inefficient
reactions; specialized tags
Ref.
[21,22]
Fusion of a relatively large tag
required
[23,24]
[25–
28]
[29]
*The list summarizes the most common surface functionalities for protein attachment. Other specific, bioorthogonal methods for chemical coupling of proteins can be
found in [30]. Further specialized approaches are given in the main text.
GFP: Green fluorescence protein; LSPR: Localized surface plasmon resonance; NTA: Nitrilotriacetic acid; PEG: poly(ethylene glycol).
have been exploited for site-specific protein immobilization.
[30] These reactions, however, require not only couplingspecific anchoring groups on surface but also modifications
of the protein, for example, by incorporation of non-natural
amino acids,[35] which is not yet routinely used in most
laboratories. Moreover, these chemical reactions are relatively
slow, thus requiring high protein concentrations, which in
turn increase the background. For these reasons, quasi-irreversible noncovalent interactions mediated via genetically
encoded tags are currently favored as they allow highly efficient and specific protein capturing to surfaces. A large
proportion of noncovalent protein immobilization
approaches relies on either the very rapid and extremely
high-affinity interaction between biotin and streptavidin (or
the related proteins), or the complexation of immobilized
transition metal ions by oligohistidine-tagged proteins.[21]
In the first case, the surface is functionalized with streptavidin or related proteins, and the protein of interest modified
with biotin by means of chemical or biochemical biotinylation are readily immobilized to the surface (FIGURE 1F). For
4
site-specific immobilization of oligohistidine-(His)-tagged
proteins, functional moieties of iminodiacetic acid or nitrilotriacetic acid (NTA) are coupled to surfaces for immobilizing
transition metal ions such as Zn(II), Cu(II), Ni(II) and Co
(II).[36] More stable and stoichiometrically defined tethering
of histidine-tagged protein was achieved by using multivalent
chelators with two to four NTA moieties grafted onto different scaffolds,[24] with tris-NTA exactly matching the stoichiometry of the frequently applied hexahistidine-tag (FIGURE
1G). A broad spectrum of surface architectures functionalized
with tris-NTA have been successfully implemented, yielding
quasi-irreversible, site-specific protein immobilization.[24]
These stable complexes, however, can be rapidly dissociated
by competitors of imidazole at millimolar concentration,
providing means for rapidly switching this interaction
under mild conditions.
Driven by the increasing demand for site-specific proteinlabeling techniques, protein immobilization via enzymatic
reactions has gained much attention. In these methods,
small-molecule ligands are covalently immobilized onto the
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Functional protein micropatterning
surface. Enzymatic reactions successfully exploited for sitespecific protein immobilization include phosphopantetheinyl
transfer,[25] transpeptidation [26] as well as enzymatic suicide reactions [27,37] (FIGURE 1H). For the assembly of
protein arrays, highly multiplexed protein immobilization is
required, and therefore orthogonal coupling approaches are
desired. For this purpose, antibodies against the proteins of
interest have been most frequently applied. For instance,
monoclonal antibodies modified with biotin were chosen
for site-specifically anchoring a variety of target proteins on
the surface.[38] However, antibody binding to proteins often
blocks active sites and thus considerably bias functional
assays. In other attempts, the huge multiplexing capability
of oligonucleotide pairing has been exploited. Single-stranded
DNA with different oligonucleotide sequences were coupled
to the surface in a spatially resolved manner and proteins
modified with the complementary strands were captured to
the surface via sequence-specific DNA hybridization, thus
enabling in situ assembly of protein arrays.[39]
3.
Spatially resolved protein immobilization
The repertoire of techniques for spatial organization of proteins on surfaces in micrometer and nanometer dimensions is
rapidly expanding in line with the rapidly growing application of protein arrays. Micro- and nanopatterned surface
modification can be achieved by self-assembly as exploited
for block copolymer micelle nanolithography.[40] While
such bottom-up techniques are powerful for reaching nanometer resolution, the ability to control and vary geometries
is rather limited. Therefore, top-down approaches are
desired, which can be achieved either by local deposition of
materials on the surface (soft lithography techniques) or by
photochemical manipulation (photolithography techniques).
Most frequently used techniques are briefly presented here as
comprehensive in-depth background has been reviewed
recently.[21,41]
3.1
Local deposition by soft lithographic techniques
Piezoelectric dispensing by exploiting ink-jet technology has
been a very straightforward approach to the assembly of
spatially resolved surface modification, which has been
exploited for generating the first-generation protein arrays.
[2] While this technology is low-cost, robust and mature,
features below 10 µm can only be obtained with relatively
high technological efforts.[42] In the late 1990s, the concept
of soft lithography for spatially resolved deposition of (bio)
molecule onto surfaces with submicrometer resolution was
introduced by George M Whitesides and coworkers to cover
a set of microfabrication methods using molds, stamps or
channels made by elastomeric materials.[43] Soft lithography
is highly versatile as very different compounds can be directly
transferred onto surfaces. The most commonly used elastomeric material is PDMS. The currently most broadly applied
soft lithographic technique is microcontact printing (µCP),
where the patterns of a master PDMS stamp coated with ink
are transferred to the substrate surface through face-to-face
contact (FIGURE 2A). By using stamps with different featured
sizes, patterning resolution down to tens of nanometers has
been obtained by µCP [43] (FIGURE 2B). µCP has been very
broadly applied for patterning proteins and other biomolecules in very different formats.[44,45] Applications include
direct deposition of proteins, for example, antibodies or
streptavidin onto reactive or adhesive surfaces. However,
this approach is not well compatible with most proteins as
the printing process is prone to protein denaturation.
Therefore, µCP of functional groups for site-specific protein
capturing is preferred, which has been extensively applied to
alkylthiolates on gold surfaces to form micropatterned SAM.
[43] Likewise, µCP of biotin to an activated PEG polymer
brush on glass surface has been implemented to prepare
protein microarrays.[46] Approaches for Ni-NTA-based protein patterning by µCP of reactive alkyl thiols followed by
chemical coupling of NTA [23], as well as direct µCP or by
backfilling of NTA derivatives have been described.[11]
Traditional µCP is designed to generate binary micropatterns. Substantially higher multiplexing has been achieved by
using microscopic or nanoscopic stamps (“pens”) for locally
depositing compounds (“inks”). This has been initially
implemented by dip-pen nanolithography (DPN) using the
tip of an atomic force microscope (AFM) as a pen to directly
create patterns with inks on the substrate [48] (FIGURE 2C).
With the options of using different proteins as inks, this
method offers a great flexibility for making multiplexed
protein arrays with length scale down to 50 nm on gold
surface. Since different types of tips and substrates are implemented lately, it is also named as scanning probe lithography.
DPN had been used for protein nanopatterning, for example,
depositing antibodies on hydrophilic surface by DPN has
been successfully demonstrated [51] as well as specific capturing of His-tagged proteins into nanopatterns.[49] By the
combination of block copolymer micelle nanolithography
and DPN, single-molecule protein arrays were obtained.
[52] Several further techniques based on nano-manipulation
via the AFM tip have been devised, including native protein
nanolithography.[53]
However, preparation of protein nanoarrays by this
method is slow since writing proteins with nanometer profiles has to be carried out one by one. To overcome this
limitation, functional immobilization of proteins on PEGmodified SAM surfaces by parallel DPN was developed,
which helps improving the throughput.[54] More recently,
Si-based nanostencil was introduced to make protein nanoarrays as “nanostencil lithography.”[55] This method can be
deemed as a variation of parallel DPN. Instead of using tips
to dip the ink, a nanostencil with ink reservoir was applied
on the surface for allowing protein inks being patterned on
substrate through apertures. Multiplexed DPN of lipid
bilayer was realized on glass substrates with subcellular
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Figure 2. Examples of soft lithography and dip-pen nanolithography (DPN) for preparing protein microarrys. A. Scheme of
microcontact printing. Polydimethylsiloxane (PDMS) stamp soaked with poly-(L)-lysine-PEG-HaloTag® ligand (PLL-PEG-HTL)
contacts a plasma-cleaned glass substrate (i), thus transfer the PLL-PEG-HTL patterning to the surface (ii). Backfilling with PLLPEG-OMe blocks the uncoated regions (iii). The obtained micropatterned surface is used for affinity-capturing HaloTag-fused
proteins (iv). B. Scheme of nanocontact printing. A planar PDMS stamp is incubated with a protein solution, blow-dried (i),
and quickly brought into contact with a photopolymer (Norland Optical Adhesive 63) replica that was plasma activated, and
separated to lift-off proteins in the contact areas (ii). The flat PDMS stamp is then printed onto a glass slide (iii) and
separated, producing protein nanopatterns on the slide (iv). (Adapted with permission from John Wiley and Sons.[47]) C.
Surface nanopatterning by DPN. Top: principle of surface deposition of chemical “ink” adsorbed onto an atomic force
microscope tip. (Reprinted with permission from the American Association for the Advancement of Science.[48]) Bottom:
functional protein nanoarray fabricated by DPN and protein capturing. (Reprinted with permission from the American
Chemical Society.[49]) D. Multiplexed protein immobilization by polymer pen lithography (PPL). Top: scheme depicting the
deposition of different biomolecules adsorbed onto a polymer pen array. Bottom: multicolor micropatterns generated by PPL
with fluorescent lipids. (Reprinted with permission from John Wiley and Sons.[50])
dimensions (down to 200 nm), which was functionalized
with proteins for T-cell activation.[56] An even more rigorous development into parallel deposition has been achieved
by polymer pen lithography (PPL).[57] Rather than AFM
tips, this technique employs elastomeric polymer molds comprising an array of pyramid-shaped “pens” (FIGURE 2D).
After inking these pens with the desired compounds, these
are transferred onto surfaces by means of a piezoelectric
system, which allows control of lateral position as well as
the size of deposited features via the axial force. Thus, huge
throughput of the fabrication process was achieved with
150,000 features per second for sub-100-nm structures.[57]
In combination of multiplexed inking of polymer pen arrays,
highly multiplexed deposition of relatively complex features
has been demonstrated.[50,58]
3.2
Photolithographic techniques
Photolitographic protein micropatterning is typically based
on a photosensitive molecular layer directly grafted on a
biocompatible surface coating. Typical components are
6
nitroveratryl group, azide, tetrazine, C=C bond containing
compounds and maleimide. Photolithographic modification
by means of illumination through a photomask or spatially
controlled focused light is employed for generating micropatterns. Due to the diffraction limitation of light, conventional photolithography obtains patterns down to a length
scale in the range of hundreds of nanometers. By exploiting
the concept of stimulated emission depletion, resolution
beyond the diffraction limit down to ~40 nm was achieved,
[59] which has been used for fabricating protein nanoarrays.[60]
The simplest approaches for photolithographic micropatterning are based on photodestruction of functional groups
on the surface. This can be done by directly destroying
capturing groups such as NTA, which can be photodegraded
by a light-induced Fenton reaction.[61] Alternatively, chemical groups for coupling of functional groups can be eliminated by light. Thus, photolithographic patterning of
maleimide-modified surface has been introduced [13],
which are used for surface functionalization via thiols
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Functional protein micropatterning
(FIGURE 3A). A strikingly high signal to background was
achieved, which facilitated single-molecule studies in microarrays requiring high contrast (FIGURE 3B). Maleimide patterning also obtained very homogenous patterning of
polymer-supported membranes that allowed assessing diffusion and interaction of reconstituted transmembrane proteins
on the single molecule level without biasing their diffusion
and interaction properties as resolved by single molecule
imaging [62] (FIGURE 3C, D).
Photolithographic patterning based on azide functionalization
has gained considerable attention nowadays as the azide is a small,
photosensitive moiety and widely used in azide-alkyne click reaction. Extracellular matrix (ECM) proteins containing RGD or
REDV peptides as well as the unnatural amino acid azidophenylalanine (pN3-Phe) were spreaded on glass coverslides and crosslinked by UV-illumination through a photomask. By washing
with 6 M guanidine hydrochloride, the protein was selectively
removed in the non-illuminated regions, yielding guided
cell adhesion these micropatterned surfaces.[63] Direct
photopatterning of biomolecules via azide groups, that is, positive
photolithography, were successfully demonstrated.[64] With the
ability to site-specifically introduce amino acids with clickable
side chains into recombinant proteins, exciting applications of
these approaches for protein patterning can be envisaged.
More versatile and sophisticated surface modification can be
achieved by
photocaging of functional
groups.
o-Nitroveratryloxycarbonyl (NVOC) is a very common photosensitive moiety applied for caging amine groups on the surface [65] (FIGURE 3E). A general photopatterning strategy for
binary surface functionalization using NVOC contains two
steps: uncaging the NVOC-protected amino groups with a
photomask under UV irradiation, followed by chemical or
biochemical functionalization of the amine groups in the
uncaged areas for capturing proteins (FIGURE 3F).[66] This
approach has been successfully applied for site-specific noncovalent and covalent protein immobilization [25,66] (FIGURE
3G). Likewise, photolithographic writing of SNAP-tag fusion
proteins was achieved using caged benzylguanine.[28]
Figure 3. Micropatterned surface functionalization by photolithography. A–D. Surface micropatterning based on maleimide
photodestruction. Spatially resolved surface functionalization was used for single-molecule protein-binding assays. (A, B:
Adapted with permission from the American Chemical Society.[13] C, D: Adapted with permission from John Wiley and Sons.
[62]) E–G. Binary surface patterning based on caging of surface amines. E. The structure of the photoprotection group onitroveratryloxycarbonyl (NVOC). F. Scheme of photolithographic patterning of lipid anchors on PEGylated surface. Top to
bottom: UV light decages NVOC-protected amine groups via a photomask, followed by coupling of functional groups type A.
A second UV exposure without mask decages the remaining NOVCs for sequential coupling of functional groups type B. G.
Binding experiment with binary micropatterned surface comprising biotin and tris-NTA functionalities. Reversible protein–
protein interaction with a soluble receptor domain is demonstrated by fluorescence imaging. (Adapted with permission from
the American Chemical Society.[25])
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While these photolithographic techniques are powerful for
spatially organizing one or two different proteins on surfaces,
higher multiplexing requires techniques for directly targeting
proteins into microscopic structures. Photolithographic
“writing” of proteins has been achieved by different
approaches. Nitroveratryl groups have also been applied for
photocaging biomolecules. By local uncaging of surface NiNTA complexed with photocleavable oligohistidine peptides,
in situ micropatterning of His-tagged proteins on surfaces by
laser lithography was achieved with diffraction-limited resolution.[67] Likewise, caged glutathione was employed for
micropatterning of proteins fused to glutathione
S-transferase,[68] a common protein used for affinity purification. These techniques very elegantly allow multiplexed
writing and erasing of proteins on surfaces, yet suffer from
instabilities and relatively high background due to the noncovalent nature of the interactions. More robust laser lithography is achieved by photocatalysed formation of covalent
bonds. Thus, protein micropatterning by means of photoinitiated thiol-ene click reactions has been implemented.[69]
Another click reaction based on tetrazine-norbornene chemistry was recently reported for forming cell-laden hydrogels.
Duplexed protein patterning in this “click gel” was realized
by taking advantage of orthogonal thiol-ene photoreaction
and Diels–Alder reaction of tetrazine-norbornene.[12]
Related to photolithographic techniques, also electrons
provide versatile means for spatially resolved chemical manipulation. Electron beams (e-beams) can be focused to spot
sizes of a few nanometers, making e-beam lithography a
powerful way for fabricating nanoarrays. By e-beam-controlled reduction and cross-linking of aromatic SAMs, nanopatterned amino groups on gold surfaces were obtained for
further biofunctionalization and nanopatterning of biomolecules at the submicron scales.[70] Multiplexed protein patterning with nanometer scale by e-beam lithography was
achieved by depositing functionalized PEG dendrimers,
yielding a line width of ~100 nm.[71] Alternatively, electron-beam-induced deposition has been used for depositing
carbon nanostructures onto a PEG polymer brush for subsequent adsorption of proteins with very high contrast.[72] It
should be noted, however, that application of these methods
is limited to conductive substrates.
4.
Applications for drug discovery
4.1
Profiling protein–protein interaction networks
Early applications of large-scale protein arrays have been
focused on protein interaction analyses, which have had
substantial impact in proteomic research for drug discovery
and, in particular, for personalized medicine.[73] Compared
to affinity-capturing techniques coupled with mass spectrometry (AC/MS) and two-hybrid technologies, the advantage
of protein microarrays is that they contain nearly all proteins
coded by whole genome, and they can be assayed
8
simultaneously in vitro. The first proteome chip contained
94% (~5800 of 6200) of the yeast proteins with N-terminal
GST-His-tag tandem affinity purification tag.[34] Since
then, this technology has become an important tool in several
fields of drug discovery [74] and protein microarrays of
kinases, phosphatases and proteases have become available.
[75] High-throughput global identification of protein kinase
substrates and protein kinase–phosphatase interaction networks have emerged.[76,77] Such global network mapping
is very important for clinical diagnosis because the anomaly
regulation of protein kinase activity is strongly related to
diseases such as cancer, and the inhibitor or activators for
these interactions are important drug candidates.
Fluorescence detection techniques are currently the preferred
readout methods as they provide unrivaled signal-to-background ratios with a sensitivity down to the level of single
molecules.[78] However, recent development of ultrasensitive label-free detection by LSPR offers interesting options,
[15,79] as often the labeling is a critical and time-consuming
step in assay development.
A key shortcoming of state-of-the-art protein array technology is the necessity to produce and purify proteins prior
to deposition on the surface, a very demanding and timeconsuming process. Owing to the fragile folding, storage of
proteins and protein arrays often leads to loss of function
and therefore in situ assembly of protein arrays is highly
desired. For this reason, current developments in the field
aim for closer linking protein production and assembly into
arrays, for example, by local in vitro translation on micropatterned support [80,81] or by in situ protein capturing
from cells.[29] For plasma membrane receptors, which are
key drug targets, yet are often very difficult to isolate, livecell protein capturing has been implemented [22] (also see
Section 4.3). Recently, this technology has been adapted to
allow multiplexed protein interaction analysis inside a living
cell.[82] To this end, an antibody array fabricated by DPN
was used to micropattern cytosolic proteins through an
engineered transmembrane linker, allowing for monitoring
activation of protein kinase A upon G-protein-coupled
receptor (GPCR) stimulation.
4.2
Screening GPCR ligand binding
GPCRs represent the largest family of therapeutic targets.[5]
These receptors are the targets of more than 50% of the
current pharmaceutical agents on the market, with a global
market reaching ~100 billion USD in 2013.[83] Over a long
time, GPCR drug discovery by high-throughput screening
(HTS) for hit identification has remained the major focus of
drug discovery worldwide.[84] Commonly used HTS assays
for GPCR ligand binding and functional assays include the
key detection points in GPCR signaling pathway, such as
detection of ligand binding, GTPγS binding, cAMP, IP3/IP1,
Ca2+ flux, β-arrestin recruitment, receptor internalization and
receptor dimerization.[85]
Expert Opin. Drug Discov. (2015) 11(1)
Downloaded by [Universität Osnabrueck], [Jacob Piehler] at 02:57 05 December 2015
Functional protein micropatterning
Over the past decade, versatile techniques for functional
micropatterning of membrane proteins have been established,
which facilitate in vitro HTS of GPCR ligand binding.
[16,86] GPCR arrays of the β1-adrenergic receptor, the
neurotensin receptor (NTR1) and the dopamine receptor
based on microspotted lipid membrane have been successfully fabricated. Specific ligand binding to these GPCR
microarrays was confirmed and IC50 value of neurotensin
to NTR1 down to 2 nM was measured by competitive ligand
chasing.[87] In the following work reported by Pfizer Inc.
(New York, NY, USA) and Corning Inc. (New York, NY,
USA), multiplexed GPCR microarrays of α-adrenergic receptors were developed for ligand-binding assays based on 125Ilabeled agonist clonidine. A 200- to 400-fold decreased sample
preparation was sufficient for analysis, which is very useful for
HTS.[88] Another type of GPCR microarray was achieved by
embedding membrane proteins into liposomes prepared from
Sf9 cellular membrane extracts. Site-directed immobilization
of the liposomes on protein repelling surface was achieved by
hybridization of cholesterol-modified oligonucleotides between
the liposomes and the surfaces. The functionalities of GPCR
models in liposomes, for example, H1-histamine receptor and
the M2-muscarinic receptor, were confirmed by ligand-binding assays and G-protein signaling assays under a microarray
reader format.[19,20] This liposome-based strategy yielded
impressive performance and can be very efficient for HTS of
potential GPCR ligands since the proof of concept of singleliposome arrays has been established by using µCP.[89] To
further simplify the sample preparation of GPCR microarray
and effectively screen GPCR drugs in native cellular environments, live-cell microarrays in the format of well-plate, micropatterned surface, and microfluidic systems have been
developed.[90]
4.3
Probing signal transduction in live cells
Large-scale monitoring of signal transduction pathways
stands as the largest challenge in drug discovery, as almost
all known diseases exhibit dysfunctional aspects in signal
transduction networks,[91] and therefore the vast majority
of drug target are part of these.[5] Many signaling pathways
have been reasonably mapped at molecular level, uncovering
a highly complex network of subtle and interdependent cross
talk. The integration of this complexity into cellular decision
making is still largely unclear. Therefore, the overall potency
of a drug has to be verified under physiological conditions.
Monitoring signaling pathways and screening the perturbations of drugs to these pathways with multiplexed assays thus
has unique potential to bridge the gap between molecularlevel assays and the animal tests.
4.3.1
Cell adhesion and integrin signal transduction
The interaction and migration of cells within tissues play
a key role in stem cell differentiation and organogenesis,
but also in tumor development and metastasis. Promising
new drug targets such as the Wnt/β-catenin pathway
involved in controlling these processes are currently emerging.[92] Micro- and nanopatterned surface functionalization offers unique means to mimic and study these
extremely complex processes in vitro and thus to identify
key molecular and cellular determinants and their potential manipulation by drugs.[93] Cell adhesion and apoptosis on micropatterned RGD peptides [94] and ECM
proteins [95] have been examined to elucidate the
mechanism of integrin signal transduction, which have
shown determinant functions for regulating cell survival,
differentiation and growth. By using copolymer micelle
nanolithography, induced cell polarization and migration
were examined in detail by spacing adhesive ligands in a
gradient in nanoscale [94] (FIGURE 4A). Interestingly,
these experiments revealed that inducing cell adhesion
on ordered RGD nanopattern requires the RGD peptide
being spaced below 70 nm. This critical distance confirms that the formation of stable focal adhesion and
actin fiber networks have a distance threshold of
70 nm. Hierarchical RGD micro-/nanopattern designed
using a similar strategy was recently used for investigation
of stem cell differentiation.[96] Strikingly, growth of
mammalian cells on micropatterned surface showed preserved left–right patterning, a phenomenon termed as
“chirality”.[97] Chirality has also been detected for
other single-cell types and multicellular structures,
which highlights the possibility of chiral morphogenesis
during stem cell differentiation.
4.3.2
Receptor tyrosine kinase signal transduction
Receptor tyrosine kinases (RTKs) are major regulatory
hubs controlling cell growth and differentiation, and therefore play a prominent role in tumor development. For this
reason, several RTKs have become key drug targets.[100]
Closer studies revealed an enormous complexity of RTK
activation,[101] demanding new tools for testing the molecular mechanisms responsible for inhibition. Live-cell
micropatterning (cf. Section 4.1) has opened unique avenues to study RTK signaling in situ. For this purpose, cells
are cultured on micropatterned monoclonal antibodies
against the target receptor, which will be captured and
thus spatially rearranged within the plasma membrane.
[22] Thus, interactions with other receptor subunits and/
or cytosolic effector proteins can be readily monitored.
[22,98,102] By using tag-specific surface capturing rather
than antibodies, generic and robust surface micropatterning for probing assembly and signaling of RTK and related
receptors in living cells was recently implemented.[98]
These surfaces allow control of cell attachment via RGD
peptides and assembly of signaling complexes via receptors
fused to the HaloTag (FIGURE 4B, C). Thus, protein–
protein interactions involved in receptor dimerization and
recruitment of cytosolic effector proteins could be quantified.[98] By using micropatterned lipid bilayers, cell–cell
Expert Opin. Drug Discov. (2015) 11(1)
9
Downloaded by [Universität Osnabrueck], [Jacob Piehler] at 02:57 05 December 2015
You & Piehler
Figure 4. Live-cell micropatterning for mapping signaling pathways. A. Scheme of probing cell polarization and migration
on micropatterns prepared by copolymer micelle nanolithography. The scanning electron microscopy image shows an
ultrasmall cellular protrusion. Inset highlights the cRGDfK-functionalized gold nanoparticles which are adhesion patches
for single integrin receptors.[94] (Reprinted with permission from the American Chemical Society.[94]) B, C. Micropatterning
of signaling complexes in living cells. B. Scheme depicting a binary RGD- and HTL-micropatterned surface used for probing
type I interferon signaling in live cells. Cells are attached to the RGD patterns via focal adhesion. Interferon receptor subunit
IFNAR2 fused with HaloTag was captured into the HTL-functionalized regions. C. Fluorescence images depicting processes
involved in the assembly of active signaling complexes in micropatterns: ligand-induced receptor dimerization on the plasma
membrane (left image); interaction with the cytosolic Janus kinases (center) and recruitment of cytosolic effector proteins.
(Adapted with permission from The Rockefeller University Press.[98]) D. Reorganization of ligand-stimulated EphA2 by
micropatterned ephrin-A1–functionalized supported membranes. Bright field images (left) and epifluorescence images of
labeled EphA2 (right). Scale bar: 5 µm. (Reprinted with permission from the American Association for the Advancement of
Science.[99]) E. Single-cell pull down for probing the kinetics and the stoichiometry of protein complexes. (Adapted with
permission from the American Chemical Society.[29])
interactions can be mimicked to identify the determinants
of cellular recognition processes. Thus, immune synapse
formation on surfaces was achieved on micropatterned
surface which mimics the antigen-presenting cells and
signaling pathways involved in T-cell activation were
probed.[17] The spatial control of EphA2 RTK signal
activation during cell–cell contact formation was quantitatively explored by a similar approach [99], revealing a
striking correlation between spatial receptor organization
and invasion potential of different mammary epithelial cell
lines (FIGURE 4D). Further exciting possibilities are opened
by combination of these approaches with spatial control of
cell adhesion using RGD nanopatterns.[103]
10
5.
Conclusions
In the past decade, spatially controlled protein immobilization has become a versatile and reliable technology with
numerous existing and potential applications in drug discovery. Key challenges for combining biocompatible surface
architectures and bioorthogonal coupling reactions with
functional micropatterning have been resolved. To this
achievement, development of affinity tags and bioconjugation techniques for site-specific protein modification has
been instrumental. Thus, maximized protein functionality
has been achieved in artificial environments for reliably probing the interactions between proteins and protein substrates.
Expert Opin. Drug Discov. (2015) 11(1)
Functional protein micropatterning
Downloaded by [Universität Osnabrueck], [Jacob Piehler] at 02:57 05 December 2015
The sensitivity of surface-based detection techniques down to
the single-molecule level by both fluorescence-based and
label-free imaging techniques nowadays allows enormous
miniaturization and parallelization for quantitative binding
and activity assays. With these tools, the scope of drug
discovery is profoundly expanded to reach whole proteomes.
Yet, manufacturing of high-density multiplexed protein
arrays remains challenging because of the limited stability of
proteins. Moreover, membrane proteins as a key class of drug
targets are typically not well covered by whole proteome
analysis techniques as they require dedicated approaches
toward functional immobilization.
6.
Expert opinion
During the past decade, drug discovery has become a much
more demanding and multifaceted process. Key challenges
include more efficient target identification, development and
testing macromolecular drugs as well as coping with the
complex demands of personalized medicine. Robust and
user-friendly protein-based assay technologies providing
high level of multiplexing and flexible design will play a
key role in addressing these challenges. Conventional protein
arrays are, meanwhile, commercially available from various
suppliers. These are typically designed for proteome-wide
screens with the aim to cover a large proportion of a given
proteome. For drug discovery, however, more complex, dedicated and adaptable assay formats will be requested for functional analysis of a subset of interesting protein candidates.
Implementation of such assays will profit from several major
biotechnological innovations, which are currently emerging.
One major current limitation in multiplexed protein assay
development results from the limited availability of specific
binders with well-characterized protein recognition properties.
While in the past monoclonal antibodies has been the solution
of choice, alternative approaches for generating high-affinity
binders are now available, which have the advantage of being
much more readily accessible. Next to recombinant antibody
fragments, which are directly obtained by in vitro selection,
simpler scaffolds such as nanobodies or DARPins have been
designed,[104] which can be readily produced by recombinant
protein expression or by in vitro translation. As a fully synthetic alternative, aptamer technology has the potential to play
an important part in this field.[105] However, considerable
and concerted efforts will be required to provide a large and
validated repertoire of recombinant or synthetic binders to the
scientific community.[106] As a second important technology
for site-specific protein attachment, incorporation of non-natural amino acids into recombinant protein is coming of age,
being available for different cell types and providing a large
variety of possible modifications. After further optimization of
the efficiency and selectivity of coupling reactions, this tool
will be the key for generic site-specific immobilization and
labeling of proteins on very small scale.
Functional reconstitution and micropatterning of membrane proteins – the most important class of drug targets –
on surfaces has been achieved by membrane architectures such
as vesicles and solid-supported membranes, as well as nanodisk
technology.[107] These approaches depend on isolation of
membrane proteins by detergent solubilization, which requires
time-consuming identification of a suitable detergent for a
specific membrane protein. As the functions of membrane
proteins are inextricably linked to lipid environment [7],
appropriate selection of lipids for functional reconstitution is
required. Currently, techniques for more direct transfer of
membrane proteins to surfaces are emerging, which do not
require the extraction from their lipid environment. Promising
strategies include budding of proteoliposomes from plasma
membranes [89] as well as extraction of membrane proteins
within their lipid environment by amphipathic polymers (lipodisq).[108] Successful extraction of GPCRs along with endogenous lipids in lipodisqs was recently demonstrated in a
detergent-free manner,[109] highlighting the potentials for a
wide application in drug discovery. As a promising alternative
strategy, cell-free production of membrane proteins by in vitro
translation opens exciting possibility to in situ reconstitute
GPCR into suitable membrane architectures.[110]
In general, more closely interfacing protein production and
array fabrication will be an important trend in this field. Next
to generating protein arrays by local in vitro translation of
cDNA libraries (cf. Section 4.1), strategies for capturing proteins directly from cells offer promising opportunities. In
particular for diagnostic screening required for personalized
drug selection, assays need to be performed from minute
sample quantities, ideally from a single cell. Single-cell analysis,
moreover, opens new avenues to deconvolute the cellular
heterogeneity.[111] Cell-dependent variations of stem cell signaling and differentiation responses were examined by large
scale (~2000 cells) single-cell western blotting (scWestern)
using an open-microwell array.[112] This approach has
already been applied to demonstrate the possibility for quantifying cell-to-cell variance to pharmaceutical stimuli.[113]
Alternative strategies aim for performing protein assays directly
on the cell culture substrate. In case of plasma membrane
protein such as receptors and transporters, capturing to surfaces within living cells opens exciting possibilities (cf. Section
4.3.2). In-cell micropatterning of soluble proteins is possible
[82], yet requires genetic manipulation. For exploiting the
growing availability of high-affinity binders and the very
high sensitivity of surface-sensitive detection, single-cell pulldown assays are highly promising, which have been recently
demonstrated for recombinant proteins [29] (FIGURE 4E). By
taking advantage of efficient cell capturing to surface in combination with local high-density arrays of binders in close
proximity to captured cell, this approach allows efficient isolation of proteins from primary cells. Thus, a broad application of ultrahigh-sensitive HTS in the format of single cell–
single molecule can be envisioned, which ultimately will fuel
drug development for personalized medicine.
Expert Opin. Drug Discov. (2015) 11(1)
11
You & Piehler
Financial & Competing Interests Disclosure
Part of this work was funded by the Deutsche
Forschungsgemeinschaft (DFG). J Piehler is the co-inventor
of tris-NTA patent EP1778651 entitled “Multivalent chelators for modifying and organizing of target molecules”
(WO2005EP08124/DE20041038134/US20040614950P).
The authors have no other relevant affiliations or financial
involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or
materials discussed in the manuscript apart from those
disclosed.
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Affiliation
Changjiang You & Jacob Piehler†
†
Author for correspondence
Department of Biology, Division of Biophysics,
University of Osnabrück, Osnabrück 49076,
Germany
E-mail: [email protected]
15