Lawn assay for compounds that affect enzyme activity or bind to target molecules

A lawn assay is described for determining compounds that affect enzyme activity or that bind to target molecules. Compounds to be screened are cleaved, and diffused from solid supports into a colloidal matrix. Enzymatic catalysis or binding to target molecules by the compounds is carried out in the matrix. Active compounds are found by monitoring a photometrically detectable change in a substrate, coenzyme, or cofactor involved in the enzymatic reaction, or in a labeled ligand bound to the target molecule, that produces a zone of activity associated with the compounds.

FIELD OF THE INVENTION 
This invention relates to assays that screen many compounds for their 
effect on enzyme activity or their ability to bind to target molecules. 
BACKGROUND OF THE INVENTION 
To find lead compounds for drug discovery programs, large numbers of 
compounds are often screened for their activity as enzyme inhibitors or 
receptor agonists/antagonists. Large libraries of compounds are needed for 
such screening. As a result of developments in this field, it is now 
possible to simultaneously produce combinatorial libraries containing 
hundreds of thousands of small molecules for screening. With the 
availability such libraries, however, has come a need for large scale, 
rapid screening methods. 
The use of microtiter plates has significantly improved screening of 
combinatorial libraries. The plates allow assays to be miniaturized and 
have provoked the development of automated liquid handling and detection 
instruments to improve throughput and reproducibility. Robotics have 
allowed integration of compound arraying, microtiter plate handing, liquid 
handling, data acquisition and data processing. Even when facilitated by 
robotics, however, the steps of weighing, distributing, dissolving and 
serially diluting compounds in microtiter plates are time consuming. 
The availability of combinatorial libraries on polymer beads have 
simplified these processes. By synthesizing sufficient compound on each 
bead for a few assays, compound handling is reduced or eliminated. 
Furthermore, thousands of beads can be arrayed in a given assay, and the 
compounds on the beads detached by photolysis, or other cleavage methods, 
into the assay in the concentration desired. Assaying library beads in 
standard microtiter plates therefore results in substantial improvement 
over the conventional assaying of bulk synthesized compounds. 
The advantages of the bead format, however, have not been fully realized. 
In particular, scanning thousands of microtiter plate wells for positive 
results can be time consuming. Also, when beads are assayed in a 
microtiter well, only low concentrations of compounds are obtained since 
each bead contains only a small amount of compound. There is a need, 
therefore, for a more efficient method of screening large combinatorial 
libraries on beads, or other solid supports. There is also a need for a 
method which can assay higher concentrations of compounds on beads. 
WO94/02515 describes screening a library of beads immobilized on a 
substrate, such as agarose. Molecules are partially cleaved from the beads 
and diffuse through the substrate, where they contact receptors on the 
surface of melanocytes. This causes pigment aggregation or dispersion 
within the cells, providing a signal that indicates where molecules have 
bound. WO94/02515 also describes determining immunological binding by 
diffusing labeled molecules in a gel, and contacting the molecules with 
antibodies on a plate below the gel, or in a second gel. Unbound molecules 
must be washed away, and the label detected. WO 94/02515 does not describe 
any method for detecting molecules that affect the activity of enzymes. It 
also does not describe any method for detecting binding to proteins 
without washing steps, which are particularly difficult to perform when 
using gels. In addition, it does not describe any method for screening 
compounds for binding to receptors that is not dependent on a biological 
response within a cell. 
SUMMARY OF THE INVENTION 
The present invention relates to a lawn assay for identifying compounds 
that affect enzymatic catalysis, or that bind to target molecules. The 
assay involves the steps of: 
a. providing an enzyme or a target molecule; 
b. where an enzyme is provided, further providing a substrate for the 
enzyme, and where a target molecule is provided further providing a 
labeled ligand bound to said target molecule; 
c. providing a plurality of solid supports, each of the supports having 
multiple copies attached thereto by a cleavable linker of a compound to be 
screened for its effect on enzyme catalysis or its ability to bind to the 
target molecule; 
d. contacting the solid supports with a colloidal matrix and cleaving the 
compounds from the supports, either before or after the contacting step, 
so that the compounds diffuse into the colloidal matrix; 
e. carrying out the enzymatic catalysis or binding of compounds to target 
molecule in the colloidal matrix; and 
f. monitoring a photometrically detectable change in: 
(1) the substrate, or a coenzyme or enzyme cofactor involved in the 
enzymatic reaction, or 
(2) in the labeled ligand to determine a zone of activity in the matrix 
associated with one or more of the compounds.

DETAILED DESCRIPTION OF THE INVENTION 
All patent applications, publications, or other references that are listed 
herein are hereby incorporated by reference. 
In one embodiment of this invention, a library of solid supports, 
preferably beads, is screened for the ability of compounds on the supports 
to affect the activity of an enzyme. Using the invention, supports 
containing the active compounds are quickly and easily located merely by 
viewing zones of inhibition in a matrix. In this embodiment, the solid 
supports are contacted with a colloidal matrix, such as agarose. The 
compounds are linked to the supports by a cleavable linker and released, 
e.g., by exposure to light. As they slowly diffuse out of the solid 
supports, zones of high concentration of the compounds are created in the 
supports' immediate vicinity. The compounds contact enzyme contained in 
the matrix. Substrate is contacted with the matrix and reacts with the 
enzyme. Conversion of substrate to product is measured by monitoring a 
photometric change in the substrate, or in a coenzyme or cofactor involved 
in reaction. For example, the substrate can be fluorogenic, i.e., becoming 
fluorescent when converted to product. In this case, compounds that are 
active inhibitors of the enzyme reaction are detected as dark zones of 
inhibition. The less active, or inactive, compounds are contained in the 
lighter areas. 
Using this assay, positive results from an assay of a combinatorial library 
can be detected very quickly. Furthermore, compound activity can be 
quantitated by e.g., comparing the sizes of zones of activity. Once zones 
of activity have been determined, the relevant supports at the center of 
the zones can be located and the active compounds on those supports 
identified. The invention thus allows large libraries of compounds to be 
quickly and easily screened. Very little effort is required to array the 
solid supports or assay the compounds released from the supports. 
In another embodiment, the lawn assay is used to determine compounds that 
bind to a target molecule, and thereby affect a detectable signal 
generated by a labeled ligand bound to the target molecule. This assay 
allows screening of compounds that, e.g., act as agonists or antagonists 
of a receptor, or that disrupt a protein:protein interaction. It also 
allows detection of binding to DNA, RNA, or complex carbohydrates. For 
example, neurokinin receptor binds to a 7-nitrobenz-2-oxa-1,3-diazol-4-yl 
(NBD)-labeled peptide ligand. The labeled ligand has the following 
formula: PhCO-2,4-diaminobutyric 
acid(gamma-NBD)ala-D-trp-phe-D-pro-pro-Nle-NH2. NBD is a fluorophore, and 
binding of the labeled ligand to the neurokinin receptor increases NBD's 
fluorescence. When a compound displaces the NBD-labeled ligand from the 
neurokinin receptor, fluorescence of the NBD fluorophore is reduced (G. 
Turcatti, H. Vogel, A. Chollet (1995) Biochemistry 34, 3972-3980). A 
library of solid supports can be screened for compounds that bind to 
neurokinin receptor in a colloidal matrix using the method of the 
invention. Active compounds are found in zones of decreased fluorescence. 
As another example, a radioligand (tritium or .sup.125 Iodine-labeled) can 
be used to screen for compounds binding to a receptor with the assay of 
invention by using Scintillation Proximity Assay beads (SPA.TM., Amersham 
Corp.) or scintillant coated plates (Flashplates.TM., Dupont NEN Research 
Products). Receptor is bound to SPA.TM. beads or to a Flashplate.TM. 
surface and radiolabeled ligand in a colloidal matrix is allowed to 
interact with the receptor. This interaction brings the radiolabel in 
close proximity with the scintillant and results in a scintillation 
signal. The signal can be detected using x-ray film, or other commercially 
available film that is specifically designed to detect tritium dependent 
scintillations. Compounds released into the matrix from the solid supports 
that bind to receptor and displace the radioligand reduce the 
scintillation signal, i.e., result in a zone of reduced scintillation. The 
receptor used in the assay can be e.g., membrane-bound, tethered to a 
solid phase, or solubilized. 
When using the assay to find compounds that affect enzyme activity, it is 
preferred that the substrate or the product of the enzymatic reaction 
generate a detectable signal. The difference in signal between the 
substrate and product should be significant. It is particularly preferred 
to use a substrate which generates little or no signal, and which converts 
to a product which generates a strong signal. If the substrate produces 
detectable signal which cannot be distinguished from that of the product, 
it can create background noise, thereby reducing the overall sensitivity 
of the assay. For this reason, non-fluorescent substrates that convert to 
fluorescent products, i.e., fluorogenic substrates, are preferred. One 
well known fluorogenic substrate is fluorescein diacetate, which converts 
to fluorescein in the presence of an esterase, such as carbonic anhydrase. 
Other fluorogenic substrates include 7-amino-trifluoromethyl coumarin 
(AFC), 4-trifluoromethylumbelliferyl (HFC), 7-amino-4-methylcoumarin (AMC) 
and 4-methoxy-2-naphthylamine (MNA). 
Alternately, a fluorescent substrate can be used that converts to a product 
having different excitation and emission characteristics. By using 
band-pass filters so that only the product is excited and detected, the 
substrate can be effectively screened out. An example of such a 
fluorescent substrate is peptidyl-aminomethylcoumarin, which is converted 
by an appropriate protease, such as thrombin, to free aminomethylcoumarin. 
The free aminomethylcoumarin excites and emits at different wavelengths 
than does the peptidyl-aminomethylcoumarin (S. Kawabata et al. (1988) Eur. 
J. Biochem. 172, 17). 
It is also possible to use a substrate containing internally quenched 
fluorophores that become fluorescent when converted to product. Such 
quenching reactions are well known (E. Matayushi et al. Science 247, 954). 
For example, a peptide substrate can be produced having two fluorophores 
at opposite ends, one absorbing the fluorescence of the other. The 
substrate therefore emits a negligible amount of light. Upon cleavage of 
the peptide by a suitable protease, the absorbing fluorophore is released 
and no longer quenches the other fluorophore, resulting in an increase in 
fluorescence. One such substrate is 4(dimethylaminophenylazo)-benzoic acid 
(DABCYL)-Gabu-glu-arg-met-phe-leu-ser-phe-pro-5-(2-aminoethyl)amino!napth 
alene-1 sulfonic acid (EDANS), which when cleaved by an aspartyl protease 
(e.g., plasmepsin 11 of Plasmodium falciparum) becomes fluorescent. In 
screening a library of aspartyl protease inhibitors using the assay of the 
invention, those that are active inhibit cleavage of the substrate, 
allowing quenching to be maintained. Active compounds are found in dark 
zones of inhibition. 
Fluorescence can be detected, e.g. using a field format fluorescence 
detection instrument, such as FluorImager.TM. from Molecular Dynamics. 
This type of fluorimeter is capable of determining fluorescence over a 
large area. It is also possible to detect fluorescence using a CCD camera 
and to transfer the image data to a computer. The image can be generated 
by illumination of the fluorophore with light of the wavelength that 
specifically excites it. Detection can be optimized by using a bandpass 
filter between the camera and the assay that is specific for the emission 
wavelength of the fluorophore. 
Assays that measure a change in fluorescence are preferred as they are 
believed to result in the greatest sensitivity. Any method, however, can 
be used that measures a change in signal from one of the compounds 
involved in the reaction as a result of conversion of the substrate to 
product, or displacement of the labeled ligand from the target. An example 
of an assay for compounds that affect a chromogenic substrate, 
p-nitrophenylphosphate, is described in the examples. It is also possible, 
for example, to measure a change in absorbance. For example, NADP is a 
common cofactor in many enzymatic reactions. Absorbance changes as NADPH 
is converted to NADP by, for example, neutrophil NADPH oxidase (such as 
during an oxidative burst associated with an immune response). This change 
can be monitored to determine zones of inhibition for compounds that 
inhibit this and other enzymes that use NADP, NADPH, NAD, and NADH as 
co-factors. The sensitivity of assays of the invention that measure a 
change in absorbance is believed to generally be lower than those that 
measure a change in fluorescence. 
Other examples of detectable changes resulting from conversion of substrate 
to product include chemiluminescent changes and scintillation changes. 
Scintillation changes can be detected as described above for receptor 
binding with the exception that a substrate is attached to the scintillant 
(i.e., to the bead or plate containing scintillant). For example, a 
radioactive reagent, such as tritiated farnesyl pyrophosphate, can be 
added to the substrate by an enzyme such as farnesyl protein transferase. 
Transferase inhibitors prevent addition of the tritiated farnesyl 
pyrophosphate to the substrate, resulting in a reduction in detectable 
scintillations; i.e., transferase inhibitors are found in zones of reduced 
scintillation. In an alternative assay, removal of the radioactive portion 
of a substrate attached to the scintillant, such as by cleaving with a 
protease, releases the radiolabeled portion (i.e., moves it away from the 
scintillant). In such an assay, protease inhibitors cause an increase in 
scintillation, i.e., are found in zones of increased scintillation. As 
noted above, the scintillation signal can be detected using x-ray film, or 
film that is specifically designed to detect tritium dependent 
scintillations. 
In the embodiment of the invention for assaying binding to a target 
molecule, a labeled ligand provides a signal that indicates such binding. 
The label is preferably a fluorescent moiety that alters its signal as a 
result of target molecule binding. Examples of such fluorescent moieties 
are NBD and 5-(dimethylamino)-1-naphthalenesulfonyl (Dansyl) chloride. 
Colloidal matrixes that are useful in the invention include silica gel, 
agar, agarose, pectin, polyacrylamide, gelatin, starch, gellan gum, 
cross-linked dextrans (such as Sephadex.TM.) and any other matrix that 
allows diffusion of compound from the solid supports in a limited region. 
Low melting-temperature agarose is preferred, generally in an amount of 
0.5-2.0%, wt./vol. The colloidal matrix can be chosen to obtain a desired 
rate of diffusion. It is generally preferred to use a matrix that allows a 
high concentration of compounds to be easily obtained. 
In carrying out the invention to determine compounds that affect enzyme 
activity, the solid supports are preferably embedded in a matrix 
containing the relevant enzyme. Following cleavage, compound diffuses from 
the support into the matrix and contacts the enzyme. Substrate is then 
added and, as it diffuses into the colloidal matrix, active compounds 
inhibit conversion to product. By following such a procedure, compounds to 
be screened are allowed to interact with enzyme before the enzyme contacts 
substrate. This is believed to be advantageous because it allows compounds 
the best opportunity to inhibit the enzyme, and thus results in the 
clearest zone of inhibition. 
It is also possible, however, to embed the solid supports in a matrix that 
contains dispersed substrate. Following cleavage, the matrix can be 
contacted with enzyme. This procedure is not believed to be as sensitive 
since the compounds may not efficiently bind to the enzyme. 
To practice the invention, however, it is not necessary that the solid 
supports be embedded in the matrix. They can also be applied to the 
matrix's surface and the compounds allowed to diffuse into the matrix. 
This can be done, for example, by arraying the solid supports on the 
surface of a stretched sheet of plastic film (e.g., Parafilm.TM.), and 
then applying the sheet to the surface of the matrix. 
In assaying for compounds that affect enzyme activity, it may be desirable 
to use two colloidal matrixes. For example, one matrix can contain enzyme 
and beads and the other can contain substrate. Contacting the surfaces of 
the matrixes to each other allows the substrate to come into contact with 
the enzyme. However, any suitable method may be used to contact the 
substrate, enzyme, and compounds in the colloidal matrix. For example, it 
is possible to add a solution of substrate over the surface of a matrix 
containing enzyme and embedded supports. Adding solution is preferred 
when, e.g., the substrate interferes with detection. Solution containing 
the substrate can be removed prior to determining the zones of activity. 
When using the assay of the invention to screen for binding to a target 
molecule, there is generally no need for more than one matrix. A matrix 
contains the target molecule bound to the labeled ligand which emits a 
detectable signal indicating binding to the target molecule. Compounds 
from the solid supports are diffused into the matrix, preferably from 
embedded supports using photolysis. Alternatively, however, labeled ligand 
can be diffused into the matrix from a second matrix (or liquid layer) 
after release of the compounds in the matrix. This allows the compounds to 
contact the receptor before interaction with the labeled ligand, which can 
be advantageous. 
Compounds can be cleaved from the solid supports either before or after the 
supports are contacted with the colloidal matrix. For example, solid 
supports may contain acid cleavable linkers, as further described below. 
These linkers can be cleaved in a gaseous acidic atmosphere before placing 
the supports on the matrix. The compounds, although cleaved, remain on the 
surface of the supports and diffuse into the matrix when the supports are 
placed on it. It is even possible to cleave the compounds prior to pouring 
low-melt liquid agarose over the solid supports. While some of the 
compounds will be washed away, sufficient compound can remain on the 
support's surface to result in a recognizable zone of activity. 
Where the compounds are cleaved after the beads are embedded in the 
colloidal matrix, it is preferred to use photolysis, e.g., cleaving by 
exposure to UV light. By adjusting light exposure, it is possible to 
control the amount of compound that diffuses into the matrix. If more 
light is applied, by increasing intensity or duration, more cleavage 
results, in turn releasing more compound into the matrix. This allows the 
amount of active compound released to be adjusted, so that zones of 
activity are only produced for compounds that are most active. The amount 
of compound released can also be optimized to produce zones that are most 
distinct. 
Any suitable solid support can be employed in the method of the invention. 
Such supports include beads, pellets, disks, fibers, and gels. They also 
include particles such as cellulose beads, controlled pore-glass beads, 
silica gels, and polystyrene beads (optionally cross-linked with 
divinylbenzene and optionally grafted with polyethylene glycol and 
optionally functionalized with amino, hydroxy, carboxy, or halo groups). 
Additional supports include grafted co-poly beads, poly-acrylamide beads, 
latex beads, dimethylacrylamide beads (optionally cross-linked with 
N,N'-bis-acryloyl ethylene diamine), glass particles coated with 
hydrophobic polymers, etc., (i.e., having a rigid or semi-rigid surface) 
and soluble supports such as low molecular weight non-cross-linked 
polystyrene. Divinylbenzene-crosslinked, polyethyleneglycol-grafted 
polystyrene type beads are preferred, such as TentaGel S-NH.sub.2 .RTM. 
beads (Rapp Polymere, Tubingen, Germany). 
The solid supports can be in a random arrangement, or in an ordered one. 
Preparing a random arrangement of solid supports requires little effort. 
For example, a library of beads can be suspended in a solvent, such as 
ethanol, and deposited on the bottom of a petri plate. After the solvent 
has completely evaporated, a layer of agarose containing the relevant 
enzyme or target molecule can be poured over the beads. On the other hand, 
an ordered array can be used to space beads apart and allow easier 
identification of those that are active. In one example of an ordered 
array, beads are arrayed on a rigid template, such as a thin glass disk 
having tapered holes. The tapered holes are sized to allow only single 
beads to settle into them. Beads are suspended in a solvent, such as 
ethanol, and washed over the top of the template to fill each hole with 
one bead. The beads can then be cleaved in the dry state, and the template 
set down on the colloidal matrix. Capillary action wets the beads, 
facilitating diffusion of the cleaved compounds into the matrix. Zones of 
activity can be observed immediately below beads containing active 
compounds. It is possible to remove the template prior to detecting zones 
of activity if an image of the template on the matrix is made. This image 
can later be used to correlate the zones of inhibition in the matrix with 
the positions of beads on the template. 
Ordered arrays also may be useful in identifying the compounds on supports 
that are associated with zones of activity. Specifically, the array can be 
ordered so that the position of the solid support on the array corresponds 
to the identity of the compound. Thus, once an assay has been carried out, 
and the position on the array determined for a support carrying an active 
compound, the identity of that compound can be easily determined. 
Preferably, however, the identity of active compounds is determined using 
the encoding system described in WO 94/08051 and in parent applications 
Ser. Nos. 08/436,120 now abandoned and 08/239,302 now abandoned. In this 
system, chemical tags encoding the identity of the compounds are applied 
to the solid supports. The identity of the compound on a given support can 
be determined by detaching the chemical tags from the support, identifying 
the tags by, e.g., gas chromatography, and correlating the pattern of tags 
with the identity of the compound. Once a zone of activity is found, the 
bead at the center of the zone can be extracted, and the identity of the 
compound on the bead decoded by this method. 
The assay is preferably carried out so that there is slow diffusion of the 
compound from the solid support following cleavage. This results in a high 
concentration of compound in the vicinity of the bead. Thus very little 
compound is required to cause a distinct zone of activity. Most of the 
compound remains on the support for any subsequent assays that are 
required. Such further assays may be needed if more than one solid support 
is found in the zone of activity. It may then be necessary to retest the 
supports from the zone to determine which releases the active compound. 
Reassaying may be required as a matter of course if many thousands of 
beads are screened at high density. Reassaying may also be desirable to 
test for selectivity, i.e. to determine which active compounds are 
inactive in a second assay that tests for a different property. 
With combinatorial libraries containing thousands of related compounds, 
many compounds may be found that have some degree of activity. It 
therefore may be to useful to use the assay of the invention to 
distinguish the most potent compounds. In the assay, if the amount of 
compound released from each support is approximately the same, potent 
compounds have a detectable effect further from the bead than weak 
compounds do, at any given time. Thus, the more active compounds create a 
larger zone of activity. Furthermore, the zone of activity of the most 
active compounds lasts longer. Thus, it is possible to quantitate the 
activity of the compound eluted from the solid support by the size of the 
zone of activity, as well as by the duration of the zone following 
cleavage. 
Reducing photolysis time reduces the amount of compound released from the 
support. As the concentration of the compounds is lowered, those that are 
less active become more difficult to detect. As a result, the number of 
active compounds drops. In experiments described in the Examples, 
compounds that were detectable at the shortest elution times, i.e., that 
were most potent, were also identified as most potent using conventional 
solution-phase screening. The activity of the inhibitors was found to 
correlate with the size and duration of the zone of activity: the most 
potent compounds produced the largest zones for the longest time, for any 
given amount of photolysis. 
When assaying a library containing many active compounds, it may be 
desirable to screen using a low density of solid supports, i.e., a low 
number of supports per cm.sup.3 of matrix. While requiring more assays to 
screen the entire library, it is less likely that supports will have to be 
retested to determine which contains the active compound. The present 
inventors believe that screening a large library containing many active 
members at a low density is often more efficient than screening at high 
density, since rescreening supports is time consuming. The optimum density 
for screening can be determined for a given library by comparing the 
throughput in the initial assay with the effort required to retest active 
supports. Other factors which affect optimum screening density include the 
cost of the target and the size of the library. 
When several large libraries are available for testing, it may be 
advantageous to incompletely evaluate each library by "scouting" each at 
high density for active compounds. Screening at high density allows one to 
statistically evaluate the number and potency of active compounds in each 
library. Libraries which contain the most active compounds can be more 
thoroughly tested. 
If the proportion of active compounds screened in the assay is high, a 
second assay of the active compounds may be performed to choose those that 
should be further evaluated. The second assay can determine whether there 
is cross reactivity with other targets, i.e., a "selectivity screening". 
For example, a given library of compounds can be screened for activity 
against HIV protease, a member of the aspartyl protease family, using 
DABCYL-gAbu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS. Compounds found active 
in the initial assay can be counter-screened against a second, different 
aspartyl protease, such as cathepsin D. Alternately, all compounds 
screened in the assay for activity against HIV protease could be 
simultaneously screened in the counter assay. 
In the embodiment of the invention that tests for compounds that affect 
enzyme activity, it is preferred to test for inhibitors. It is also 
possible, however, to test for compounds that interfere with proteins that 
inhibit enzyme activity. In such an assay, the most active compounds 
prevent enzyme inhibition, resulting in more enzymatic catalysis. Thus, 
when a fluorogenic substrate is used active compounds result in a brighter 
zone of activity. For example, P16 is a known protein inhibitor of 
cyclin-dependent kinase-4 (Cdk-4). Using the assay of the invention, 
Cdk-4, Cyclin C1, p16, a fluorogenic substrate and a library of beads to 
be screened can be included in a layer of low-melt agarose. Following 
photocleavage, and after allowing sufficient time to convert substrate to 
product, the gel can be subjected to an electrophoretic separation. 
Product migrates to the anode, where it is preferably trapped on an anode 
filter. The location of product on the filter indicates the position in 
the gel of compound that disrupts p16 inhibition of Cdk4. 
In another embodiment of the invention, an electrophoretic procedure is 
used to separate substrate from product to increase the sensitivity of the 
assay. In this embodiment, a substrate is used which changes charge when 
converted to product. An example of such a substrate is the peptide 
leu-arg-arg-ala-ser-leu-gly attached to a fluorophore, sold commercially 
as Pep-Tag.TM. (Promega Corp.). Protein kinase A (PKA) phosphorylates this 
substrate, which has net .sup.+ 1 charge, to form a phosphopeptide which 
has a net .sup.- 1 charge. A lawn assay is performed in which PKA is 
contacted in a colloidal matrix with substrate and a library of potential 
inhibitors. An electrophoretic separation is then carried out across the 
width of (i.e., perpendicular to) the matrix. The phosphopeptide (i.e., 
product) moves towards the anode, and the dephosphopeptide (i.e., 
substrate) moves towards the cathode. If a membrane is applied to one or 
both sides of the matrix during electrotransfer, electroblotting can be 
achieved. For example, the phosphopeptide can be electroblotted to a 
suitable membrane, such as an Immobilon.TM. CD membrane. Alternately, the 
dephosphopeptide can be electrotransferred to an appropriate paper, such 
as Whatman.TM. 3MM paper. In another embodiment, the substrate and product 
can be chosen so that one is neutral and one is charged. Application of 
the electrophoretic field will remove the charged moiety. The resulting 
matrix will contain only the neutral moiety, thereby allowing detection of 
compounds that affect the conversion to product. The position of the bead 
containing the active compound can be determined by fluorescent imaging of 
the substrate or product, using, e.g., photography or video imaging. This 
technique increases sensitivity of the lawn assay by separating 
fluorescent substrate from fluorescent product, concentrating the 
fluorescent image, and by eliminating compounds from the matrix that might 
cause background signal. Other protein kinases and phosphatases such as 
protein kinase C, cyclin dependent kinases, MAP kinases, and inositol 
monophosphatase can also be used with appropriate substrates in this 
method. A protease can also be screened by this method by using a 
substrate consisting of an appropriate peptide linked to a labelling 
moiety, such as a fluorophore. The peptide sequence is chosen so that the 
substrate and product will migrate differentially in an electric field. 
The compounds to be tested are linked to the solid support through a 
cleavable linker. In a preferred embodiment, the linker is photocleavable. 
This is especially advantageous where the support is cleaved after being 
embedded in a matrix. Photocleavable linking groups include: 
##STR1## 
Where a photocleavable linker is used, the supports should be protected 
from light prior to use. 
Other examples of linkers and the relevant cleavage reagents are described 
in WO 94/08051 and parent applications Ser. Nos. 08/436,120 and 
08/239,302, which have been incorporated by reference herein. 
Photocleavable linkers are further described in Barany et al. (1985) J. 
Am. Chem. Soc. 107, 4936. Acid cleavable linkers are further described in 
Chao et al. (1994) J. Am. Chem. Soc. 116, 1746. Ammonia cleavable linkers 
are described in Kaiser et. al. (1982) J. Org. Chem. 47, 3258. Acid 
cleavable linkers and ammonia cleavable linkers allow cleavage by exposing 
the solid supports to a gaseous atmosphere, e.g. before the supports are 
applied to the colloidal matrix. 
Enzymes that can be used in the assay include, but are not limited to, the 
following: 
Acid Phosphatase 
Activated Protein C 
Alkaline Phosphatase 
Aminopeptidases B & M 
Amyloid A4-Generating Enzyme 
Angiotensinase 
Aryl Sulfatase 
.beta.-Galactosidase 
.beta.-Glucosidase 
.beta.-Glucuronidase 
Calpains I & II 
Cathepsins B, C, D, & G 
Cholinesterase 
Chymotrypsin 
Collagenase 
Dipeptidyl Peptidases I-IV 
Elastase 
Endothelin Converting Enzyme 
Factor Xa 
Factor XIa 
Factor XIIa Df-Protease 
Furin 
.gamma.-Glutamyltranspeptidase 
Granzymes A & B 
HIV Protease 
IL-1B Convertase 
Kallikrein 
Lysozyme 
Mast Cell Protease 
Peroxidase 
Plasmin 
Prohormone Convertase 
.gamma. ANP Precursor Processing Enzyme 
Renin 
Spleen Fibrinolytic Proteinase 
Staphylocoagulase 
Thrombin 
Tissue Plasminogen Activator 
Trypsin 
Tryptase 
Urokinase 
The invention is further illustrated by the Examples below, which are 
intended to exemplify the invention, not limit its scope. 
EXAMPLES 
Methods and Materials 
The lawn assay was performed in Petri plates using two layers of agarose, 
each about 1.5 mm thick. The first layer contained TentaGel S-NH.sub.2 
.TM. beads and enzyme. The TentaGel S-NH.sub.2 .TM. beads had compounds to 
be screened attached thereto by a o-nitrobenzyl photocleavable linker and 
chemical tags attached for identifying the compounds, prepared according 
to methods described in WO 94/08051, parent applications Ser. Nos. 
08/436,120 and 08/239,302, and H. Nestler et al. (1994) J. Org Chem. 59, 
4723. The beads were either placed on the Petri plate and agarose poured 
over them, or beads and agarose were first mixed and then poured together 
onto the plate. A second layer of agarose containing the fluorescein 
diacetate was contacted with the first layer to initiate the reaction. 
More specifically: 50 mM sodium phosphate, pH 7.4, was used as a buffer and 
all solutions equilibrated in a 37.degree. C. water bath immediately prior 
to initiation of the assay. 0.1 mL of 5.3 .mu.M bovine carbonic anhydrase 
(Sigma Chemical Co.) was diluted in 2.15 mL of buffer, and 1.25 mL of 2.5% 
low-gelling agarose added (SeaPlaque.TM., FMC BioProducts). Library beads 
suspended in methanol were added to a 6 cm polystyrene petri plate and, if 
necessary, distributed with a flat pipette tip. After evaporation of the 
methanol, the agarose solution was poured over the beads and allowed to 
gel at room temperature for 2-3 minutes. (Alternatively, dry beads can be 
added to a mixing tube, and then enzyme and agarose added; the mixture is 
then vortexed and poured.) The plate was then placed under a long wave 
(360 nm) UV lamp (Blackray.TM. UVP, Inc.) for from 5 sec to 1 hour. After 
irradiation, 0.01 mL of fluorescein diacetate (10 mM in DMF, Molecular 
Probes, Eugene, Oreg.) was combined with 2.25 mL buffer and 1.25 mL of 
2.5% agarose and poured over the first agarose layer. Detection was 
achieved by illumination using a short wavelength UV lamp (UVX, 254 nm) 
and image capture using a Sony.RTM. CCD camera coupled to a Macintosh.RTM. 
computer with NIH Image software obtained from the National Institutes of 
Health. 
Fluorescein diacetate was hydrolyzed to produce fluorescein as the reaction 
proceeded. The plate then became significantly brighter except in the 
vicinity of beads that released inhibitors, thereby forming zones of 
inhibition. Beads at the center of these zones were removed with a hollow 
glass tube, or a spatula, and washed in methanol/methylene chloride (1:1), 
or with hot water (80.degree. C.), to remove most of the agarose. After a 
final rinse in methanol, beads were either retested in a separate assay 
using the methods described above to confirm activity, or analyzed to 
determine the relevant compound structures by tag decoding. The tag 
decoding methodology used is described in WO 94/08051, U.S. patent 
application Ser. Nos. 08/436,120 and 08/239,302, and Nestler et al. 
Example 1 
Compound Elution 
Compound PC463767 linked to library beads through a photocleavable linker 
were released, in triplicate, under 365 nm light. Light exposure ranged 
from 0.5 to 24 hours. Vials each contained 20 beads in 100 .mu.l of 
acetonitrile. After photoelution, 20 .mu.l of solution containing the 
photoeluted compound was removed from each vial and analysed by reverse 
phase HPLC. The amount released over time, in pmol/bead, is shown in FIG. 
1. Release was found to be a time dependent event. 
Example 2 
Assay of Two Known Inhibitors 
In this example, two compounds were tested for inhibition of carbonic 
anhydrase by the lawn assay of the invention. Carbonic anhydrase 
inhibitors are useful in treating e.g., glaucoma. Results were compared 
with those obtained using a conventional solution phase assay. 
It is known that there is a high correlation between compounds that inhibit 
binding of dansylamide to carbonic anhydrase and those that inhibit 
conversion of fluorescein diacetate to fluorescein by carbonic anhydrase. 
This is believed to result from dansylamide and fluorescein diacetate 
occupying the same active site (a zinc atom) on carbonic anhydrase. The 
solution phase assay measured inhibition of dansylamide binding. The lawn 
assay measured inhibition of the conversion of fluorescein diacetate to 
fluorescein. 
Two aryl sulfonamide-containing compounds (compounds "I" and "II") were 
synthesized on TentaGel.RTM. beads (Rapp Polymere) and assayed in the 
standard solution-phase assay and in the assay of the invention. Compounds 
containing aryl sulfonamide substituents are known to be potent inhibitors 
of carbonic anhydrase. In the solution phase assay, Ki's were determined 
to be 4 and 660 nM for compounds I and II respectively. 
In the assay of the invention, beads containing each compound were embedded 
in agarose in a series of petri plates. The right side of each plate 
contained beads with compound 1, and the left side contained beads with 
compound II. Separate plates were irradiated for 2.5, 5, 10, 20 and 30 
minutes. FIGS. 2A-2C respectively show the zones of inhibition that 
resulted in the plates at 5 minutes, 15 minutes, and 30 minutes following 
photolysis. The more potent inhibitor of carbonic anhydrase (compound 1) 
showed a clear zone of inhibition after only 2.5 minutes of photolysis. 
The weaker inhibitor (compound II ) caused only a weak zone of inhibition 
after five minutes of photolysis. Ten minutes of photolysis was required 
to obtain a distinct zone. The clearest zones of inhibition were observed 
at the shortest time after photolysis. Zones at five minutes after 
photolysis were all sharper than at 15 minutes after photolysis. At 30 
minutes after photolysis, all zones were much less distinct; some zones 
(for compound II) had disappeared. 
In a second experiment, a plate containing beads with compounds I and II 
was irradiated for a predetermined period of time. The size and duration 
of the resulting zones of inhibition were determined. Results are shown in 
FIG. 3. The right side of the plate contained beads with compound 1, and 
the left side beads with compound II. The zones resulting from compound I 
were larger than those resulting from compound II. Furthermore, the zones 
for compound I could be observed for a longer time: signal from compound I 
persisted for more than two hours (although the zones became very diffuse) 
while signal for compound II all but disappeared after 90 minutes. In 
addition, zones of inhibition for compound I were more distinct, i.e., 
there was greater contrast between the zones and the surrounding areas. 
Example 3 
Assay of Two Combinatorial Libraries 
Two combinatorial libraries were evaluated by the assay of the invention 
for esterase activity in converting fluorescein diacetate to fluorescein. 
The same libraries were also evaluated by the solution-phase method (for 
determining displacement of dansylamide). The libraries contained 
compounds encompassed by the general formulae shown in FIGS. 4A and 4B. 
The library encompassed by the formula in FIG. 4A contained 1,171 
dihydrobenzopyran compounds. The dihydrobenzopyran library contained one 
aryl sulfonamide substituent at R2' out of 31 possible substituents. The 
library encompassed by the formula in FIG. 4B contained 6,727 
acylpiperidine compounds. The acylpiperidine library was synthesized in 
two parts. One half of the library (3,472 compounds) contained no aryl 
sulfonamides. In the other half of the library, three of 15 substitutions 
at R3 were aryl sulfonamides. These substitutions were a 4-sulfonamide 
substituted phenyl, a 4-chloro, 3-sulfonamide substituted phenyl, and a 
2,4-dichloro, 3-sulfonamide substituted phenyl. 
Assays were carried out to identify the most active compounds. In the 
solution phase assay, dansylamide concentration was increased until only a 
few members of the library were effective at displacement. 
In the assay of the invention, following dispersal of beads in the agarose 
matrix and photolysis to release compound, enzyme inhibitors were detected 
as dark zones surrounding specific beads. As carbonic anhydrase converted 
fluorescein diacetate (non-fluorescent) to fluorescein (fluorescent), the 
agarose matrix increased significantly in fluorescence intensity. 
Inhibitors released from beads prevented this conversion and thus caused a 
dark zone of inhibition around the bead. Beads were then removed for 
reassay or for decoding. Reassay was necessary if more than one bead was 
detected at the center of the zone of inhibition. 
The amount of photolysis was adjusted to 5 seconds. At this duration, only 
a few compounds were released in sufficient quantity to inhibit esterase 
activity. The result was a concentration-dependent competition between 
library compounds and substrate. 
As noted above, the dihydrobenzopyran library contained one aryl 
sulfonamide substituent at R2' out of 31 possibilities, amounting to about 
3% of the library. Using the assay of the invention, approximately 3% of 
the compounds were found to be active. Decoding indicated that all of the 
active compounds were aryl sulfonamides. The most active aryl sulfonamides 
in the lawn assay were found to be very similar in structure to those 
found to be most active in the solution-phase assay. Reducing the amount 
of dansylamide was not found to effectively distinguish the most active 
compounds in either assay. 
When the library of acylpiperidine compounds was assayed according to the 
invention, no active compounds were found in the part of the library that 
contained no aryl sulfonamide substitutions. Similarly, no compounds in 
this part of the library were found to be active using the solution-phase 
assay. In the other half of the library, 25% of the compounds containing 
aryl sulfonamides at R3 were found to be active using 20 minutes of 
photolysis. The same library was also screened using 2 minutes of 
photolysis, to decrease the level of activity of the compounds. All active 
structures contained an aryl sulfonamide substitution. The most potent 
compounds were structurally similar to those that were also found most 
potent in the solution phase assay. At the shortest photolysis times, 
4-sulfonamide substituted compounds predominated among the active 
compounds. Similarly, in the solution phase assay, the most potent 
compound contained a 4-sulfonamide substitution. The most active compounds 
in both the lawn assay and the solution phase assay contained hydrophobic 
amino acid chains at R2 (i.e., containing valine, leucine and/or 
phenylalanine residues). In both assays, moderately active compounds 
contained similar substituents at R2, including glutamine. In both assays, 
active compounds contained 2 or 3 substituted hydroxymethyl piperidine at 
the R1 position. 
These results showed that the assay of the invention allowed the rapid and 
accurate detection of enzyme inhibitors released from the libraries of 
beads. 
Example 4 
Lawn Assay for Inhibitors of Inositol Monophosphate 
An assay of the invention for inhibitors of inositol monophosphate is 
carried out in the same manner as described above for carbonic anhydrase 
inhibitors, with the following substitutions: The buffer used is 20 mM 
Tris, 1 mM EGTA, pH 7.8. The agarose layer contains 1 mg/mL of recombinant 
human inositol monophosphate, purified from E. coli, and 10 mM MgCl.sub.2. 
The substrate is methylumbelliferyl phosphate (Sigma Chemical Company, St. 
Louis Mo., M-8883), CSPD (Tropix, Bedford Mass.) or CDP-Star (Tropix). 
CSPD and CDP-Star are chemiluminescent substrates. The preferred substrate 
is CSPD. Inositol monophosphate is believed to be the molecular target of 
Lithium therapy in bipolar disease. 
Example 5 
Lawn Assay for Compounds that Affect Tyrosine Phosphatase 
An assay of the invention chromogenically assays compounds for their affect 
on the catalytic domain of human SHPTP1, a protein tyrosine phosphatase 
(D. Pei et al. (1993) PNAS 90, 1092) using p-nitrophenylphosphate as a 
substrate. This enzyme is assayed as described above for carbonic 
anhydrase, with the following substitutions. The buffer used is 100 mM 
N,N-bis(2-hydroxyethyl)glycine, pH 8. The first (lower) agarose layer 
contains 0.5 mg/mL recombinant human SHPTP1 catalytic domain, purified 
from E. coli, and the substrate is 4-nitrophenyl phosphate (Sigma Chemical 
Corp.). Enzyme activity corresponds with the release of the 
4-nitrophenolate anion (.lambda..sub.max 400 nm, .epsilon. 18,300 M.sup.-1 
cm.sup.-1), which appears as a yellow color on a clear background. Areas 
where affectors of the SHPTP1 catalytic domain are found are distinguished 
by either clear zones of inhibition or more colored zones of stimulation.