DRUG DISCOVERY - NIH Clinical Centerclinicalcenter.nih.gov/.../slides/Discovery08-09oneslide.pdf ·...

DRUG DISCOVERY

Edward A. Sausville, M.D., Ph.D.Professor of Medicine

Associate Director for Clinical ResearchMarlene & Stewart Greenebaum Cancer Center

University of Maryland at BaltimoreMarch 19, 2009

OUTLINE OF PRESENTATION

• General Introduction

• Definition of Drug Targets

• Generating Diversity

• Definition of Lead Structures

• Qualifying Leads for Transition to Early Trials

DRUG DISCOVERY:WHERE HAS IT WORKED?

Nature 384 suppl 11:5, 1996

Majority of Drug Targets:

- G-Protein Coupled Receptors- Nuclear (Hormone) Receptors- Ion Channels- Enzymes

% Top Sales

181016

~50

Problem:How to choose target likely to succeedespecially if directed at new target(e.g. protein-protein interactions)?

DRUG DISCOVERY:A SUCCESSION OF STYLES

Antiquity to 1960s:Mixtures of natural products vs. bioassays(e.g., digitalis, rauwolfia, penicillins, anthracyclines,vinca, taxol, camptothecins)

1930s to present:Pure compounds vs. bioassays(e.g., sulfas, diuretics, hypoglycemics, antiHBP)

1960s to present:Pure compounds vs. pure enzymes(e.g., ACE inhibitors, cholesterol-lowering statins,RT and protease inhibitors)

1980s to present:Combinatorial methods to bring mixtures of compoundsvs. many targets

WHY COMPOUNDSFAIL AND SLOW DOWN IN DEVELOPMENT

Modern Drug DiscoveryJanuary/February 1999Modern Drug Discovery, 1999, 2 (1), 55-60.Copyright © 1999 by the American Chemical Society

Reasons for failure Reasons for slowdown

• Toxicity, 22%• Lack of efficacy, 31%• Market reasons, 6%• Poor biopharmaceutical

properties, 41%

• Synthetic complexity• Low potency• Ambiguous toxicity finding• Inherently time-intensive

target indication• Poor biopharmaceutical

properties

TRADITIONAL PHARMACEUTICAL R&DSuffers High Attrition*

* Tufts CSDD, H&Q 1998; The Pfizer Journal, 1/2000

Target ID Synthesis/Screening

Target ValidationLead Optimization

Preclinical Ph1 Ph2 Ph3 Filed

100-150 Screens

103-105

Compoundsper Screen

“Hits” to“Leads” “Drugs”“Drug Candidates”

1-10K Hits

100 Leads

12

4.8 3.6 1.7 1Research Development

TRADITIONAL PHARMACEUTICAL R&DCostly* and Time Consuming**

* Lehman Brothers, 1997; ** Tufts CSDD

Target ID Synthesis/Screening

Target ValidationLead Optimization

Preclinical Ph1 Ph2 Ph3 Filed

Lead DiscoveryResearch

6 Years

Drug Development

8.9 Years

$230m+71m

+56m+169m

+169m+44m

$608m*






• Qualifying Lead for Transition to Early Trials

TWO CONTRASTING DRUG-DISCOVERY “PHILOSOPHIES”

• “EMPIRICAL”: Recognize initial drug leadby functionally useful effect-E.g. : penicillin (anti-bacterial effect)

rauwolfia (anti-hypertensive)taxol (anti-tumor)digoxin (cardiotonic / antiarrythmic)

• “RATIONAL”: Recognize drug by design or screenagainst biochemical target’s function-E.g.: HIV-protease inhibitor (anti-infection)

metoprolol (anti-hypertensive)methotrexate (anti-tumor)

“EMPIRICAL” DRUG DISCOVERYSCREEN

BIOLOGICAL ACTIVITY(in vitro/in vivo)

PHARMACOLOGY

CHEMISTRY

OPTIMIZED SCHEDULE (in vivo)

IND-DIRECTED TOX/FORMULATION

PHASE I: DOSE/SCHEDULE HUMAN PHARM/TOX

PHASE II: ACTIVITY

PHASE III: COMPARE WITH STANDARD

PROBLEMS WITH EMPIRICAL MODELS• Lead optimization difficult without known biochemical

target--How to optimize?

• Value of screen depend on predictive value ofscreening model with biology of disease-E.g.: acid hypo-secretion or H2 receptor binding assay

HIGHLY correlate with useful anti-ulcer Rx-Counter E.g.: anitumor activity in > 33% mouse modelsof cancer have at best 50% chance of >1 P2 trial fornon=targeted cancer Rx’s

• Divorced from mechanism: an intriguing lead mustbe “deconvolutedh

N

N

N

NH

O

OHHO

NHHOOH

Me( CH2)8CH=CHCH=CHC(O)NHCH2C(O)NH

N

N

N

NH

O

OHHO

NHHOOH

Me( CH2)8CH=CHCH=CHC(O)NHCH2C(O)NH

KRN5500

N

N

N

NH

O

OHHO

NHHOOH

H2 NCH2C(O)NHH3C(CH2)8CH=CHCH=CHCO2H

Deacylation SAN-Gly

Protein Synthesis

EFFECT OF KRN5500 ON COLO-205 ATHYMIC MOUSE XENOGRAFTS

Med

ian

Tum

or W

eigh

t (m

g)

Day Posttumor Implantation

500045004000350030002500200015001000500

08 12 15 18 22 26 29 33 36 41

vehicle13.5 qdx533.5 q4dx322.4 q4dx320 qdx550 q4dx330 qdx5

+

X

0.1

1

10

0 1 2 3 4 5

KRN5500 PLASMA CONCENTRATIONS ON EFFECTIVE SCHEDULE(20 MG/KG/D) IN MICE

Pla

sma

Con

cent

ratio

n (μ

M)

Time (days)

SUMMARY OF KRN-5500 PHASE I

• 26 patients as IV once per day over 5 days

• Dose limiting toxicity = interstitial pneumonitis

• MTD = 2.9 mg/M2/d x 5

• Achieve only 0.75 - 1 μM at 3.7 mg/M2/d x 5

• 4/6 patients with >25% incr Cmax havegrade 4 toxicity

Data of J. P. Eder, DFCI

“RATIONAL” DRUG DISCOVERY

TARGET-DEPENDENT IN VIVO MODEL

IND DIRECTED TOX/FORM

PHASE I: DOSE/SCHEDULE: HUMAN PHARM/TOX;? AFFECT TARGET

PHASE II: ACTIVITY = ? AFFECT TARGET

PHASE III: COMPARE WITH STANDARD; STRATIFY BY TARGET?

PHARMACOLOGY(to affect target)

CHEMISTRY

MOLECULAR TARGET SCREENBiochemicalEngineered cellAnimal (yeast/worm/fish)

bcr-abl AS TARGET: RATIONALE

• Apparently pathogenetic in t9:Q22 (Ph+) CML/ALL

• Absence in normal tissues

• Modulate signal transduction events downstream

Maintenance of chronic phaseAdjunct to bone marrow transplantation

bcr-abl FUSION PROTEIN

bcr SH2 SH2 V SH2/SH3 kinase NT DNA Actin

bcr

autophosphorylation

Phosphorylation ofother substances

McWhirter JR, EMBO 12:1533, 1993

EXAMPLE OF “RATIONAL” APPROACH:bcr-abl directed agents

OH

OH

NHCHOOH

OH

N

HO

OH

CO2H

HO

HO

OH

OH

OH

OH

NH

CO2Me

HN CN

NHN

CN

NH2

N

N

N

NH NH

O

N

NMe

Me

erbstatin lavendustin piceatannol

AG957 AG1112

CGP 57148B = STI571

Naturalproductempiric lead

1st generationsynthetic

2nd generationsynthetic;in clinic

00.20.40.60.8

11.21.41.61.8

0 5 10 15 20

Tum

or W

eigh

t (g) control mice 3x160 mg/kg oral

STI571: An oral in vivo bcr-abl kinase inhibitor

N

N

N

NH NH O

Me

N

NMe

Tyr phosphorylation in vivo

le Coutre et al, JNCI 91:163, 1999

Antitumor activity in vivo

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9

% P

hosp

hory

latio

n

Intraperitoneal Oral

0102030405060708090

100

0 10 20 30 40 50 60

% T

umor

Fre

e Su

rviv

al

KU812 control mice U937 control miceKU812 3x50 mg/kg i.p. U937 3x50 mg/kg i.p.KU812 3x160 mg/kg oral U937 3x160 mg/kg oral

(hrs)(days)

(days)

NEJM 344: 1031, 2001

EFFICACY AND SAFETY OF A SPECIFIC INHIBITOR OF THE BCR-ABLTYROSINE KINASE IN CHRONIC MYELOID LEUKEMIA

BRIAN J.DRUKER,M.D.,MOSHE TALPAZ,M.D.,DEBRA J.RESTA,R.N.,BIN PENG,PH.D.,ELISABETH BUCHDUNGER,PH.D.,JOHN M.FORD,M.D.,NICHOLAS B.LYDON,PH.D.,HAGOP KANTARJIAN,M.D.,

RENAUD CAPDEVILLE,M.D.,SAYURI OHNO-JONES,B.S.,AND CHARLES L.SAWYERS,M.D.

% in

Met

apha

se0 100 200 300 400

100

80

60

40

20

0

Ph Chromosome + Cells

Duration of Treatment with STI571(Days)

White Cell Count

(cel

ls x

10-

3/ m

m3 )

0 30 60 90 120 150

100

10

1

Duration of Treatment with STI571(Days)

* Cytogenetics Breakpoints Molecules (bcr-abl)* “Positive” selection from tumor DNA Active oncogenes

(signal transduction)* Tumor gene expression profiling (CGAP)

* Binding partners (geldanamycin, rapamycin, fumagillin)* Computational algorithm (molecule target)

* Cell metabolism / Biochemistry* Suggest single targets Inefficient; Medicinal Chemistry possible

* Libraries of molecules and precisely defined organisms

MOLECULAR TARGET DEFINITION - HOW TO?

• BIOLOGY:

• “ RETROFIT” ACTIVE MOLECULES:

• “CLASSICAL:”

• CHEMICAL GENETICS:

- COMPARE- Cluster analysis

Establishing for a cell the repertoire of genes expressed, togetherwith the amount of gene products produced for each, yields apowerful "fingerprint". Comparing the fingerprints of a normal versusa cancer cell will highlight genes that by their suspicious absence orpresence (such as Gene H ) deserve further scientific scrutiny todetermine whether such suspects play a role in cancer, or can beexploited in a test for early detection.

Normal Cell

Cancer Cell

Gene Expression: The Cell’s Fingerprint

http://cgap.nci.nih.gov

http://cgap.nci.nih.gov/

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling

Alizadeh et al, Nature 403: 503, 2000

Overall survival (yrs)

Prob

abilit

yGC B-like DLBCL Activated B-like DLBCL

All patients

All patients Low clinical risk pts

Prob

abilit

y

Overall survival (yrs)

Low clinical risk

High clinical risk

P=0.002

GC B-like

Activated B-like

P=0.05

GC B-like

Activated B-like

P=0.01

Geldanamycin

17-AAG

122750

330507

OMe

NHCH2CH=CH2

RNSC

GELDANAMYCIN: EXAMPLE OF BINDING PARTNER DEFINING TARGET

O

O

R

NH

O

OCONH2

OH

O

MeO

Me

carbamateansa ring

benzoquinone

BENZOQUINOID ANSAMYCINSINITIAL CELL PHARMACOLOGY - I

• “Reverse” transformed phenotype of src-transformed rat kidney cell line– decrease tyrosine phosphorylation of pp60src– not inhibit pp60 immune complex kinase directly but

these were inhibited from drug-treated cells– thus alter “intracellular environment” of src

• Decrease steady state phosphorylation levels to 10% of control– decrease steady state level of pp60src by 30%– accelerate turnover of pp60src

(Uehara et al, MCB 6: 2198, 1986)

(Uehara et al, Cancer Res 49: 780, 1989)

Bead 18 Atom Spacer

O

O

R4

NH

O

Me

Me

R2

OMeMeMe

H2 NC(O)O

R1MeO

GELDANAMYCIN BEADSIDENTIFY HSP90 AS BINDING PARTNER

Neckers et al, PNAS 91:8324, 1994

1 2 3 4

p90

R. Lysate

1) Bead-Geld

2) Bead-Geld + Geld

3) Bead-Geld + Geldampicin

4) Bead

X

degradation

nucleus

Xhsp90

ImmatureX

MatureX

ERPRetc

Cyclin D

Hsp 90pAKT EIF2αkinase

raferbB2EGFR

lck, met,etc

nucleus hsp90

*

X

*

A.

B.

C.

ERfolding

G0α

telomerase

hsp90

*hsp90

X-mRNA

DiversityIt is estimated

that there are 1040

compounds in all of “chemical space”.

Since the Big Bang, there have only

been 1017 seconds.

- Peter Wipf

SOURCES OF DIVERSITY

• “Natural Products” = entities derived from plants, animals, bacteria, etc. May have “ethnopharmacognosy” to suggest use- “pure compound” collections- extracts: aqueous/organic- genetically altered producer organisms

• Target non-selected chemical compound libraries-peptide / protein-non-peptide

• Target-directed chemical compound libraries- “classical” medicinal chemistry / bona fide

crystal structure - derived- “docked” lead structures into model

Natural Products: Unique arrays of the four “elements” which make a really useful drug

PhC

O

HN

H

C

HO

Ph

C

H

C

O

O

Me

Ph

AcO

C(O)OH

O

Me

Me

O

AcO

H

MeOH

O

H

Oil(high

dielectric)

Water(low

dielectric)

Acid(+)

Base(-)

Sources of “Modern Drugs”

If one looks at the current drug scene from a chemical perspective (data from

1981 – 2002) then the following slides show reasonable approximations of the

sources of drugs currently approved, World-wide, by the FDA or equivalent body.

Codes are:

N Natural Product

ND Natural Product Derivative

S* Natural Product Pharmacophore

S Synthetic Compound

B/V Biological / Vaccine

(NM) Natural Product Mimic as a subdivision

Sources of Drugs (1981-2002); Extended Subdivisions n = 1031

B12% N

5%

ND23%

S33%

S/NM10%

S*4%

S*/NM10%

V3%

B

N

ND

S

S/NM

S*

S*/NM

V

Newman et al, J. Nat. Prod., 2003, 66, 1027-1037

EXAMPLES OF NP LEAD GENERATION OF NOVEL SCAFFOLDS

Discovery of Lidocaine*Central Asian camels refused to eat a certain type of reed

*Characterization of gramine as the antifeedant principle led to the synthesis of isogramine

*Taste-test: numbness; therefore, lead for anesthetic agent development

NH

NH

NN

NH

ON

Gramine Isogramine LidocaineCourtesy of N. R. Farnsworth

Natural Product Isolation Tree

“You are what you eat”

Dolabella auriculariaDolastatins come from a Symploca species that they graze on

“Non-culturable” versus “Cultured”microbes

•The microbial World has only just been scratched. -Much less than 1% of the available organisms have even beenseen, let alone identified.

• In soil, there are estimates of > 1000 species per gram - very few can be cultured- these may not be representative of the “Soil meta-

Genome”

• Over 1000 microbes per mL of seawater can be seen and only ~ 1% can be cultured using current methods.

SOURCES OF DIVERSITY

• “Natural Products” = entities derived from plants, animals, bacteria, etc. May have “ethnopharmacognosy” to suggest use- “pure compound” collections- extracts: aqueous/organic- genetically altered producer organisms

• Target non-selected chemical compound libraries-peptide / protein-non-peptide

• Target-directed chemical compound libraries- “classical” medicinal chemistry / bona fide

crystal structure - derived- “docked” lead structures into model

TRIPEPTIDE COMBINATORIAL LIBRARY

after R. Houghten, 1999

X X X

Four amino acids in each position43 = 64

A = AlanineR = ArginineT = ThreonineW = Tryptophan

NUMBER OF PEPTIDESPOSSIBLE WITH INCREASING LENGTH


Length Peptide Number

O = Individual Defined Amino Acid

2345678

4008,000

160,0003,200,000

64,000,0001,280,000,000

25,600,000,000

Ac – OO – NH2

Ac – OOO – NH2

Ac – OOOO – NH2

Ac – OOOOO – NH2

Ac – OOOOOO – NH2

Ac – OOOOOOO – NH2

Ac – OOOOOOOO – NH2

IC50 OF MIXTURES

Log Concentration

A single activecompound:

IC50 = 1.0 nM

A single 1.0 nMactive compound

+ 9 inactives:IC50 = 10 nM

A single 1.0 nMactive compound+ 9,999 inactives:IC50 = 10,000 nM

100

75

50

25

0-12 -10 -8 -6 -4 -2-11 -9 -7 -5 -3

COMBINATORIAL LIBRARIES:THE MIXTURE QUESTION


NaturalProductExtracts

SyntheticCombinatorial

Mixtures

Direct screening of compound mixturesDiscovery of highly active compoundsEqual concentrations of compoundsChemical structures knownSynthetic pathway knownStructure – activity relationship known

YesYesYesYesYesYes

YesYesNoNoNoNo

NON-PEPTIDE “COMBINATORIAL” STRATEGIESCOMBINE “SCAFFOLDS” (OR “BACKBONES”)

WITH “FUNCTIONAL GROUPS”

Synthesis Synthesis

“Scaffold” Functional Groups

The Chemical Generation of Molecular Diversity fromhttp://www.netsci.org/Science/Combichem/feature01.html

O-FG2 (O-FG4)

O-FG3FG1-O

THE RULE OF FIVE

• More than 5 H-bond donors• Molecular weight >500• c log P > 5• Sum of N’s and O’s (a rough measure

of H-bond acceptors) > 10

Modern Drug DiscoveryJanuary/February 1999Modern Drug Discovery, 1999, 2 (1), 55-60.Copyright © 1999 by the American Chemical Society

An awareness tool for discovery chemists:Compounds with two or more of the followingcharacteristics are flagged as likely to havepoor oral absorption


COMBINATORIAL LIBRARIES OF BICYCLIC GUANIDINES FROM REDUCED ACYLATED DIPEPTIDES

NH

R1

NHHN

R2

R3

NH2

N

N

N +

R3

R2

R1

H

R1 x R2 x R3 = 49 x 51 x 42 =104,958 compounds

1. CSIm2

2. HF/anisole

BIOASSAYS(READY APPLICATION OF SOLUBLE LIBRARIES)

• Soluble Acceptors- antibodies- enzymes

• Membrane-bound Receptors- tissue homogenate- functional cell based

• Microorganisms: Disruption of Function- bacteria- fungi- virus

• Differentiation- stem cells

• In Vivoafter R. Houghten, 1999


POSITIONAL SCANNING BICYCLIC GUANIDINE LIBRARY (κ RECEPTOR)

1/pe

rcen

t bou

nd

R1 Position0.200.150.100.05

0 5 15 25 35 451 10 20 30 40 50R2 Position

0.140.120.100.080.060.040.02

0 55 65 75 85 9551 60 70 80 90 100

0.200.150.100.05

0

R3 Position

105 115 125 135102 110 120 130 140






• Qualifying Lead for • Transition to Early Trials

" RATIONAL":

-Structure based design

-Biochemical Screen

-Target-driven

Cell-based Screen

"EMPIRICAL"

-Bioassay of effect

NMR-BASED SCREENING

Hajduk et al, J Med Chem 48: 2518, 2005

1. Screen “fragment” like molecules with “leadlike” properties (MW <300; ClogP ~1.5)

2. Characterize binding and portion of molecule to which they bind

3. Ligands with weak affinities can be defined (~KD = 5mM)

4. Lead to high affinity binders through iterative screening

5. Can label protein of interest with isotopes “sensitive” to ligand effects (e.g. N15) and utilize proton resonances of drug to simultaneously allow definition of ligand and receptor binding sites

NMR AS MEANS OF DEFINING BINDING SITES

Horwitz et al, Biochemistry 16: 3641, 1977

E.G., BLEOMYCIN BIMDING TO DNA

BUILDING A DRUG LEAD

Target

Screen 1

Library 1

Lead 1Kd1

Library 2

Lead 2Kd2

Kd3 = K1K2

Library 3

Successive iterations“build”

more potent Kd

AFFINITIES OF

SELECTED BIARYL COMPOUNDS FOR BCL-XL

Petros et al, J Med Chem 49: 656, 2006

SECTION FROM A 15N HSQC SPECTRUM OF BCL-XL IN THE PRESENCE AND

ABSENCE OF COMPOUND


alone (white)2 mM biaryl acid 1(cyan)2 mM biaryl acid 1 and 5 mM naphtholderivative 11 (pink)

SUPERPOSITION OF SEVEN LOW-ENERGY STRUCTURES CALCULATED FOR

BCL-XL COMPLEXED TO 1 AND 11


THREE DIMENSIONAL VIEW OF GELDANAMYCIN BINDING POCKET IN AMINO TERMINUS OF HSP90

Stebbins et al, Cell 89:239, 1997

17-AAG BINDS TO HSP90 & SHARES IMPORTANT BIOLOGIC ACTIVITIES WITH GELDANAMYCIN

dose (nM)

erbB

2 (%

of b

ase

line)

Raf

-1 (%

of b

ase

line)

Schulte & Neckers, Cancer Chemother Pharmacol 42: 273, 1998

120

100

80

60

40

20

00 1 10 100 1000 10000

17-AAGGA

120

100

80

60

40

20

00 1 10 100 1000 10000

17-AAGGA

dose (nM)

p185erbB2

17-AAG GA (μM)

0.03

0.1

0.3

0.5

2 0.03

0.1

0.3

0.5

2

cont

rol

Raf-1

17-AAG GA (μM)

0.03

0.1

0.3

0.5

2 0.03

0.1

0.3

0.5

2

cont

rol







" RATIONAL":


-Biochemical Screen

-Target-driven

Cell-based Screen

"EMPIRICAL"

-Bioassay of effect

MM

G2G2 SS

G1G1

RbRb

RbRb

Cyclin DCyclin D

PP

E2FE2F

E2FE2F

Cyclin ECyclin E

Cdk2Cdk2Cyclin ACyclin A Cdc25ACdc25A

Cdk2Cdk2Cyclin ACyclin A

Cyclin A/BCyclin A/B

Cdk1Cdk1

Cyclin A/BCyclin A/BCdk1Cdk1

Cdc25BCdc25B

Cdc25CCdc25CPP

PP

Cdc25ACdc25A

Cdk2Cdk2Cdk4Cdk4

??

Cell cycle regulation by Cdc25 phosphatases

CancerCausing

Regulation of Cell Cycle Progression by Regulation of Cell Cycle Progression by Cdc25: Cdk ActivationCdc25: Cdk Activation

CdkCdkCyclinCyclin

InactiveInactive InactiveInactive ActiveActive

T14 Y15

T161 CdkCdkCyclinCyclin

T14 Y15

T161

PP PP

PP

CdkCdkCyclinCyclin

T14 Y15

T161

PP

Myt1Myt1

CAKCAK

Wee1Wee1 Cdc25Cdc25

CDC25 Phosphatases and Cancer

• CDC25A and B overexpressed in many cultured cancer cell lines.

• Cdc25A suppresses apoptosis.• Overexpression of CDC25A or B has been detected in

human breast, head and neck, cervical, skin, lymph, lung and gastric cancers.

• Human CDC25A & B cooperated with Ha-RasG12V and CDC25A cooperated with Rb -/- in the oncogenic focus transformation of mouse embryonic fibroblasts and tumor formation in nude mice. Thus, Cdc25A & B may be human oncogenes.

Method for identifying Cdc25 phosphatase inhibitors

GST-Cdc25 in assay buffer

Fluorescein diphosphate

Incubate 1h

RT

Read product (fluorescein monophosphate)

on cytoflour II

Chemical Screening Chemical Screening ApproachApproach

• Targeted Array Libraries• Diverse Chemical Libraries

-7 -6 -5 -4 -30

25

50

75

100 Cdc25AVHRPTP1B

Log [Compound 5] M

Perc

ent I

nhib

ition

C.

Compound 5 inhibits Cdc25Compound 5 inhibits Cdc25

Cdc25B2 Ki ~ 2 μM

+En-SH + OHHS

Cdc25A Cpd 5 Adduct Mercaptoethanol

O

O

SO H

3

O

O

S-En3

MWmonoisotopic = 248.1 Da

% In

tens

ity

12000 60000m/z

0

50

10025444

12738

50978[M+2H]+2

[M+H]+1

[2M+H]+1

25444

12000 60000m/z

0

50

100

% In

tens

ity

25288

[M+2H]+212644

50576[2M+H]+1

[M+H]+125288

Lixia Pu

MALDIMALDI--TOF ANALYSESTOF ANALYSESCompound 5 binds tightly to the catalytic domain of Cdc25ACompound 5 binds tightly to the catalytic domain of Cdc25A

DMSO

Compound 5

Compound ValidationCompound Validation

Cellular: Cell Cycle Biochemical: Substrate phosphorylationGenetic: Chemical complementation

tsFT210 Cell SystemtsFT210 Cell System

tsFT210 cellsCdk1 mutants

G1 G2/M

32o 17 h

39.4o 17 h

No functional Cdk1

Functional Cdk1

Compound 5 causes G2/M arrestCompound 5 causes G2/M arrest

2C 4C

39.4 oC17 h

A

rela

tive

cell

num

bers

32.0 oC

B

6 h, 32.0 o C

+DMSO

C

+Nocodazole1 μΜ

D

+Compound 520 μM

F

+Compound 510 μM

E

+Compound 2620 μM

I

+Compound 2720 μM

HG







" RATIONAL":


-Biochemical Screen

-Target-driven

Cell-based Screen

"EMPIRICAL"

-Bioassay of effect

• The transcription factor C/EBPα plays key roles in regulation of differentation of various cell lineages (adipocytes, keratinocytes, etc.)

• Mutations in CEBPA (the gene coding for C/EBPα ) are associated with development of AML [t(8;21) - subtypes M1 and M2]

• CEBPA knock-out mice show no mature neutrophils

• Conditional expression of CEBPA is sufficient to trigger neutrophilic differentiation

• Pharmacologic modulators of CEBPA could act as differentiationinducers and thus limit proliferation of AML cells

C/EBPα AS A TARGET FOR DEVELOPMENTOF NOVEL CANCER THERAPEUTICS

4 x C/EBP TKmin.

luciferase

CTCGAGAAGGTGTTGCAATCCCCAGCG

CTCGAG AAGGTGTTcaccaaCCCAGC AAGGTGTTcaccaaCCCAGC GTCGAG AAGGTGTTcaccaaCCCAGC AAGGTGTTcaccaaCCCAGC GTCGAC

XhoI

Sal I

Xho I

CEBP Reporter Construct*

*Host cell for this construct is U-937

CEBPA Assay TimelineDispense CellsTo Assay Plates

TransferTest SamplesTo Assay Plates

Incubate Plates At 37oC, 5% CO2, Ambient O2

Add “Bright-Glow”Luciferase Reagent

Read Assay PlatesOn Luminometer

Incubate Plates at RT5% CO2, Ambient O2

Remove PlatesFrom Incubator*

24 Hours 24 Hours One Hour

*Sister plates processed for Alamar blue toxicity assay

C/EBPa Training Set: 1st Run compared to 2nd Run % Induction

y = 1.3969x + 15.927R2 = 0.8584

-200

-150

-100

-50

0

50

100

150

200

-150 -100 -50 0 50 100 150 200

% Induction 1st Run

% In

duct

ion

2nd

Run

Correlation Coefficient = .9265

C/EBPα Training Set 1 uM Results

30

40

50

60

70

80

90

100

110

-100 -50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800

% of Control Induction (relative to .625 uM ~ 100%)

% A

lam

ar F

luor

esce

nce

INACTIVE

TOXIC

619179 Rhodium Complex

102816 Azacitidine

360036 Neolignan

from Clerodendron

Inerme

338259Lycobetaine

693322

224131N-(phosphoacetyl)-L-aspartate (PALA)

*Data averaged from two independent assays

*

C/EBPa Screen: % Concentration Response Graphs % Induction (relative to .625 uM retinoic acid induction) for seven select compounds

-200-100

0100200300400500600700800900

10001100120013001400150016001700

10uM 5uM 2.5uM 1.25uM .625uM .313uM .156uM .078uM .039uM .019uM

Concentration

% o

f Con

trol

Indu

ctio

n

619179

102816

360036

.625

uM

Ret

inoi

c A

cid

NHHN

HN

HNNH

NH

Rh3+

3Cl-

N

O

N

N

O

HOCH2

HO OH

NH2

O

O

O

O

O

O

O

Me

Me

Me

Me

Me

Me

Categories of Confirmed Actives in CEPBα HTS

• β-adrenergic agonists• Toxic compounds (stress signaling)• Retinoids• HDAC Inhibitors• Novel Drug Lead - Sterol mesylate

C/EBPa Frequency of Fold Induction for OSR Compounds in HTS0 7 11 11 15 22 19 41 52 72 11

019

131

871

3 1572

3433

6372

1034

115

136

1983

622

474

2027

715

104

9196

5104

2605

1330

640

280

135

81 35 25 15 19 6 9 3 3 4 4 3 0 3 0 2 2 1 0 00

5000

10000

15000

20000

25000

-1.7

-1.6

-1.5

-1.5

-1.4

-1.3

-1.2

-1.2

-1.1

-1.0

-0.9

-0.9

-0.8

-0.7

-0.6

-0.6

-0.5

-0.4

-0.3

-0.3

-0.2

-0.1 0.0

0.0

0.1

0.2

0.3

0.3

0.4

0.5

0.6

0.6

0.7

0.8

0.9

0.9

1.0

1.1

1.2

1.2

1.3

1.4

1.5

1.5

1.6

1.7

1.8

1.8

1.9

2.0

Fold Induction

Freq

uenc

y

(Total of 135640 Compounds Tested)

0 C

ompo

unds

Und

er -1

.7

8 C

ompo

unds

Ove

r 2

NSC67657 when screened at 1.0µM scored 1.4 fold induction relative to RA control

-20

0

20

40

60

80

100

120

10uM 5uM

2.5u

M

1.25

uM

.625

uM

.313

uM

.156

uM

.078

uM

.039

uM

.019

uM

289336

57654

59264

62786

89747

C/EBPa % Induction Dose Response Curves%

Indu

ctio

n (T

est –

Cel

ls O

nly)

/(Ret

–C

ells

Onl

y)*1

00

Concentration of Test Compounds

289336

57654

59264

62786

89747

Rimiterol HBr

Epinephrine

DL-Isoproterenol HCl

NSC 67657, a novel sterol mesylate inducer of CEBPαwith potential anti-leukemic activity

• Identified in a DTP high-throughput screen of > 140,000 compounds• Induced CEBP-luciferase activity at low concentrations: 50% activation at 40 nM• Induced differentiation in U937cells as measured by CD11b or CD11c antigens or NBT staining• Induced morphologic differentiation in HL60 cells•Induced cell surface markers of monocytic differentiation in AML patient blasts ex vivo

Basis for Interest

Max Fold Induction 1.6 at 10 μM

EC50 > 10 uM

Secondary testing of NSC67657 in C/EBPa (U937) cells

Dose-dependent increase of luciferase reporter activity (max. 1.6 fold)

Based on control induction of retinoic acid (1µM)

Activity occurs at non-toxic concentrations

1/2Max.

Untreated control -largely myeloblasts

40x

Evidence for Morphologic Differentiation in HL60 Cells

1 mM ATRA – Reduced cell numbers,

segmented and cells resembling neutrophils

(arrow)

20 mM NSC 67657 –Reduced cell numbers,

segmented and cells resembling neutrophils

(arrow)

GENERATION OF SAR AROUND STEROID MESYLATE LEAD

• Related compounds available from the DTP Repository were tested in concentration-response format

• No compounds with comparable activity were found (most were completely inactive)

• Three compounds which showed some activity provided an initial SAR model

Key: up regulated genedown regulated geneno changeunexpressed gene or

missing data point

CD14CD14

CES1CAMP

CCL4

COX8A

CCL2

CCL3

NSC 67657 ATRA

120h incubation

When compared to ATRA treated cells, several genes of the monocyte/ macrophage lineage were uniquely up regulated by NSC 67657.

Hierarchical cluster of 51 genes dysregulated >3 fold over control by NSC 67657 in HL60 cells.

COX8A: cytochrome c oxidase subunit 8ACD14 : surface protein preferentially expressed on monocytes/macrophages.CCL4: chemokine (C-C motif) ligand 4;macrophage inflammatory proteinCCL3: chemokine (C-C motif) ligand 3; macrophage inflammatory protein.CES1: carboxylesterase 1 (monocyte/macrophage serine esterase 1)CAMP: cathelicidin antimicrobial peptideCCL2: chemokine (C-C motif) ligand 4;macrophage inflammatory protein

DMSO Control 2µM ATRA 20µM NSC 67657

ATRA induces differentiation (measured by NBT reduction after 7 days) in both HL60 and NB4 cell lines, while NSC 67657 induced differentiation only in HL60 cells. This supports the monocyte/ macrophage lineage specific differentiation proposed from the gene expression studies

HL60 cells: Can differentiate to either

granulocytes or monocyte/macrophages

DMSO Control 2µM ATRA 20µM NSC 67657

NB4 cells:Can only differentiate

into granulocytes

NSC 67657 induces differentiation in different cell lines compared to ATRA

CD18CD14Key:

Control (DMSO)

NSC 67657 (20μM)

& ATRA (2 &10μM)

NB4 cells

HL60 cells

CD14 CD18

NSC 67657No effect

NSC 67657Induces CD14

NSC 67657 induces a different pattern of cell surfacemarkers compared to ATRA

NSC 67657 induced CD14 expression only in HL60, not NB4 cells. ATRA does not induce CD14 expression in either cell line (5 day incubation).

CD14 CD18

INITIAL STRUCTURE-ACTIVITY MODEL







" RATIONAL":


-Biochemical Screen

-Target-driven

Cell-based Screen

"EMPIRICAL"

-Bioassay of effect

NCI IN VITRO DRUG SCREEN

1985 Hypothesis:

Emerging Realities:

• Cell type specific agents• Activity in solid tumors

• Unique patterns of activity, cut across cell types

• Correlations of compound activity

ANDCell type selective patterns found

- relate to molecular “target” expression- generate hypothesis re: molecular target

NCI IN VITRO CANCER CELL LINE SCREEN• 60 cell lines

• 48 hr exposure; protein stain O.D.

(8 breast, 2 prostate, 8 renal, 6 ovary, 7 colon,6 brain, 9 lung, 8 melanoma, 6 hematopoietic)

O.D.

Time

Control

“GI50” = 50% inhibit

“TGI” = 100% inhibit

“LC50” = 50% kill

All Cell Lines

Log10 of Sample Concentration (Molar)

Per

cent

age

Gro

wth

100

50

0

-50

-100 -9 -8 -7 -6 -5 -4

National Cancer Institute Developmental Therapeutics ProgramDose Response Curves

NSC: 643248-Q/2 (a rapamycin) Exp. ID: 9503SC35-46

PATTERN RECOGNITION ALGORITHM:COMPARE

• Goal: COMPARE degree of similarity of a new

• Calculate mean GI50, TGI or LC50• Display behavior of particular cell line as deflection

• Calculate Pearson correlation coefficient:

compound to standard agents

1 = identity ; 0 = no correlation

resistant mean sensitive

from mean

Taxol Halichondrin B Daunorubicin

Topoisomerase II

Leukemia

NSCLC

Small Cell Lung

Colon

CNS

Melanoma

Ovarian

Renal

AGENTS WITH SIMILAR MECHANISMS HAVESIMILAR MEAN GRAPHS

Tubulin

THE COMPARE ALGORITHMSeed: Rubidazone

16401182151

123127665934

DiscreetDiscreet267469305884665935668380639659644946254681

DiscreetDiscreet180510

DiscreetDiscreet

1.0000.9210.9150.8910.8800.8670.8650.8650.8640.8610.8540.8500.8480.8470.8430.8420.8370.833

RubidazoneDaunomycinAdriamycinEpipodophyllotoxin analogueGyrase-To-TOPO analogueAMSA analogueDeoxydoxorubicinAcodazole HCLEpipodophyllotoxin analogueAzatoxin analogueAdriamycin analogueEpipodophyllotoxin analogueDaunomycin analogueEpipodophyllotoxin analogueEpipodophyllotoxin analogueDaunomycin analogueEpipodophyllotoxin analogueGyrase-To-TOPO analogue

RELATIVE EGF RECEPTOR mRNA EXPRESSION

Bre

ast

Pro

stat

e

Ren

al

Ova

rian

Mel

anom

a

CN

S

Col

on

NS

CLC

Leuk

emia

Rel

ativ

e E

xpre

ssio

n

COMPARE ANALYSIS:EGF RECEPTOR

40,421 COMPOUNDS IN THE NCI DATABASE

RANK CORRELATION CHEMICAL NAME1

2

7

88

0.71

0.66

0.57

0.43

TGFα-PE40

Toxin-Δ53L, MW=43KEGFR Tyrosine Kinase Inhibitor

EGFR Tyrosine Kinase Inhibitor

DRUG TARGET CLUSTERINGSREVEAL CLUES TO MECHANISM

Nature Genetics 24: 236, 2000; http://dtp.nci.nih.gov5FU/DPYD L-Asparaginase/ASNS

GOALS OF PRECLINICAL DRUG STUDIES

• IND = “Investigational New Drug” application = approval by FDA to conduct human studies; main criterion : SAFETY AND LIKELY REVERSIBLE TOXICITY = allows start of Phase I trials

• NDA = “New Drug Application” = basis for sale to public; main criteria: SAFETY AND SOME MEASURE OF EFFICACY = result of Phase II/III trials

Regulatory framework

COMPONENTS OF AN IND

• “Form 1571”

• Table of Contents

• Intro Statement / Plan

• Investigator Brochure

• Clinical Protocol

• Chemistry, Manufacture, Control

The goal of the pre-clinical process

• Pharmacology/ Toxicology

• Prior Human Experience

• Additional Info - Data monitoring, Quality Assurance

OBJECTIVES OF PRECLINICAL PHARMACOLOGY STUDIES FOR

ANTI-NEOPLASTIC DRUGS

• Development of Sensitive Analytical Methods for Drugs in Biological Fluids & Tissues

• Determine In Vitro Stability and Protein Binding• Determine Pharmacokinetics in Rodents (& Dogs)• Identification and Analysis of Metabolites• Define Optimal Dose Schedule and Blood Sampling

Times• Define CP and/or AUC with Efficacy, Safety & Toxicity• Analog Evaluation - Determine Optimal Development

Candidate

OBJECTIVES OF PRECLINICAL TOXICOLOGY STUDIES

• DETERMINE IN APPROPRIATE ANIMAL MODELS:

– The Maximum Tolerated Dose (MTD)

– Dose Limiting Toxicities ( DLT )

– Schedule-Dependent Toxicity

– Reversibility of Adverse Effects

– A Safe Clinical Starting Dose

FDA PRECLINICAL PHARMACOLOGY & TOXICOLOGY REQUIREMENTS: ONCOLOGY Rx

• DRUGS– Two Species - Rodent & Non-rodent– Clinical Route & Schedule

• Follow NCI Guidelines

– Pharmacokinetics - Optional

• BIOLOGICALS– Most Relevant Species– Clinical Route & Schedule

CORRELATION BETWEEN 20S PROTEASOMEINHIBITORY POTENCY & GROWTH INHIBITION

FOR 13 DIPEPTIDE BORONIC ACIDS

Adams et al, Cancer Res 59:2615, 1999

Mea

n G

I 50

(nM

)

Ki (nM)

PS-341

PS-273

PS-293

0.1 1 10 100 1000 10000

105

104

103

102

101

100

NH

O

N

N

NH

O

BOH

OH

Correlationr2=0.92

EFFECT OF PS-341ON PC-3 TUMOR GROWTH IN MICETu

mor

Vol

ume

(% V

ehic

le)

Week

Treatment

700

600

500

400

300

200

100

00 1 2 3 4 5 6

PS-3411.0 mg/kg(n=10)

PS-3410.3 mg/kg(n=15)

Vehicle(n=15)


EFFECT OF PS-341ON 20S PROTEASOME ACTIVITY

120

100

80

60

40

20

00.1 0.3 1.0 3.0VehiclePS-341 (mg/kg)

20S

Act

ivity

(% V

ehic

le)

Mouse WBC PC-3


120

100

80

60

40

20

0

PS-341 (mg/kg) 0.3 0.6Vehicle

20S

Act

ivity

(% V

ehic

le)

PS-341: INTERSPECIES DOSE RELATIONSHIP

*In white blood cells at 1.0 h, post*In white blood cells at 1.0 h, post--dosedose

Q: Is the Q: Is the ‘‘safesafe’’ dose in animals in the efficacy dose in animals in the efficacy range for man?range for man?

Ref: Adams, et al, Cancer Res 59:2615, 1999

Species

Dose (mg/kg)

Dose (mg/m2)

% 20S Proteasome Inhibition*

Mouse 1.0 3.0 80

Rat 0.25 1.5 80

NHP 0.067 0.8 70

0.1 1 100

20

40

60

80

100

120 MDACCMSKCCMayoNYUWisconsinUNCDFCICortes

1.96 mg/m2

PS-341 (Log dose, mg/m2)

% 2

0S A

ctiv

ity

Ex Vivo Proteasome Activity:Ex Vivo Proteasome Activity:1 Hour Post Treatment1 Hour Post Treatment

ACKNOWLEDGEMENTS

NCIJ. TomaszewskiM. AlleyM. Hollingshead / S. StinsonJ. JohnsonA. Monks / N. ScudieroS. BatesD. Zaharevitz / R. GussioS. DeckerR. Shoemaker / M. Currens

J. AdamsMillenium

J. LazoU. Pittsburgh

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