# Presentation: 582730

## DRUG DISCOVERY

**Edward A. Sausville, M.D., Ph.D.**
**Associate Director for Clinical Research**
**Greenebaum Cancer Center**
**University of Maryland at Baltimore**

## 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?

**DRUG DISCOVERY:****WHERE HAS IT WORKED?**

*Nature 384 suppl 11:5, 1996*

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

## DRUG DISCOVERY:A SUCCESSION OF STYLES

**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 compounds
- 	vs. many targets

## WHY COMPOUNDSFAIL AND SLOW DOWN IN DEVELOPMENT

**WHY COMPOUNDS****FAIL AND SLOW DOWN IN DEVELOPMENT**

- Modern Drug Discovery
- January/February 1999
*Modern 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&DSuffers High Attrition*

**TRADITIONAL PHARMACEUTICAL R&D****Suffers High Attrition***

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

## TRADITIONAL PHARMACEUTICAL R&DCostly* and Time Consuming**

**TRADITIONAL PHARMACEUTICAL R&D****Costly* and Time Consuming****

** Lehman Brothers, 1997; ** Tufts CSDD*

- Lead Discovery
- Research
**6 Years**

- Drug Development
**8.9 Years**

- $608m*

## OUTLINE OF PRESENTATION

-  General Introduction
-  ***Definition of Drug Targets***
-  Generating Diversity
-  Definition of Lead Structures
-  Qualifying Lead for Transition to Early Trials

## TWO CONTRASTING DRUG-DISCOVERY “PHILOSOPHIES”

-   “EMPIRICAL”: Recognize initial drug lead
-    by functionally useful effect
-    -E.g. : penicillin (anti-bacterial effect)
-               rauwolfia (anti-hypertensive)
-               taxol (anti-tumor)
-               digoxin (cardiotonic / antiarrythmic)
-   “RATIONAL”: Recognize drug by design or screen
-     against biochemical target’s function
-     -E.g.: HIV-protease inhibitor (anti-infection)
-               metoprolol (anti-hypertensive)
-               methotrexate (anti-tumor)

## “EMPIRICAL” DRUG DISCOVERY

- SCREEN

*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 of
-     screening 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 models
-       of cancer have at best 50% chance of >1 P2 trial for
-       non=targeted cancer Rx’s
-    Divorced from mechanism: an intriguing lead must
-     be “deconvolutedh

## KRN5500

**KRN5500**

**H****3****C(CH****2****)****8****CH=CHCH=CHCO****2****H**

- Cell Membrane

- Deacylation

- SAN-Gly

- Protein Synthesis

## EFFECT OF KRN5500 ON COLO-205 ATHYMIC MOUSE XENOGRAFTS

- Median Tumor Weight (mg)

- Day Posttumor Implantation

- 5000
- 4500
- 4000
- 3500
- 3000
- 2500
- 2000
- 1500
- 1000
- 500
- 0

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

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

- Plasma Concentration (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 M at 3.7 mg/M2/d x 5
-   4/6 patients with >25% incr Cmax have

- grade 4 toxicity

*Data of J. P. Eder, DFCI*

## “RATIONAL” DRUG DISCOVERY

- PHARMACOLOGY
- (to affect target)

- CHEMISTRY

- MOLECULAR TARGET SCREEN

- Biochemical
- Engineered cell
- Animal (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 phase
- Adjunct to bone marrow transplantation

## bcr-abl  FUSION PROTEIN

- bcr

- SH2

- SH2

- V

- SH2/SH3

- kinase

- NT

- DNA

- Actin

- bcr

- autophosphorylation

- Phosphorylation of
- other substances

*McWhirter JR, *EMBO* 12:1533, 1993*

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

- erbstatin

- lavendustin

- piceatannol

- AG957

- AG1112

**CGP 57148B = STI571**

- Natural
- product
- empiric lead
- 1st generation
- synthetic
- 2nd generation
- synthetic;
- in clinic

## STI571: An oral in vivo bcr-abl kinase inhibitor

**STI571: An oral in vivo bcr-abl kinase inhibitor**

**Tyr phosphorylation *****in vivo***

*le Coutre et al, JNCI 91:163, 1999*

**Antitumor activity *****in vivo***

- (hrs)

- (days)

- (days)

## EFFICACY AND SAFETY OF A SPECIFIC INHIBITOR OF THE BCR-ABLTYROSINE KINASE IN CHRONIC MYELOID LEUKEMIA

**EFFICACY AND SAFETY OF A SPECIFIC INHIBITOR OF THE BCR-ABL****TYROSINE 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.

## MOLECULAR TARGET DEFINITION - HOW TO?

- *  “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, together

- with the amount of gene products produced for each, yields a
- powerful "fingerprint". Comparing the fingerprints of a normal versus
- a cancer cell will highlight genes that by their suspicious absence or
- presence (such as Gene H ) deserve further scientific scrutiny to
- determine whether such suspects play a role in cancer, or can be
- exploited 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/

- <date/time>

**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)

- Probability

- All patients

- All patients

- Low clinical risk pts

- Probability

- Overall survival (yrs)

## GELDANAMYCIN: EXAMPLE OF BINDING PARTNER DEFINING TARGET

**GELDANAMYCIN: EXAMPLE OF BINDING PARTNER DEFINING TARGET**

## BENZOQUINOID ANSAMYCINSINITIAL 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)*

## 18 Atom Spacer

**18 Atom Spacer**

## GELDANAMYCIN BEADSIDENTIFY HSP90 AS BINDING PARTNER

*Neckers et al, PNAS 91:8324, 1994*

**1)  Bead-Geld**
**2)  Bead-Geld + Geld**

**3)  Bead-Geld + Geldampicin**
**4)  Bead**

## *

- X

- degradation

- nucleus

- X

**hsp**
**90**

- Immature
- X

- Mature
- X

**ER**
**PR**
**etc**

**Cyclin D**

**Hsp 90**

**pAKT**

**EIF2******
**kinase**

**raf**

**erbB2**
**EGFR**
**lck, met,**
**etc**

- nucleus

**hsp**
**90**

- X

**A.**

**B.**

**C.**

- ER

- folding

**G****0******

**telomerase**

**hsp**
**90**

**hsp**
**90**

- X-mRNA

## OUTLINE OF PRESENTATION

-  General Introduction
-  Definition of Drug Targets
-  ***Generating Diversity***
- Definition of Lead Structures
- Qualifying Lead for Transition to Early Trials

## Slide 32

## 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

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

## 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 

***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**

***Courtesy of N. R. Farnsworth***

## Natural Product Isolation Tree

## “You are what you eat”

***Dolabella auricularia***** **
**Dolastatins 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 been**
**   ****seen, 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

**TRIPEPTIDE COMBINATORIAL LIBRARY**

*after R. Houghten, 1999*

- X X X

- Four amino acids in each position
- 43 = 64

- A = Alanine
- R = Arginine
- T  = Threonine
- W = Tryptophan

## NUMBER OF PEPTIDESPOSSIBLE WITH INCREASING LENGTH

**NUMBER OF PEPTIDES****POSSIBLE WITH INCREASING LENGTH**

*after R. Houghten, 1999*

- O = Individual Defined Amino Acid

## IC50 OF MIXTURES

**IC****50**** OF MIXTURES**

- Log Concentration

## COMBINATORIAL LIBRARIES:THE MIXTURE QUESTION

**COMBINATORIAL LIBRARIES:****THE MIXTURE QUESTION**

*after R. Houghten, 1999*

## NON-PEPTIDE “COMBINATORIAL” STRATEGIESCOMBINE “SCAFFOLDS” (OR “BACKBONES”)WITH “FUNCTIONAL GROUPS”

- The Chemical Generation of Molecular Diversity from
*http://www.netsci.org/Science/Combichem/feature01.html*

## THE RULE OF FIVE

**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 Discovery
- January/February 1999
*Modern 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 following
- 	characteristics are flagged as likely to have
- 	poor oral absorption

## COMBINATORIAL LIBRARIES OF BICYCLIC GUANIDINES FROM REDUCED ACYLATED DIPEPTIDES

*after R. Houghten, 1999*

**COMBINATORIAL LIBRARIES OF BICYCLIC GUANIDINES FROM REDUCED ACYLATED DIPEPTIDES**

## BIOASSAYS(READY APPLICATION OF SOLUBLE LIBRARIES)

**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 Vivo*

*after R. Houghten, 1999*

## POSITIONAL SCANNING BICYCLIC GUANIDINE LIBRARY ( RECEPTOR)

*after R. Houghten, 1999*

**POSITIONAL SCANNING BICYCLIC GUANIDINE LIBRARY (******** RECEPTOR)**

- 1/percent bound

## OUTLINE OF PRESENTATION

-  General Introduction
-  Definition of Drug Targets
-  Generating Diversity
***Definition of Lead Structures***
- Qualifying Lead for Transition to Early Trials

-         -***Structure based design***
- -Biochemical Screen
- -Cell-based Screen

## NMR-BASED SCREENING

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

- Screen “fragment” like molecules with “leadlike” properties   (MW <300; ClogP ~1.5)
- Characterize ***binding*** and portion of molecule to which they bind
- Ligands with weak affinities can be defined (~KD = 5mM)
- Lead to high affinity binders through iterative screening
- 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

## BLEOMYCIN METAL & DNA BINDING DOMAINS

- Metal Binding/
- Oxygen Activation

- DNA Binding

*Sausville et al;*
*Biochem 17: 2740, 1978*

- DNA* • BLM • Fe(III)

- •OH+OH-

- H2O2

- •O2-

- •O2-+2H+

- O2

- H2O2

- DNA • BLM • Fe(II)

- DNA • BLM

- DNA + BLM

- Fe(III)

- Fe(II)

- •O2-+2H+

- H2O2

- •O2-

- O2

- ascorbate
- ascorbate•

- RS-
- RS•

*OR*

*OR*

*OR*

## 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 1
- Kd1

- Library 2

- Lead 2
- Kd2

- 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 

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

- alone (white)
- 2 mM biaryl acid **1** (cyan)
- 2 mM biaryl acid **1** and 5 mM naphthol derivative **11** (pink)

## SUPERPOSITION OF SEVEN LOW-ENERGY STRUCTURES CALCULATED FOR BCL-XL COMPLEXED TO 1 AND 11 

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

## 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

- <date/time>

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

- dose (nM)

- erbB2 (% of base line)

- Raf-1 (% of base line)

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

- dose (nM)

## OUTLINE OF PRESENTATION

-  General Introduction
-  Definition of Drug Targets
-  Generating Diversity
***Definition of Lead Structures***
- Qualifying Lead for Transition to Early Trials

-       -Structure based design
-    -***Biochemical Screen***
- -Cell-based Screen

## Cell cycle regulation by Cdc25 phosphatases

- <date/time>

**M**

**G2**

**S**

**G1**

**P**

**E2F**

**E2F**

**Cdk2**

**Cyclin A**

**Cdk2**

**Cyclin A**

**Cyclin A/B**

**Cdk1**

**Cyclin A/B**

**Cdk1**

**Cdc25B**

**Cdc25C**

**P**

**P**

**Cdk2**

**Cdk4**

**?**

**Cell cycle regulation by Cdc25 phosphatases**

**Cancer**
**Causing**

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

- <date/time>

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

**Cdk**

**Cyclin**

**Inactive**

**Inactive**

**Active**

- T14

- Y15

- T161

**Cdk**

**Cyclin**

- T14

- Y15

- T161

**Cdk**

**Cyclin**

- T14

- Y15

- T161

**Myt1**

**CAK**

**Wee1**

**Cdc25**

## 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 Approach

-  Targeted Array Libraries
-  Diverse Chemical Libraries

## Slide 68

## Compound 5 inhibits Cdc25

**Compound 5 inhibits Cdc25**

- Cdc25B2 Ki ~ 2 M

## <footer>

- <date/time>

## Compound Validation

** ****Cellular: Cell Cycle **
** ****Biochemical: Substrate phosphorylation**
** ****Genetic: Chemical complementation**

## tsFT210 Cell System 

- tsFT210 cells
- Cdk1 mutants

**G1   G2/M**

**32****o**** 17 h**

**39.4****o**** 17 h**

**No functional Cdk1**

**Functional Cdk1**

## Compound 5 causes G2/M arrest

## OUTLINE OF PRESENTATION

-  General Introduction
-  Definition of Drug Targets
-  Generating Diversity
***Definition of Lead Structures***
- Qualifying Lead for Transition to Early Trials

-     -Structure based design
- -Biochemical Screen
- -***Cell-based Screen ***

## C/EBP AS A TARGET FOR DEVELOPMENT

**  ****Mutations in CEBPA (the gene coding for C/EBP******** ) 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 differentiation inducers and thus limit proliferation of AML cells**

**C/EBP******** AS A TARGET FOR DEVELOPMENT**
** ****OF NOVEL CANCER THERAPEUTICS**

## CEBP Reporter Construct*

**4 x C/EBP**

**TK**
**min.**

**luciferase**

_**CTCGAG**_** AAGGTGTTcaccaaCCCAGC AAGGTGTTcaccaaCCCAGC GTCGAG **
**AAGGTGTTcaccaaCCCAGC AAGGTGTTcaccaaCCCAGC **_**GTCGAC**_

**XhoI**

**Sal I**

**Xho I**

- *Host cell for this construct is U-937

## CEBPA Assay Timeline

- Dispense Cells
- To Assay Plates

- Transfer
- Test Samples
- To Assay Plates

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

- Add “Bright-Glow”
- Luciferase Reagent

- Read Assay Plates
- On Luminometer

- Incubate Plates at RT
- 5% CO2, Ambient O2

- Remove Plates
- From Incubator*

- 24 Hours

- 24 Hours

- One Hour

- *Sister plates processed for Alamar blue toxicity assay

## Slide 78

## *

***Data averaged from two independent assays**

*****

## Slide 80

## C/EBPa % Induction Dose Response Curves

**C/EBPa % Induction Dose Response Curves**

**% Induction (Test – Cells Only)/(Ret – Cells Only)*100 **

**Concentration  of Test Compounds**

**57654**

**59264**

**62786**

**89747**

**Rimiterol HBr**

**Epinephrine**

- DL-Isoproterenol HCl

## 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

**AND**

- Cell 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**

**“****GI****50****” = 50% inhibit**

**“****TGI” = 100% inhibit**

**“****LC****50****” = 50% kill**

## All Cell Lines

- Log10 of Sample Concentration (Molar)

- Percentage Growth

## 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

## AGENTS WITH SIMILAR MECHANISMS HAVESIMILAR MEAN GRAPHS

- Taxol

- Halichondrin B

- Daunorubicin
- Topoisomerase II

- Leukemia
- NSCLC
- Small Cell Lung
- Colon
- CNS
- Melanoma
- Ovarian
- Renal

**AGENTS WITH SIMILAR MECHANISMS HAVE****SIMILAR MEAN GRAPHS**

- Tubulin

## THE COMPARE ALGORITHMSeed: Rubidazone

- 164011
- 82151
- 123127
- 665934Discreet
- Discreet
- 267469
- 305884
- 665935
- 668380
- 639659
- 644946
- 254681
- Discreet
- Discreet
- 180510
- Discreet
- Discreet

- 1.000
- 0.921
- 0.915
- 0.891
- 0.880
- 0.867
- 0.865
- 0.865
- 0.864
- 0.861
- 0.854
- 0.850
- 0.848
- 0.847
- 0.843
- 0.842
- 0.837
- 0.833

- Rubidazone
- Daunomycin
- Adriamycin
- Epipodophyllotoxin analogue
- Gyrase-To-TOPO analogue
- AMSA analogue
- Deoxydoxorubicin
- Acodazole HCL
- Epipodophyllotoxin analogue
- Azatoxin analogue
- Adriamycin analogue
- Epipodophyllotoxin analogue
- Daunomycin analogue
- Epipodophyllotoxin analogue
- Epipodophyllotoxin analogue
- Daunomycin analogue
- Epipodophyllotoxin analogue
- Gyrase-To-TOPO analogue

## RELATIVE EGF RECEPTOR mRNA EXPRESSION

- Breast

- Prostate

- Renal

- Ovarian

- Melanoma

- CNS

- Colon

- NSCLC

- Leukemia

- Relative Expression

## COMPARE ANALYSIS:EGF RECEPTOR

*40,421 COMPOUNDS IN THE NCI DATABASE*

**RANK**

**CORRELATION**

**CHEMICAL NAME**

- 1
- 2
- 7
- 88

- 0.71
- 0.66
- 0.57
- 0.43

- TGF-PE40
- Toxin-53L, MW=43K
- EGFR Tyrosine Kinase Inhibitor
- EGFR Tyrosine Kinase Inhibitor

## DRUG TARGET CLUSTERINGSREVEAL CLUES TO MECHANISM

*Nature Genetics 24: 236, 2000; http://dtp.nci.nih.gov*

## WHAT TO DO AFTER THE LEAD COMPOUND IS IDENTIFIED?

-  If “*empirical” *lead, OPTIMIZE: schedule; formulation vs. biological effect after identifying a “predictive” model; ideally bring series of close analogs through pharmacology and perhaps early toxicology
-  If “*rational”* lead, capitalize on target-directed effects in optimizing process
- IN EITHER CASE, KEY TASKS ARE: ACTIVITY; PHARMACOLOGY; FORMULATION; SAFETY TESTING

## OUTLINE OF PRESENTATION

-  General Introduction
-  Definition of Drug Targets
-  Generating Diversity
-  Definition of Lead Structures
*** ******Qualifying Lead for Transition to Early Trials***

## 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 PROTEASOMEINHIBITORY POTENCY & GROWTH INHIBITIONFOR 13 DIPEPTIDE BORONIC ACIDS

- <date/time>

**CORRELATION BETWEEN 20S PROTEASOME****INHIBITORY POTENCY & GROWTH INHIBITION****FOR 13 DIPEPTIDE BORONIC ACIDS**

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

- Mean GI50 (nM)

- Ki (nM)

- Correlation
- r2=0.92

## EFFECT OF PS-341ON PC-3 TUMOR GROWTH IN MICE 

- Tumor Volume (% Vehicle)

- Week

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

## EFFECT OF PS-341ON 20S PROTEASOME ACTIVITY

- 20S Activity (% Vehicle)

- Mouse WBC

- PC-3

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

- 20S Activity (% Vehicle)

##    PS-341:  INTERSPECIES DOSE RELATIONSHIP

- <date/time>

-    PS-341:  INTERSPECIES DOSE RELATIONSHIP

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

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

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

## Ex Vivo Proteasome Activity:1 Hour Post Treatment

**1.96 mg/m****2**

**PS-341 (Log dose, mg/m****2****)**

**% 20S Activity**

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

## ACKNOWLEDGEMENTS

**NCI**

-  J. Tomaszewski
-  M. Alley
-  M. Hollingshead / S. Stinson
-  J. Johnson
-  A. Monks / N. Scudiero
-  S. Bates
-  D. Zaharevitz / R. Gussio
-  S. Decker
-  R. Shoemaker / M. Currens

- J. Adams
-       *Millenium*
- J. Lazo
-       *U. Pittsburgh*