COMMUNICATION
Activity-based high-throughput profiling of metalloprotease inhibitors
using small molecule microarrays{
Jun Wang,a Mahesh Uttamchandani,b Li Ping Suna and Shao Q. Yao*abc
Received (in Cambridge, MA, USA) 27th October 2005, Accepted 1st December 2005
First published as an Advance Article on the web 4th January 2006
DOI: 10.1039/b515278j
specificity) against metalloproteases, we find the approach to be
well-suited for high-throughput discovery of potential MMP
inhibitors. Previously, SMM had only been used successfully for
activity-based profiling of substrate specificity, rather than
inhibition, of proteases.3 Our present work thus adds ‘‘inhibitor
fingerprinting’’ to the expanding repertoire of SMM applications.
Our SMM strategy works by precoating a glass slide with a
fluorogenic enzyme substrate (bodipy FL casein in our case4),
followed by programmed spotting of mixtures of the target enzyme
and an inhibitor, in individual nanodroplets, to predefined
locations on the surface. Upon incubation and detection by
fluorescence, relative potency of all spotted inhibitors is immedi-
ately revealed and simultaneously compared (Scheme 1). This
strategy thus takes advantage of the parallel and miniaturized
aspect of microarray, together with quantitative fluorescence
readouts attainable from an enzyme-sensitive surface in response
to a protease/inhibitor mixture (in a dose-dependent manner).
Tagging of the enzyme with a fluorophore is not necessary, thus
allowing it to be evaluated in its native form and in real time.
Diamond et al. recently developed an SMM system by printing
chemical libraries in glycerol droplets followed by introduction of
an enzyme onto the slide with aerosol spray.5 The strategy,
however, has limited applications due to the need of glycerol
droplets (which inhibit enzymatic reactions and prevent long-term
slide storage) and complicated aerosol set-ups. Our approach needs
only a conventional arrayer for spotting and standard bioconjuga-
tion chemistry for surface derivatization, making it adaptable by
most researchers for high-throughput inhibitor screening.
We herein describe a high-throughput small molecule micro-
array (SMM) method that enables quick and cost-effective
identification of potent inhibitors of metalloproteases in an
activity-dependent manner, thereby offering a rapid means for
inhibitor discovery and profiling.
Microarray-based technologies have received much attention due
to their enormous potential in high-throughput screening.1 Of the
various platforms available, small molecule microarray (SMM)
emerges as an important tool for rapid screening of large chemical
libraries.2 This method generally involves immobilization of
chemicals in addressable grids on a glass slide, followed by
screening with a fluorescently tagged protein. The critical
limitation of SMM lies not so much in hit identification as in hit
validation. This is because most SMM screening methods rely on
non-covalent ligand–protein interactions, which invariably intro-
duce false positives as a result of inconsequential affinity between
the ligand and non-targeted regions of the protein. Without time-
consuming validation, it remains unconfirmed whether any of the
initial ‘‘hits’’ detected are relevant to the desired biological context.
Enzymes play a key role in virtually every biological process.
They have long been considered valuable drug targets for potential
treatments of major human diseases. Matrix metalloproteases
(MMPs) for example, are a family of zinc-containing proteases
which have been implicated in diseases such as arthritis,
Alzheimer’s disease and cancer. There is thus considerable interest
in developing highly potent and selective inhibitors that target
different human MMPs.
We aim to develop an SMM technology suitable for high-
throughput identification of potential inhibitors on the basis of
their ability to directly inhibit the catalytic activity of an enzyme,
thus doing away with tedious hit validation processes. Herein, we
explore one such platform which enables simultaneous evaluation/
comparison of hundreds (or thousands) of small molecule
inhibitors against an enzyme in an activity-based manner
(Scheme 1). By demonstrating its utility in profiling potent
hydroxamate-containing inhibitors (for both their potency and
Thermolysin and collagenase, two well-characterized metallo-
proteases, were chosen in our study, as they exhibit similarity to
many vertebrate metallopeptidases, in particular to those of the
MMP family.6 Earlier reports showed that both enzymes, like
most MMPs, show substrate specificity at P9 sites, with a strong
preference of hydrophobic residues at their P19 position.6b Little is
known, however, about their specificities at P29 and P39 positions.6c
We therefore synthesized a 400-member small molecule library
with
the
scaffold
HONH-Suc(2-iBu)–P29–P39–Gly–Gly–
Lys(biotin)–CONH2, as shown in Fig. 1. Each inhibitor in the
library comprises a succinic hydroxamate ‘‘warhead’’ (a highly
potent zinc-binding group against metalloproteases), in which the
P19 residue was maintained as an isobutyl group throughout. The
design was based on the structures of Marimastat, Batimastat, and
GM6001, three broad-spectrum potent hydroxamate inhibitors of
MMPs (See Supporting Information{). With variations across P29
and P39 positions in the library, we aimed to profile both the
potency and selectivity of individual members against different
metalloproteases, in particular MMPs. A flexible linker and biotin
aDepartment of Chemistry, National University of Singapore, 3 Science
Drive 3, 117543, Singapore. E-mail: chmyaosq@nus.edu.sg;
Fax: (+65) 67791691; Tel: (+65) 68741683
bDepartment of Biological Sciences, National University of Singapore,
117543, Singapore
cNUS MedChem Program of the Office of Life Sciences, National
University of Singapore, 117543, Singapore
{ Electronic supplementary information (ESI) available: Procedures for
the synthesis and characterization of inhibitors, microarray and microplate
experiments, as well as kinetic experiments and computer modeling. See
DOI: 10.1039/b515278j
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 717–719 | 717