Structure-based drug design enables conversion of a DFG-in binding CSF-1R kinase inhibitor to a DFG-out binding mode

Article abstract of DOI:10.1016/j.bmcl.2010.01.078

The work described herein demonstrates the utility of structure-based drug design (SBDD) in shifting the binding mode of an HTS hit from a DFG-in to a DFG-out binding mode resulting in a class of novel potent CSF-1R kinase inhibitors suitable for lead development.

Full text of DOI:10.1016/j.bmcl.2010.01.078

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1543–1547
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based drug design enables conversion of a DFG-in binding
CSF-1R kinase inhibitor to a DFG-out binding mode
*
Marvin J. Meyers , Matthew Pelc, Satwik Kamtekar, Jacqueline Day, Gennadiy I. Poda, Molly K. Hall,
Marshall L. Michener, Beverly A. Reitz, Karl J. Mathis, Betsy S. Pierce, Mihir D. Parikh, Deborah A. Mischke,
Scott A. Long, John J. Parlow, David R. Anderson, Atli Thorarensen
Pfizer Global Research & Development, St. Louis Laboratories, 700 Chesterfield Parkway West, Chesterfield, MO 63017, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The work described herein demonstrates the utility of structure-based drug design (SBDD) in shifting the
binding mode of an HTS hit from a DFG-in to a DFG-out binding mode resulting in a class of novel potent
CSF-1R kinase inhibitors suitable for lead development.
Received 3 December 2009
Revised 13 January 2010
Accepted 14 January 2010
Available online 21 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
cFMS
CSF-1R
Kinase
DFG-in
DFG-out
Structure-based drug design
Colony-stimulating factor-1 receptor (CSF-1R or cFMS) is a
receptor tyrosine kinase whose expression is restricted to macro-
phages, osteoclasts and trophoblasts and which is uniquely
responsible for mediating the growth differentiation and survival
effects of monocyte colony stimulating factor-1 (CSF-1).1 CSF-1 sig-
nals through its receptor by means of ligand-induced dimerization
and subsequent autophosphorylation. Macrophage proliferation,
activation and survival are believed to be important for the pro-
gression of diseases such as rheumatoid arthritis (RA), osteoporo-
sis, inflammatory bowel disease, and cancer. Thus, a reduction in
the number of synovial macrophages through inhibition of CSF-
1R signaling is expected to be therapeutic in RA, related inflamma-
tory diseases, and cancer.1,2
There are a number of CSF-1R inhibitors with in vivo antiinflam-
matory efficacy reported in the literature, including 1,2a–c 2,2d
Ki20227,2e,f and GW2580.2g,h As a part of our program targeting
CSF-1R for RA, our internal kinase-targeted compound library
was screened measuring the inhibition of autophosphorylation of
CSF-1R in a cell-based assay.3 Compounds 3a and 3b were identi-
fied as potent inhibitors of CSF-1R (IC50 = 40 nM and 57 nM,
respectively). However, the high molecular weights (MW = 524–
525) and poor ligand efficiencies (LE = 0.27–0.28) of these two hits
required significant structural modifications to maximize their po-
tential as leads. The work described herein demonstrates the utility
of structure-based drug design (SBDD) in shifting the binding
mode of this series from DFG-in to a DFG-out binding mode result-
ing in novel potent CSF-1R kinase inhibitor leads.
In our preliminary evaluation of this series, we were surprised
to discover that compound 3a was found to bind CSF-1R in a
classical DFG-in binding mode (Fig. 2a).4 Dramatic conformational
differences in the juxtamembrane domain and activation loops are
apparent when this DFG-in structure is compared with prior DFG-
out CSF-1R structures, with some residues displaced by over 30 Å.
Indeed, superposing the DFG-out and -in structures shows that
elements of the juxtamembrane domain in the former (especially
residues 550–554) mimic elements of the activation loop in the lat-
ter (residues 797–804). This is the first example of a DFG-in bind-
ing mode for a CSF-1R kinase inhibitor.5,2i It superposes well on the
structure of the active conformation of cKit bound to ADP (RCSB
code 1PKG).
Several known selective CSF-1R inhibitors bind the enzyme in a
DFG-out mode. For example, GW2580 is exquisitely selective for
CSF-1R (Fig. 2b).2g,h,5b An important feature of this co-crystal struc-
ture is the bidentate hydrogen bonding network made by the oxy-
gen atoms of the methoxy and benzyloxy groups of the ‘DFG-out
Tail’ to the backbone amide NH of D796. We hypothesized that this
is an important feature for locking in the DFG-out binding mode
revealing access to the deep pocket.
* Corresponding author. Tel.: +1 314 604 2344.
E-mail addresses: marvin.j.meyers@pfizer.com, mrvmyrs@gmail.com (M.J.
Meyers).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.078

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M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1543–1547
We desired a chemical series with a DFG-out binding mode,
which would lock the CSF-1R kinase into an inactive confirma-
tion.6a,b DFG-out inhibition is expected to increase the biochemical
efficiency, defined as how effectively the binding of an inhibitor to
the target results in blocking a physiological response.6c,d Further-
more, the likelihood for kinase selectivity is enhanced by a DFG-
out binding mode as only a subset of kinases can achieve DFG-out
conformations. We hypothesized that we could transform this ami-
nopyrazine class of inhibitors into a class of DFG-out binders. An
overlay of the crystal structures of 3a and GW2580 (Fig. 2c) illus-
trates the overlap of the hinge binding group aminopyrazine in 3a
and the corresponding diaminopyrimidine group in GW2580. Since
the basic amine head group of 3a reaches out to solvent, apparently
contributing little to binding affinity, we hypothesized that the
head group could be truncated to a simple aromatic group (Step 1
in Fig. 1). If this could be accomplished, then the DFG-out tail group
found in GW2580 could be incorporated (Step 2 in Fig. 1) giving hy-
drid compound 4 with a DFG-out binding mode, a hypothesis sup-
ported by molecular docking studies (Figs. 1 and 2d).
Compound 3a and 3b, while quite potent, have high molecular
weights (MW) resulting in relatively poor ligand efficiencies (LE).7
In order to transform the dichlorobenzyl ether group into the high-
er MW DFG-out tail group, which was anticipated to be necessary
to bind into the DFG-out deep pocket, we initiated an effort to
truncate the aryl amide head group. In addition to reducing MW,
we desired to remove the basic amine as it puts this series at risk
for binding the hERG channel.8
Compounds 8a–c were prepared as shown in Scheme 1.
Selective nucleophilic attack by benzylic alcohols or amines on
dibromoaminopyrazine 5 results in bromopyrazine intermediate
6 which undergoes Suzuki coupling with aromatic boronic acids
and esters such as pyrazole boronate 7 to give final products 8a–
b. Other aromatic and heteroaromatic boronic acids and esters
were also prepared in a library format (data not shown).
Figure 2. Binding surfaces are colored by electrostatic potential. (a) X-ray crystal
structure of compound 3a in CSF-1R in a DFG-in binding mode at 2.5 Å resolution
(Ref. 4; RCSB code 3LCD). (b) X-ray crystal structure of GW2580 in CSF-1R in a DFG-
out binding mode (Ref. 5). (c) Overlay of compound 3a (yellow) and GW2580
(green) X-ray crystal structures in CSF-1R. (d) Molecular docking of hypothetical
hybrid (pink) of compound 3a and GW2580 in the X-ray crystal structure of
GW2580/CSF-1R.
We were delighted to find that the entire aryl amide head group
from compounds 3a–b could be truncated to a simple methyl
pyrazole as shown in compounds 8a–c (Table 1). Although
9- and 30-fold less potent than the hits 3a–b, compounds 8a–b
are significantly more ligand efficient (LE) than the screening hits
Figure 1. CSF-1R inhibitors. HTS hits and hybrid SBDD approach to designing a
DFG-out kinase inhibitor.

M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1543–1547
1545
Table 1
Truncation of HTS hits
Dof. % inh.b
a
Compd
X
R
CSF-1R IC50 (nM)
MW
LE
c Log P
LipE
3a
3b
8a
8b
8c
NH
O
NH
O
—
—
Cl
Cl
H
40
57
345
1700
1990
524
525
348
349
315
0.28
0.27
0.38
0.34
0.35
5.5
5.7
3.7
3.9
3.2
1.9
1.5
2.8
1.9
2.5
78
91
6
7
7
O
a
Ref. 3.
b
Dof. = dofetilide-Cy3B competitive binding assay, % inhibition at 10
l
M of compound. See Ref. 8c.
due to their reduced MWs. Compound 8c illustrates the second Cl
atom was also unnecessary for CSF-1R potency. In terms of c Log P
and lipophilic efficiency (LipE),9 compounds 8a and 8c are an
improvement of approximately one order of magnitude.
Furthermore, the hERG potential of this series was significantly re-
duced as evidenced by the reduction in inhibition of the binding of
fluorescently tagged dofetilide to the hERG channel.8c As a result,
compound 8c was then utilized as the core pharmacophore onto
which the DFG-out tail group was incorporated.
The requisite extended benzyl alcohols 10a–b, 16, and 17 and fi-
nal potential DFG-out binding hybrid inhibitors 12a–b and 18a–b
were prepared as shown in Schemes 2 and 3. Benzaldehyde 9 was
alkylated with para-methoxybenzyl chloride and then reduced with
sodium borohydride to give benzyl alcohol 10a. Pyridine derivative
Scheme 1. Reagents and conditions: (a) X = NH2; Na2CO3, NMP, 165 °C; (b) X = OH;
KOtBu, THF, 60 °C; (c) Pd(PPh3)2Cl2, 2 M Na2CO3, DME, 80 °C.
Scheme 3. Reagents and conditions: (a) 4-methoxybenzyl alcohol, NaH, DMF; (b)
(i) n-BuLi, THF, À60 °C; (ii) DMF; (c) NaBH4, THF, MeOH. (d) (i) mCPBA, CH2Cl2; (ii)
NaOMe, MeOH; (e) 5, KOtBu, THF, 60 °C; (f) 7, Pd(PPh3)2Cl2, 2 M Na2CO3, DME,
80 °C; (g) 5, Cs2CO3, DMF, 70 °C.
Scheme 2. Reagents and conditions: (a) KOH, acetonitrile, reflux; (b) NaBH4, MeOH,
dioxane; (c) TMSCHN2, MeOH, toluene; (d) KOtBu, THF, 60 °C; (e) 7, Pd(PPh3)2Cl2,
2 M Na2CO3, DME, 80 °C.

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M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1543–1547
10b was prepared by O-methylation of pyridone 1110 with trimeth-
ylsilyldiazomethane. Benzyl alcohols 10a–b were then reacted with
dibromoaminopyrazine 5, followed by Suzuki coupling.
a dramatic 36-fold enhancement in CSF-1R potency over 8c, pre-
sumably properly accessing the deep binding pocket revealed in a
DFG-out binding mode. Similarly, pyridine variants of this
methoxyphenyl-PMB tail were also quite potent (12b and 18a).
In contrast to GW2580, these analogs have an additional oxygen
atom in the linker between the hinge binding aminopyrazine and
the DFG-out tail group, yet still inhibit CSF-1R with a high degree
of potency. Pyridine analog 18b demonstrates that the OCH2 linker
can be shortened to a single oxygen atom, analogous to the 1-atom
linker length of the GW2580 scaffold, resulting in a slight improve-
ment in CSF-1R potency.
To ascertain whether compounds 12a–b and 18a–b were in-
deed binding in a DFG-out mode, compound 12b was co-crystal-
lized with CSF-1R. A relatively low resolution structure of 12b
with CSF-1R showed that 12b was indeed binding in a DFG-out
mode (Fig. 3).4 Although hydrogen bonding interactions cannot
be definitively determined given the resolution of the crystallo-
graphic data, the methoxy and benzyloxy oxygen atoms are near
D796 in a manner similar to GW2580. A hydrogen bonding interac-
tion with this residue is likely a key requirement for binding and
accessing the DFG-out deep pocket.
Pyridine derivatives 16 and 17 were prepared starting with
5-bromo-2-chloro-3-methoxypyridine (13) as shown in Scheme
3. Displacement of the chloride with 4-methoxybenzyl alcohol
followed by lithium–halogen exchange and trapping with DMF
gives aldehyde 15. Aldehyde 15 was reduced with sodium borohy-
dride to give alcohol 16. Alcohol 16 was reacted with 5 followed by
Suzuki coupling to give pyridine analog 18a. Baeyer–Villiger rear-
rangement of aldehyde 15, followed by hydrolysis in methanolic
sodium methoxide gave phenol 17. Nucleophilic displacement in
the presence of cesium carbonate followed by Suzuki coupling
gave analog 18b.
Table 2 shows potency of compounds 12a–b and 18a–b.
Incorporation of the GW2580-like DFG-out tail (12a) resulted in
By transforming a series of DFG-in CSF-1R inhibitors to DFG-
out inhibitors, we have established a potent series more suitable
for lead development. In contrast to 3a–b, which were less attrac-
tive in terms of MW, LE, LipE, and hERG potential, 12a–b and
18a–b have significantly reduced MW, c Log P, improved LipE,
and reduced hERG potential (Tables 1 and 2). For example, LipE’s
were improved 2–3.5 orders of magnitude and MW’s were re-
duced into the 425–450 range. Furthermore, the potency and li-
gand/lipophilic efficiency enhancements were made in a more
relevant cell-based assay system as opposed to a biochemical en-
zyme assay.
In summary, we have successfully utilized SBDD to transform a
classical DFG-in binding mode HTS hit into a class of potent DFG-
Figure 3. X-ray crystal structure of compound 12b in CSF-1R at 3.4 Å resolution in a
DFG-out binding mode (Ref. 4; RCSB code 3LCO).
Table 2
DFG-out binding leads
NH2
N
R
N
N
N
a
Compd
CSF-1R IC50 (nM)
MW
447
LE
c Log P
LipE
3.5
O
O
O
O
O
12a
56
0.30
3.8
O
O
O
O
O
12b
18a
143
30
448
448
434
0.28
0.31
0.34
3.1
3.1
2.9
3.7
4.4
5.0
N
N
O
O
O
N
O
O
18b
12
O
a
Ref. 3.

M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1543–1547
1547
incubated at room temperature for 60–90 min. Plates were washed three
times with wash buffer (TBS + 0.1% Tween 20), and 40 L of cell lysate was
transferred to the ELISA capture plate and incubated for 2 h at room
temperature. Plates were again washed three times with wash buffer, and
out binding CSF-1R inhibitors with potential to be developed into
viable CSF-1R kinase inhibitor drug candidates.
l
50
mL in Superblock was added to the plate and incubated for 30 min at room
temperature. Plates were washed three times with wash buffer, and 50 L/
well of TMB solution (Bio-Rad, Cat# 172-1066) were added and plates were
incubated at room temperature for 1–10 min (incubation time varies
depending on color development). The color reaction was then stopped by
lL/well HRP-PY20 antibody (Santa Cruz, cat# SC508 Hrp) diluted to 1 lg/
Acknowledgments
l
The authors thank Daniel Walker and Michael Ennis for critical
review of this manuscript, and the Advanced Photon Source and
the staff at beamline 21ID-D for their help in the collection of dif-
fraction data.
addition of 50
lL/well of 0.1 N H2SO4 and plates were read on a
spectrophotometer with 450 nm OD.
4. Compound 3a bound to CSF-1R was crystallized following the protocol of
Schubert et al. (Ref. 5a). CSF-1R protein for the co-crystal with 12b was
obtained following the protocol of Emerson et al. (Ref. 5b). 11 mg/mL protein in
20 mM HEPES pH 7.5, 300 mM NaCl, 5 mM DTT was incubated with 4 mM
References and notes
1. Pixley, F. J.; Stanley, E. R. Trends Cell Biol. 2004, 14, 628.
inhibitor at 4 °C overnight followed by the addition of 1
from Roche (per 50 L sample) and further incubated at 22 °C for 24 h. The
limited proteolysis was quenched with the addition of 0.5 L of 5 mg/mL
leupeptin. Hanging drop vapor diffusion experiments were set up by mixing
2.0 L of protein solution with equal volumes of well solution (22.5–35% PEG
lg Arg-C purchased
2. (a) Patch, R. J.; Brandt, B. M.; Asgari, D.; Baindur, N.; Chadha, N. K.; Georgiadis,
T.; Cheung, W. S.; Petrounia, I. P.; Donatelli, R. R.; Chaikin, M. A.; Player, M. R.
Bioorg. Med. Chem. Lett. 2007, 17, 6070; (b) Illig, C. R.; Chen, J.; Wall, M. J.;
Wilson, K. J.; Ballentine, S. K.; Rudolph, M. J.; DesJarlais, R. L.; Chen, Y.;
Schubert, C.; Petrounia, I.; Crysler, C. S.; Molloy, C. J.; Chaikin, M. A.; Manthey, C.
L.; Player, M. R.; Tomczuk, B. E.; Meegalla, S. K. Bioorg. Med. Chem. Lett. 2008, 18,
1642; (c) Meegalla, S. K.; Wall, M. J.; Chen, J.; Wilson, K. J.; Ballentine, S. K.;
DesJarlais, R. L.; Schubert, C.; Crysler, C. S.; Chen, Y.; Molloy, C. J.; Chaikin, M. A.;
Manthey, C. L.; Player, M. R.; Tomczuk, B. E.; Illig, C. R. Bioorg. Med. Chem. Lett.
2008, 18, 3632; (d) Huang, H.; Hutta, D. A.; Rinker, J. M.; Hu, H.; Parsons, W. H.;
Schubert, C.; DesJarlais, R. L.; Crysler, C. S.; Chaikin, M. A.; Donatelli, R. R.; Chen,
Y.; Cheng, D.; Zhou, Z.; Yurkow, E.; Manthey, C. L.; Player, M. R. J. Med. Chem.
2009, 52, 1081; (e) Uemura, Y.; Ohno, H.; Ohzeki, Y.; Takanashi, H.; Murooka,
H.; Kubo, K.; Serizawa, I. J. Neuroimmunol. 2008, 195, 73; (f) Ohno, H.; Uemura,
Y.; Murooka, H.; Takanashi, H.; Tokieda, T.; Ohzeki, Y.; Kubo, K.; Serizawa, I. Eur.
J. Immunol. 2008, 38, 283; (g) Conway, J. G.; McDonald, B.; Parham, J.; Keith, B.;
Rusnak, D. W.; Shaw, E.; Jansen, M.; Lin, P.; Payne, A.; Crosby, R. M.; Johnson, J.
H.; Frick, L.; Lin, M.-H. J.; Depee, S.; Tadepalli, S.; Votta, B.; James, I.; Fuller, K.;
Chambers, T. J.; Kull, F. C.; Chamberlain, S. D.; Hutchins, J. T. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 16078; (h) Conway, J. G.; Pink, H.; Bergquist, M. L.; Han, B.;
Depee, S.; Tadepalli, S.; Lin, P.; Crumrine, R. C.; Binz, J.; Clark, R. L.; Selph, J. L.;
Stimpson, S. A.; Hutchins, J. T.; Chamberlain, S. D.; Brodie, T. A. J. Pharmacol. Exp.
Ther. 2008, 326, 41; (i) Manthey, C. L.; Player, M. L. Ann. Rep. Med. Chem. 2009,
44, 211; (j) Manthey, C. L.; Johnson, D. L.; Illig, C. R.; Tuman, R. W.; Zhou, Z.;
Baker, J. F.; Chaikin, M. A.; Donatelli, R. R.; Franks, C. F.; Zeng, L.; Crysler, C.;
Chen, Y.; Yurkow, E. J.; Boczon, L.; Meegalla, S. K.; Wilson, K. J.; Wall, M. J.; Chen,
J.; Ballentine, S. K.; Ott, H.; Baumann, C.; Lawrence, D.; Tomczuk, B. E.; Molloy,
C. J. Mol. Cancer Ther. 2009, 8, 3151.
l
l
l
4000, 0.1 M Tris–HCL pH 8.5, 0.2 M MgCl2). Crystallographic data were
integrated and scaled using the HKL2000 software package (Ref. 11a),
refinement was carried out using programs contained within the CCP4 suite
(Ref. 11b), manual rebuilding was performed with COOT (Ref. 11c), ligands
were docket with Afitt (OpenEye Scientific), and structural figures made using
PyMol (http://www.pymol.org). CSF-1R bound to 3a was refined to
a
crystallographic R value of 21.8% (Rfree 26.5%) using all the data to 2.5 Å
resolution and CSF-1R bound to 12b was refined to a crystallographic R value of
24.7% (Rfree 28.9%) using all the data to 3.4 Å resolution.
5. (a) Schubert, C.; Schalk-Hihi, C.; Struble, G. T.; Ma, H.-C.; Petrounia, I. P.; Brandt,
B.; Deckman, I. C.; Patch, R. J.; Player, M. R.; Spurlino, J. C.; Springer, B. A. J. Biol.
Chem. 2007, 282, 4094; (b) Emerson, H. K.; Musso, D. L.; Chamberlain, S. D.;
Peckham, G. E. U.S. Patent 7,491,731, 2009.
6. (a) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.;
Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan, J. Nat. Struct. Biol. 2002, 9,
268; (b) Knight, Z. A.; Shokat, K. M. Chem. Biol. 2005, 12, 621; (c) Swinney, D. C.
Nat. Rev. Drug Disc. 2004, 3, 801; (d) Swinney, D. C. Curr. Top. Med. Chem. 2006,
6, 461.
7. (a) Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430; (b)
Reynolds, C. H.; Tounge, B. A.; Bembenek, S. D. J. Med. Chem. 2008, 51, 2432; (c)
Tyrchan, C.; Blomberg, N.; Engkvist, O.; Kogej, T.; Muresan, S. Bioorg. Med. Chem.
Lett. 2009, 19, 6943.
8. (a) Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. J. Med. Chem. 2006, 17,
5029; (b) Jalaie, M.; Holsworth, D. D. Mini-Rev. Med. Chem. 2005, 5, 1083; (c)
Singleton, D. H.; Boyd, H.; Steidl-Nichols, J. V.; Deacon, M.; de Groot, M. J.; Price,
D.; Nettleton, D. O.; Wallace, N. K.; Troutman, M. D.; Williams, C.; Boyd, J. G. J.
Med. Chem. 2007, 50, 2931.
3. HEK293 Freestyle cells transiently transfected with human CSF-1R vector
(Origene, SC116882) were suspended in DMEM supplemented with 0.1%
fetal bovine serum, 1%
L-glutamine, and 0.0405% bovine albumin (Sigma #A-
8918) at 1.7 Â 106. Thirty microlitre of the cell suspension were then
9. Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.;
Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Bioorg. Med. Chem. Lett. 2009, 19,
4406.
10. Miller, W. H.; Pendrak, I.; Seefeld, M. A. WO2006/002047, 2006.
11. (a) Otwinoski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307; (b) Collaborative
Computational Project, Number 4. Acta Crystallogr., Sect. D 1994, 50, 760.; (c)
Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126.
dispensed into each well of plates containing 100 nL of compound in 100%
DMSO. After 1 h of incubation at 37 °C, cells were lysed by adding 40
2Â lysis buffer to each well and incubated for 4 min at room temperature.
Forty microlitre of lysates were then transferred to capture antibody
coated and blocked ELISA plate. c-Fms/CSF-1R(C-20) antibody (Santa Cruz,
cat.#: sc-692) was diluted to 1 g/mL in Superblock (Pierce, cat #37545),
and 50 was added to each well of goat anti-rabbit coated plates and
lL of
a
l
l
L