systematic screening of targeted chemical combinations …signaling, kinase, lipid signaling,...
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Systematic Screening of Targeted Chemical Combinations for Cancer TherapySystematic Screening of Targeted Chemical Combinations for Cancer TherapySystematic Screening of Targeted Chemical Combinations for Cancer TherapySystematic Screening of Targeted Chemical Combinations for Cancer TherapyAdrian Heilbut, Joseph Lehár, Glenn F. Short III, Grant R. Zimmermann and Curtis T. KeithAdrian Heilbut, Joseph Lehár, Glenn F. Short III, Grant R. Zimmermann and Curtis T. KeithAdrian Heilbut, Joseph Lehár, Glenn F. Short III, Grant R. Zimmermann and Curtis T. Keith
CombinatoRx Inc., Cambridge, MA 02142, USACombinatoRx Inc., Cambridge, MA 02142, USA
#1410CombinatoRx Inc., Cambridge, MA 02142, USA
#1410#1410
Abstract Systematic Multi-Target Mechanism Screen Preliminary Results and Analysis Follow-on StudiesAbstract Systematic Multi-Target Mechanism Screen Preliminary Results and Analysis Follow-on StudiesAbstract Systematic Multi-Target Mechanism Screen Preliminary Results and Analysis Follow-on Studies
Clinical experience and theoretical analyses suggest that multi-target approaches are required to Synergy Score StatisticsObjective Modulators of DNA damage response pathwaysClinical experience and theoretical analyses suggest that multi-target approaches are required to
overcome redundant and adaptive oncogenic mechanisms, and cotherapies are indeed the standard ofSynergy Score Statistics
Experimental error estimate from self combinations
Objective
Screen diverse inhibitors of cellular processes to find 9
Histogram of Additivity Volumes Combos
Combos
+ 0.1mM Topotecan
Modulators of DNA damage response pathways
DNA damage drugs remain central to cancer therapyovercome redundant and adaptive oncogenic mechanisms, and cotherapies are indeed the standard of
care for many cancers. Identification of optimal and selective combinations of the many targeted agentsExperimental error estimate from self combinations
Scores > 1 are significant at p ~99%
Screen diverse inhibitors of cellular processes to find
selective antiproliferative synergies. Probe Set Cells 8
HCT116
A549
MRC9
Combos+ 0.1mM TopotecanDNA damage drugs remain central to cancer therapycare for many cancers. Identification of optimal and selective combinations of the many targeted agents
becoming available presents a critical challenge. CombinatoRx has developed a platform for
combination high throughput screening (cHTSTM) that we have deployed to screen combinations of
Scores > 1 are significant at p ~99%~35% of Score>1 synergies are artifacts
selective antiproliferative synergies.Cytoskeleton
Pleiotropic
Viral replication
undefined
Cytoskel.
Probe Set Cells7
MRC9
self /self Can combinations selectively modulate responses?combination high throughput screening (cHTSTM) that we have deployed to screen combinations of
approved drugs for the discovery of therapeutically relevant synergies in cell based models. In addition
~35% of Score>1 synergies are artifactsScore < 1 tails dominated by artifacts
Receptor, neural
Receptor, hormone
CytoskeletonFungal cell wall
DNA damage
DNAReceptor
Cytoskel. HCT116carcinoma
6
approved drugs for the discovery of therapeutically relevant synergies in cell based models. In addition
to providing effective treatments, chemical synergies can provide information on interactions betweenScore < 1 tails dominated by artifacts1. Probe Library
DNA metabolism
DNA synthesis
Transcription, activation
Receptor, adenosine
Receptor, adrenergic
Receptor, growth factor
DNAReceptor carcinoma
(colon)5
log(
Cou
nt)
Selected 24 probes of survival and death pathways to providing effective treatments, chemical synergies can provide information on interactions between
targeted pathways, elucidating previously unappreciated connections between disease mechanisms. 430 compounds, ~250 diverse targets Transcription, chromatin
Signaling, kinase, PKC
Signaling, kinase, MAPK
Transcript.
Kinase3
4
log(
Cou
nt)
Selected 24 probes of survival and death pathways
Screening combinations in DNA damage backgroundtargeted pathways, elucidating previously unappreciated connections between disease mechanisms.Scores are correlated across cell types
430 compounds, ~250 diverse targetsProbes not biased to therapeutic targets
Transcription, machinery
Translation, ribosome
Signaling, kinase, PKA
Signaling, kinase, PKBKinaseA549 2
3 Screening combinations in DNA damage background
Here we extend systematic combination screening to probe perturbations of diverse cellular
Scores are correlated across cell types
� Cell line commonalities dominate over artifacts
Probes not biased to therapeutic targets
Even coverage of sampled mechanismsProtein processing
Signaling, kinase, lipid
Signaling, kinase, tyrosine
Protein
A549carcinoma
(lung) 1
2
� Synergies persist on top of DNA damage backgroundHere we extend systematic combination screening to probe perturbations of diverse cellular
mechanisms to discover novel pathway interactions with therapeutic potential, and to evaluate the utility� Cell line commonalities dominate over artifactsEven coverage of sampled mechanisms
Protein modification
Protein degradation
Signaling, phosphatase
Signaling, phosphodiesterase
Signaling, kinase
Protein
modif.Lipid
(lung)
-4 -3 -2 -1 0 1 2 3 40
1
� Synergies persist on top of DNA damage background
� True 3 way synergies are rare (< ~0.25%)mechanisms to discover novel pathway interactions with therapeutic potential, and to evaluate the utility
of combination effect measures for predicting mechanisms of action for novel compounds. A set of 180Significant synergies occur at ~1% rate
Proteasome
Metabolism
Metabolism, metals
Signaling, lipid
Signaling, phosphataseLipid
signalMRC9
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116 p53-/-
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
T116
-4 -3 -2 -1 0 1 2 3 4
Additivity Volume
� True 3 way synergies are rare (< ~0.25%)of combination effect measures for predicting mechanisms of action for novel compounds. A set of 180
chemical probes were selected that modulate molecular targets involved in diverse cellular functions. All Significant synergies occur at ~1% rate2. Screen Design
Metabolism, energy
Metabolism, redox
Metabolism, lipid
Signaling, apoptosis
Metab.
signalMRC9
fibroblast
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT
HCT116 HCT116 p53 -/-
Correlations
Between Cell Lines
16,110 pair wise combinations of these probes were tested at multiple concentrations and ratios using
cHTS in a proliferation assay with HCT116 human colon cancer cells. Interesting combinations were� Synergies are rare but more common than2. Screen Design Metabolism, sphingolipid
Metabolism, sterol
Metabolism, leukotrieneSignaling, intracellular
Signaling, cell cycle Metab.
IonSignaling
fibroblast
(lung)HCT
C-10331
C-10331
C-10677
C-10739
C-10896
C-11035
C-11343
C-11465
C-12220
C-12240
C-12312
C-12316
C-12317
C-12318
C-12353
C-12390
C-12395
C-12440
C-12460
C-12463
C-12477
C-12487
C-12488
C-12490
C-12505
C-10331
C-10677
C-10739
C-10896
C-11035
C-11343
C-11465
C-12220
C-12240
C-12312
C-12316
C-12317
C-12318
C-12353
C-12390
C-12395
C-12440
C-12460
C-12463
C-12477
C-12487
C-12488
C-12490
C-12505HCT116 HCT116 p53 -/-
3
ad
ditiv
ity s
co
re
Most combinations behave similarlycHTS in a proliferation assay with HCT116 human colon cancer cells. Interesting combinations were
further evaluated for tumor selectivity using additional cell lines. The screen identified both previouslyGenotype-Specific Synergiesr = 0.27±0.08
� Synergies are rare but more common than
genetic interactions (~0.5%, Tong et al. 2004)Selected 180 probes covering ~120 targetsIon transport
Signaling, ionSignaling, inflammatory
Signaling, neural IonSignalingC-10331
C-10677
C-10739
C-10896
C-110351
2
ad
ditiv
ity s
co
re
Most combinations behave similarly
across isogenic cell lines
further evaluated for tumor selectivity using additional cell lines. The screen identified both previously
reported and novel synergies and antagonisms that reflect connections between pathways relevant to
Genotype-Specific Synergies
Does synergy depend on p53 status?
genetic interactions (~0.5%, Tong et al. 2004)
� Only 1/3 of probes account for 70% of synergies
Selected 180 probes covering ~120 targetsAll pairwise combinations in sparse dose matrix
C-11035
C-11343
C-11465
C-12220
C-12240
r = 0.20±0.08
-1
0
ad
ditiv
ity s
co
re
reported and novel synergies and antagonisms that reflect connections between pathways relevant to
cancer proliferation. Synergy profiles of compounds with proximal targets were found to be correlated,
Does synergy depend on p53 status?� Only 1/3 of probes account for 70% of synergies
� Finding unexpected synergies requires
All pairwise combinations in sparse dose matrix
Two cancer and one “normal” cell lineC-12240
C-12312
C-12316
C-12317
C-12318
-2
-1
MR
C9
ad
ditiv
ity s
co
re
cancer proliferation. Synergy profiles of compounds with proximal targets were found to be correlated,
suggesting that such profiles may be used to infer mechanism of action. Combination screening data� Finding unexpected synergies requires
very large combination screens
Two cancer and one “normal” cell line� identify cancer selective synergies A549 MRC9
C-12318
C-12353
C-12390
C-12395
C-12440
r = 0.19±0.08-5 -4 -3 -2 -1 0 1 2 3
-4
-3
MR
C
r = 0.27 ± 0.08
suggesting that such profiles may be used to infer mechanism of action. Combination screening data
using cell lines analyzed in the context of emerging knowledge of cancer genotypes and expression Screened combinations of the same 24 probesvery large combination screens� identify cancer selective synergies
C-12460
C-12463
C-12477
C-12487
r = 0.19±0.08-5 -4 -3 -2 -1 0 1 2 3
HCT116 synergy scoreusing cell lines analyzed in the context of emerging knowledge of cancer genotypes and expression
profiles may lead to the development of more selective, personalized, and effective cancer therapies.
Screened combinations of the same 24 probes
384-well
Incubate 72h
Add ATP-Lite
C-12488
C-12490
C-12505
profiles may lead to the development of more selective, personalized, and effective cancer therapies.
� preliminary data suggests rare selective 3. Quality Control and Filtering
384-well
Assay Plate
Add ATP-Lite
(Luminescence)Profiles for Two Statins Correlation
No single-agent effect;
Qualitatively different synertySingle-agent effect
References:� preliminary data suggests rare selective
synergiesSynergy profiles
2
2.53. Quality Control and Filtering
Failed plates and bad wells flagged for exclusion
Correlation
HCT p53 +/+ HCT p53 -/-
1.5
Qualitatively different synerty
from secondary target
Single-agent effect
and modest synergy
Score
forB
77
Borisy AA et al. “Systematic Discovery of Multicomponent Therapeutics” PNAS 100 (13):7977 (2003)
Keith CT, Borisy AA, Stockwell BR. “Multicomponent Therapeutics for Networked Systems” Nat Rev Drug Discov. 4(1):71 (2005)
synergiesProfile = vector of all scores involving a probe 1
1.5Failed plates and bad wells flagged for exclusion1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
Score
HCT p53 +/+ HCT p53 -/-0.5
1
Score
forB
(uM)
.83
1.7
(uM)
.83
1.7Keith CT, Borisy AA, Stockwell BR. “Multicomponent Therapeutics for Networked Systems” Nat Rev Drug Discov. 4(1):71 (2005)
Zimmermann G, Lehar J & Keith CT. “Multi-target therapeutics: when the whole is greater than the sum of the parts” Drug Disc Tod 12(1): 34
Profile = vector of all scores involving a probe
Expect similar mechanism � correlated profiles0
0.5
C
D
E
F
B
C
D
E Score
inhib
-0.5
0
Score
forB
lysin (
1.41
lysin (
1.41
Zimmermann G, Lehar J & Keith CT. “Multi-target therapeutics: when the whole is greater than the sum of the parts” Drug Disc Tod 12(1): 34
Lehar J et al. “Chemical combination effects predict connectivity in biological systems” Mol Syst Biol 3:80 (2007) Eg: p53 pathway modulator + kinase inhibitorExpect similar mechanism � correlated profiles
-1.5
-1
-0.5
Intra plate artifacts quantified with control wells and G
H
I
J
E
F
G
H
Score
Kin
ase
inhib
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5-1.5
-1
r = 0.57 ± 0.06
Probe number ascapl
.1.21
ascapl
.1.21Lehar J et al. “Chemical combination effects predict connectivity in biological systems” Mol Syst Biol 3:80 (2007) Eg: p53 pathway modulator + kinase inhibitor
� potential secondary target of p53 pathway0 20 40 60 80 100 120 140 160 180
-1.5Intra plate artifacts quantified with control wells and corrected computationally
J
K
L
M
N
I
J
K
LScore for A K
inase
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5-1.5
Fa
0
N=1Fa
0
N=1
Combination High Throughput Screening� potential secondary target of p53 pathway
probe whose effect is unmasked in corrected computationally
2005 Q4 Mech Onc MTM Sparse6 / aheilbut-2006-09-06-1 / CT00032194
N
O
P
L
M
N
O
PCombination High Throughput Screening probe whose effect is unmasked in
absence of p53Cluster analysisHCT116Compounds with unstable single agent activity Plate Responses
2005 Q4 Mech Onc MTM Sparse6 / aheilbut-2006-09-06-1 / CT00032194
2005 Q4 Mech Onc MTM Sparse6 / aheilbut-2006-09-06-1 / CT00032194
P
P53 pathway inhib P53 pathway inhibabsence of p53Cluster analysis
Clustered profiles by score correlation Staurosporine tyrosine kinaseDigoxin Sodium/Potassium ATPase inhibitorW-7 Calmodulin inhibitor3-Bromopyruvate hexokinase II inhibitorMitomycin DNA cross-linker
HCT116ScoreCompounds with unstable single agent activity Plate Responses
after Quality ControlSparse Dose Matrix
P53 pathway inhib P53 pathway inhib
Cell based phenotypic assaysHighlights
Clustered profiles by score correlation
Many probes with similar annotations Sirolimus mTOR inhibitor1-[(3,4-Dichlorophenyl)methyl]-1H-indole-2,3-dione Apaf-1 activatorS-Trityl-L-cysteine Eg5 inhibitorIbudilast PDE inhibitorH-8 PKA inhibitorParthenolide NFkB inhibitorChelerythrine Chloride PKC inhibitorClomiphene Citrate Squalene epoxidase inhibitorGefitinib (Base) EGFR inhibitorChk2 Inh II Chk2 inhibitorPD169316 p38 inhibitorPrednisolone GC receptor activatorForskolin Adenylate cyclase activatorLY 294002 PI3KUCH-L1 inhibitor UCH-L1 inhibitorCladribine DNA polymerase inhibitorSB 202190 p38 inhibitorMenadione CDC25 phosphatase inhibitorDeferoxamine Mesylate Iron chelator2-amino-8-hydroxyquinoline Urokinase inhibitorMG115 proteasome 26S inhibitorStaurosporine tyrosine kinaseDigoxin Sodium/Potassium ATPase inhibitor
identified and excluded from analysisCell based phenotypic assaysHighlightsMany probes with similar annotations
group togetherTunicamycin P-MurNAc penapeptide synthase; glycosyltransferase inhibitorMethylglyoxal bis(guanylhydrazone) dihydrochloride hydrate S-adenosyl-L-methionine decarboxylase inhibitorAdenosine-5'-(N-ethylcarboxamide) Adenosine receptor agonist3-Aminobenzamide PARP inhibitor; Tubulin binder; HIF-1 antagonistPurvalanol B CDK1 inhibitorRoscovitine CDK1, CDK2, CDK5 inhibitorCelastrol HSF1 inhibitor; cytokine antagonistMycophenolic Acid INPDH (inosine phosphate dehydrogenase) inhibitorC75 Fatty acid synthase inhibitorShikonin Caspase 3/8 activatorProscillaridin Sodium/Potassium ATPase inhibitorAG-1296 PDGFR inhibitor; tyrosine kinase inhibitor; Kit inhibitorDMNB DNAPK inhibitorTG003 Clk inhibitor (Clk1-5)GW9662 PPAR gamma agonist 5,6-Dichloro-1-Beta-D-Ribofuranosylbenzimidazole RNA polymerase IIEthinyl Estradiol Estrogen receptor agonistH 89 PKA inhibitorOlomoucine CDK2 inhibitorRwj-60475 PTP, CD45 inhibitorSuccinylcholine Sirolimus mTOR inhibitor1-[(3,4-Dichlorophenyl)methyl]-1H-indole-2,3-dione Apaf-1 activator
StatinsPreserve biological networks to measure effects in a disease relevant context, and provide Highlights
group together
� Profiles contain information on mechanismManumycin A Ras farnesyltransferase inhibitorTyrphostin 9 PDGFRPaclitaxel Tubulin stabilizerNocodazole Tubulin destabilizerNSC-95397 CDC25 phosphatase inhibitor Calcimycin A23187 Calcium binderTPEN Heavy metal chelatorCyclosporine Calcineurin inhibitorSB218078 Chk1 inhibitorMethotrexate Vinblastine Sulfate Tubulin destabilizerDL-PPMP glucosylceramide synthase inhibitorC6-Ceramide Sphingosine kinase inhibitorEthacrynic Acid Glutathione S-transferase inhibitorFascaplysin CDK4/Cyclin D1 inhibitorToremifene Estrogen receptor inhibitorIMD-0354 IkappaB-alpha kinase inhibitorMonorden HSP90 inhibitorGF 109203X PKC inhibitor Bax Channel Blocker Bax channel blockerLovastatin HMG-CoA reductase inhibitorCerivastatin Sodium HMG-CoA reductase inhibitorTunicamycin P-MurNAc penapeptide synthase; glycosyltransferase inhibitorMethylglyoxal bis(guanylhydrazone) dihydrochloride hydrate S-adenosyl-L-methionine decarboxylase inhibitor
Dose Response Curve
Statins
Tubulin inhibitorsan opportunity to observe target interactions.
Comprehensive, well sampled survey Chemical library
� Profiles contain information on mechanismAphidicolin DNA polymerase inhibitorAdefovir Dipivoxil Reverse transcriptase inhibitorIsotretinoin Retinoic acid receptor binderActinonin MMP, aminopeptidase M4-ANI PARP inhibitorRo106-9920 IkappaB EE ubiquitination inhibitorExo1 ARF1 binderJSH-23 NFkB translocation inhibitorSirtinol HDAC Class III inhibitor8-bromo-cAMP PKA activator; Epac activatorBaicalein LOX (5-Lipoxygenase) inhibitorFluoxetine Hydrochloride Serotonin 5-HT transporterAclarubicin DNA topoisomerase inhibitorCalmidazolium Calmodulin inhibitorSU9516 CDK inhibitorPKR inhibitor PKR inhibitorAnisomycin ribosomal peptidyl transferase inhibitor; p38 activator; JNK activator Imatinib Mesylate (free base) tyrosine kinaseMechlorethamine Hydrochloride DNA alkylatorTrichostatin A HDAC inhibitorClotrimazole Lanosterol 14-alpha-demethylase inhibitorManumycin A Ras farnesyltransferase inhibitorTyrphostin 9 PDGFRPaclitaxel Tubulin stabilizer
4. Quantifying combination effects I X 1:10?
Dose Response Curve Tubulin inhibitors
MAPK inhibitors
an opportunity to observe target interactions.
Comprehensive, well sampled survey
of antiproliferative chemical combinationsChemical library2,400+ bioactive agents include:
Topotecan Hydrochloride DNA topoisomerase I inhibitorRaltitrexed DHFR inhibitorLY 83583 Guanylate cyclase inhibitorHC Toxin HDAC inhibitorN6-Benzyladenosine Adenosine deaminase inhibitorSangivamycin Hydrate PKC inhibitorChlorpromazine Hydrochloride PDE inhibitor; phospholipase A2 inhibitor; NOS inhibitorTrans-4-iodo, 4'-boranyl-chalcone MDM2 inhibitor Cantharidic acid PP1 inhibitor; PP2A inhibitorNifedipine Calcium channel blockerAntimycin A mitochondrial complex III inhibitorMG-132 proteasome 26S inhibitor, NFkB inhibitorApigenin MAP kinase inhibitor; ODC inhibitorAloisine A CDK5/p25 inhibitorU-0126 MEK inhibitorPurvalanol A CDC2 inhibitorTerbinafine Hydrochloride Squalene epoxidase inhibitorNaftopidil adrenergic receptor alpha1 antagonistT0070907 PPAR gamma inhibitor2-methoxyestradiol PARP inhibitor; Tubulin binder; HIF-1 antagonistEtoposide DNA topoisomerase II inhibitorAphidicolin DNA polymerase inhibitorAdefovir Dipivoxil Reverse transcriptase inhibitorIsotretinoin Retinoic acid receptor binder
4. Quantifying combination effects
Different types and patterns of synergy: Effect
I
Dru
g Y 1:1?
1:10?MAPK inhibitors
of antiproliferative chemical combinations2,400+ bioactive agents include:
• approved pharmaceutical ingredientsJuglone parvulin PPIases inhibitor; RNA polymerase II inhibitorCarbobenzoxy-valinyl-phenylalaninal Calpain I inhibitorAG-825 HER2Rottlerin PKC delta inhibitor, CaMKIII inhibitorCytochalasin B Actin polymerization inhibitor; phospholipid synthesis inhibitor2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole Sphingosine kinase inhibitorIndomethacin COX inhibitorRO-20-1724 PDE inhibitorUbenimex LTA4 HydrolaseUCH-L3 inhibitor UCH-L3 inhibitorResveratrol HDAC inhibitorDoxorubicin Hcl DNA topoisomerase II inhibitorCycloheximide ribosomal peptidyl transferase inhibitor; 23S rRNAOxaliplatin DNA cross-linkerBay 41-2272 Guanylate cyclaseNSC 663284 CDC25 phosphatase inhibitor EMETINE DIHYDROCHLORIDE HYDRATE ribosomeBafilomycin A1 mitochondrial F1 ATPase inhibitorGliotoxin farnesyltransferase inhibitor3-(2-Chloro-3-indolylmethylene)-1,3-dihydroindol-2-one CDK1 inhibitorTOFA Acetyl-CoA carboxylase inhibitorTopotecan Hydrochloride DNA topoisomerase I inhibitorRaltitrexed DHFR inhibitor
Different types and patterns of synergy: Effect
Dru
g
10:1?
• approved pharmaceutical ingredients• chemical probes with known targets
Caffeine ATM inhibitor; ATR inhibitorHemicholonium Choline kinase inhibitorWedelolactone IkappaB-alpha kinase inhibitorFluconazole Lanosterol 14-alpha-demethylase inhibitorPropranolol Hydrochloride Phosphatidate phosphohydrolase inhibitor; PKC inhibitorSU11652 PDGFR inhibitor; FGFR inhibitor; VEGFR inhibitor; Kit inhibitorAkt Inhibitor IV AKT inhibitorGenistein tyrosine kinaseHydroxyurea ribonucleoside diphosphate reductase inhibitorN-((3,3,3-Trifluoro-2-trifluromethyl)propionyl)sulfanilamide Hdm2 E3 Ligase inhibitor2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole GSK-3beta inhibitorFumagillin methionine amino-peptidaseDAG Inhibitor II Diacylglycerol kinase inhibitorTacrolimus (FK-506) Calcineurin inhibitorAG-370 PDGFR inhibitorPiceatannol Syk inhibitor; Lck inhibitor; mitochondrial F1 ATPase inhibitor; RG-14620 EGFR inhibitorWortmannin PI3KFlucytosine DHFS inhibitortrans-HR22C16 Eg5 inhibitorTamoxifen Citrate Estrogen receptor inhibitor; PKC inhibitorJuglone parvulin PPIases inhibitor; RNA polymerase II inhibitorCarbobenzoxy-valinyl-phenylalaninal Calpain I inhibitor
potency shift vs. efficacy boost at unknown ratios Concentration X Drug X
RTK inhibitors
Synergistic antiproliferative combinations are rare• chemical probes with known targets
• drugs in developmentAG-490 JAK tyrosine kinase inhibitorSulfamethoxazole dihydropteroate synthase Acyclovir DNA polymerase inhibitorDNA-PK inhibitor II DNAPK inhibitorEflornithine ODC (Ornithine decarboxylase) inhibitorTO-901317 LXR agonistCarmofur DHFS inhibitorNutlin-3 MDM2 inhibitor L-P-Bromotetramisole Oxalate tyrosine kinase inhibitor; alkaline phosphatase inhibitorThapsigargin Cerulenin HMG-CoA synthetase inhibitorSP 600125 JNK inhibitorZM 449829 JAK1 inhibitor; EGFRK inhibitor; JAK3 inhibitorDexamethasone GC receptor activatorOkadaic acid, Potassium salt PP1 inhibitor; PP2A inhibitorSU1498 Flk-1GW 5074 MAPK, cRAF1Pifithrin-alpha p53 inhibitorFluorouracil DHFR inhibitorTyrphostin 46 EGFR inhibitorPinacidil potassium channel activatorCaffeine ATM inhibitor; ATR inhibitorHemicholonium Choline kinase inhibitor
Selected Hits
Co
mp
ou
nd
B (
uM
)
potency shift vs. efficacy boost at unknown ratios
Dose matrices test many doses and ratios Inhibition (%)Dose Matrix Top viewResponse Surface
RTK inhibitors
Synergistic antiproliferative combinations are rare• drugs in development• biologics (antibodies, proteins, siRNA)
FR122047 COX-1 inhibitorRolipram PDE4 inhibitorZ-VAD (OMe)-FMK caspase inhibitorSU6656 SRC tyrosine kinase inhibitorAG-490 JAK tyrosine kinase inhibitor
Probes, by profile similaritySelected Hits
Combinations for further investigation:
Co
mp
ou
nd
B (
uM
)1
0
Dose matrices test many doses and ratios Inhibition (%)Dose Matrix Top viewResponse Surface
Synergistic hits reflect both known biology and novel interactions• biologics (antibodies, proteins, siRNA)
Probes, by profile similarityCombinations for further investigation:
Co
mp
ou
nd
B (
uM
)6
32
.51
0
Response = Inhibition = (untreated-treated)/untreated
5
4
Co
nce
ntr
atio
n s
tep
Y
Co
ncen
tra
tio
n C
Y
80
90
100100
8080
90
100100
80
(%)
Synergistic hits reflect both known biology and novel interactionsDose matrix data
.synergistic, selective, novel mechanisms
Co
mp
ou
nd
B (
uM
)0
39.1
6.6
3
Response = Inhibition = (untreated-treated)/untreated 3
2
Co
nce
ntr
atio
n s
tep
Dru
g Y
Co
ncen
tra
tio
n
40
50
60
70
80
Inhibition (%
)
80
60
4040
50
60
70
80
Inhibition (%
)
80
60
40
Inh
ibitio
n I
(%
)
HCT116 Inhibition (%)DT-1032204
71 70 63
Synergistic hits reflect both known biology and novel interactionsDose matrix data Inhibition (%)
DT-1024585
81 87 89
synergistic, selective, novel mechanisms
Co
mp
ou
nd
B (
uM
)0
.01
. 03
9
2
1
0
Co
nce
ntr
atio
n s
tep
Dru
g
Co
ncen
tra
tio
n
2.50
5.00
10.00
2.50
5.00
10.00
0
10
20
30Inhibition (%
)
0.5 1
.0
1.0
20
0
2.50
5.00
10.00
2.50
5.00
10.00
0
10
20
30Inhibition (%
)
0.5 1
.0
1.0
20
0
Inh
ibitio
n
MRC9HCT116
uM)
26
79
43
71
44
70
46
63
46
Subset being followed up with DNA damage and loss of p53 Orthogonally titrated compounds generate 20
81
25
87
65
89
85
Some hits reflect known biologyCo
mp
ou
nd
B (
uM
)
Compound A (uM0 .01 .039.16 .63 2.5 10
Compare response to Loewe additivity model0
0 1 2 3 4 5
Concentration stepDrug X 0
.00
0.01
0.02
0.04
0.08
0.16
0.31
0.63
1.25
2.50
5.00
10.00
0.00
0.01
0.02
0.04
0.08
0.16
0.31
0.63
1.25
2.50
5.00
10.00
Compound B (uM) Compo
und A
(uM)
0.0 .0
3
.13 .2
5 0.5 1
.0
.03
.13.2
50.51
.0
ConcentrationXConcentration
Y
.06
.06
0.00
0.01
0.02
0.04
0.08
0.16
0.31
0.63
1.25
2.50
5.00
10.00
0.00
0.01
0.02
0.04
0.08
0.16
0.31
0.63
1.25
2.50
5.00
10.00
Compound B (uM) Compo
und A
(uM)
0.0 .0
3
.13 .2
5 0.5 1
.0
.03
.13.2
50.51
.0
ConcentrationXConcentration
Y
.06
.06
inhib
OD
C inhib
enin (u
2.9
8.8
-1.6
8.8
19
34
32
36
33
Subset being followed up with DNA damage and loss of p53 a two dimensional dose response matrix. 2
18
4.5
27
17
44
38
Some hits reflect known biology
SAMD and ODC inhibs cut off parallel metabolic
Compound A (uMCompare response to Loewe additivity model
(expected response for drug-with-itself)Concentration step
Concentration CX
ConcentrationConcentration
Y ConcentrationConcentration
Y
OD
C inhib
OD
C inhib
Apige
.98
2
=1-2
0
-1.5
-1.6
15
19
20
18
25
32
28
30
32
33a two dimensional dose response matrix.
-.4
1.7
4.7 1.7
.1
.4 15
21
33
SAMD and ODC inhibs cut off parallel metabolic
pathways in spermine biosynthesis
(expected response for drug-with-itself)IsobologramIsobologramAdditive modelExcess (data-model)Observed data O
DC
OD
C inhib
0
Methylglyoxal bis(guanylhydrazone)
0 .41 1.2 3.7 11 33
N=0 15 20 25 28 32
Synergies and antagonisms can be Methods
Methylglyoxal bis(guanylhydrazone)
0 .41 1.2 3.7 11 33
-.4 4.7 1.7 .4 15 33pathways in spermine biosynthesisSynergy Score = Volume between data and Loewe E
CY1
dditivity
1
dditivity O
DC
OD
C inhib
SAMD inhib SAMD inhibMethylglyoxal bis(guanylhydrazone)
dihydrochloride hydrate (uM)
Synergies and antagonisms can be identified over many doses and ratios. Methodsdihydrochloride hydrate (uM)Synergy Score = Volume between data and Loewe
Lin
ea
r C
Y/E
C.5
Ad
75.5
Ad
75
SAMD inhib SAMD inhib
Inhibition (%)
2
DT-1032209
-5.8 -12 40
identified over many doses and ratios.
Cell CultureHits suggest novel biological interactions
Lin
ea
r 0.
on( %
)=70.
on( %
)=7
Inhibition (%)DT-1024587
A549HCT116
Kin
ase in
hib
Kin
ase in
hib
M)17
52
-3.1
-5.8
-10
-12
-.7
40
64Quantify synergy relative to reference Cell Culture
Cell lines were obtained from ATCC and grown in RPMI-1640 media with 10% FBS, 2 mM glutamine, and 1% penicillin/streptomycin.
Hits suggest novel biological interactions
Protein modification inhibitor + Kinase inhibitorIsobologram shows amount of potency shifting
Lin
ea
r
0 0.5 1
0
Inhi biti o
0 0.5 1
0
Inhi biti o
0
1.9
-.9
7.2
16
70
76
Kin
ase in
hib
Kin
ase in
hib
490 (uM
1.9
5.8
3.3
8.2
-6.2
-7.7
-7.5
11
55
Quantify synergy relative to reference models.
Cell lines were obtained from ATCC and grown in RPMI-1640 media with 10% FBS, 2 mM glutamine, and 1% penicillin/streptomycin.
P53-null HCT116 cells were obtained from the Vogelstein Lab at Johns Hopkins.Protein modification inhibitor + Kinase inhibitor
Suggests an additional mode of regulation of Linear CX /ECX
0 0.5 10 0.5 1
-1.2
-1.5
.4
1.5
4.2
30
54
Kin
ase in
hib
Kin
ase in
hib
AG-4
.64
1
=1-2
-1.2
-.1
3.3
-8.5
-6.2
-4.1
-7.1
-.3
-7.5
3.5
6.3
50
55models.
Compounds
Suggests an additional mode of regulation of a specific kinase signaling pathway
Linear CX /ECX
∑ 1.6
-1.2 .4
3
4.2
17
54
Kin
ase in
hib
Kin
ase in
hib
0
Trichostatin A (uM)0 3e-3 .0091 .027 .081 .24
N=-1.2 -8.5 -4.1 -.3 3.5 50
cHTSTM provides a powerful tool to:CompoundsStock solutions of each compound were prepared in DMSO, and serial dilutions were created in 384 well plates using a MiniTrak
(PerkinElmer). Compounds were diluted 1:100 into culture media to create 10X stock on the day of the assay. Combinations were created by
a specific kinase signaling pathway
5. Screen Results A549 MRC9HCT116
∑
Trichostatin A (uM)0 3e-3 .0091 .027 .081 .24
0 -2.4 4.3 7.1 15 30Kin
ase in
hib
Kin
ase in
hib
Trichostatin A (uM)cHTS provides a powerful tool to:• Discover multi-target mechanisms and
(PerkinElmer). Compounds were diluted 1:100 into culture media to create 10X stock on the day of the assay. Combinations were created by
diluting (1:10) the plates containing individual compounds into assay plates.
5. Screen Results
Full screen performed in HCT116 cells;
A549 MRC9HCT116Trichostatin A (uM)
Prot. Modif inhib Prot. Modif inhib• Discover multi-target mechanisms and therapies
diluting (1:10) the plates containing individual compounds into assay plates.
Assays
Potential therapeutic combinationFull screen performed in HCT116 cells; HCT116 MRC9therapies
• Characterize patterns of drug synergyAssaysCells were seeded at 1500 cells per well (A549, HCT116) or 3000 cells per well (MRC9) in 384 well plates and allowed to recover overnight at DNA Damage Agent + Kinase Inhibitor (
uM)
340
(uM)
340
120x120 subset tested in A549 and MRC9
DN
A d
am
age
DN
A d
am
age
• Characterize patterns of drug synergyand antagonism
Cells were seeded at 1500 cells per well (A549, HCT116) or 3000 cells per well (MRC9) in 384 well plates and allowed to recover overnight at
37°C. Compounds were added and assay plates incubated at 37°C for proliferation differences to occur. Total time of compound treatment
was 72 hours. Effect on cell proliferation was quantified using ATP-Lite 1Step (PerkinElmer), and luminescence was measured using a Wallac
DNA Damage Agent + Kinase Inhibitor
Novel interaction between two targets in ipivoxil
4.4
13
ipivoxil
4.4
13
120x120 subset tested in A549 and MRC9
DN
A d
am
age
DN
A d
am
age
and antagonism
• Guide clinical cotherapy development was 72 hours. Effect on cell proliferation was quantified using ATP-Lite 1Step (PerkinElmer), and luminescence was measured using a Wallac
VictorV or Envision plate reader. Percent inhibition of proliferation was calculated compared to vehicle-treated controls.
Novel interaction between two targets in clinical development fo
vir Di
.49
1.5
3 fovir Di
.49
1.5
2
DN
A d
am
age
DN
A d
am
age
• Guide clinical cotherapy development decisions
VictorV or Envision plate reader. Percent inhibition of proliferation was calculated compared to vehicle-treated controls.
Acknowledgements
clinical development
� Selective synergy in HCT116 over MRC9
Adef
0
N=1-3
Adef
0
N=1-2
Synergy Score for the 120x120 probe subset DN
A d
am
age
DN
A d
am
age
decisionsAcknowledgements
We would like to thank John Healy and Yiqun Bai for assistance with screening, and Margaret Lee and Ricky Rickles for valuable advice.
� Selective synergy in HCT116 over MRC9Kinase inhib Kinase inhib We would like to thank John Healy and Yiqun Bai for assistance with screening, and Margaret Lee and Ricky Rickles for valuable advice.Kinase inhib
AACR Annual Meeting, Los Angeles, April 2007AACR, Los Angeles, CA, USA, April 2007AACR Annual Meeting, Los Angeles, April 2007AACR, Los Angeles, CA, USA, April 2007