drug%discovery%for%malaria%dbbs.wustl.edu/curstudents/documents/markey/2014-09-09 markey...lead...

Marvin J. Meyers, Ph.D.

Markey Pathway Washington University in St Louis

September 9, 2014

CWHM Mission: Discover and advance to the clinic affordable new therapies for unmet medical needs with an emphasis on orphan diseases,

neglected diseases, and diseases of poverty

Drug Discovery for Malaria I. Introduc6on to Drug Discovery II. Applica6on to Iden6fica6on of New An6malarial Agents

Research & Development Process

2

Target Discovery Lead Discovery Lead

Optimization Preclinical

Development

•  Disease identification & therapeutic needs

•  Target identification

•  Target validation

•  Assay development •  Hit identification •  High-throughput

screening (HTS) •  Structure-activity

relationships (SAR) •  Medicinal chemistry

“hit-to-lead” •  Mechanism of

action (MOA)

•  Medicinal chemistry optimization

•  Pharmacodynamics (PD) – drug interaction with the target

•  Pharmacokinetics (PK) – getting the drug to the target

•  Absorption, distribution, metabolism, and excretion (ADME)

•  Efficacy in animal models

•  Safety & Toxicology •  Process Chemistry

& Scale-up •  Formulation &

stability •  ADME &

Pharmacology •  Patents filed •  Regulatory package

Investigational New Drug (IND)

Application

Drug Discovery: 3-7 years

Development: 7-10 years

Research & Development Process

3

FDA Review & Approval: New Drug

Application (NDA)

Phase I Clinical Trials

Phase II Clinical Trials

Phase III Clinical Trials

Phase IV

•  Determine drug safety

•  “Is the drug safe to test?”

•  10-100 healthy volunteers

•  Unexpected side effects may be identified

•  Determine drug efficacy

•  “How well does the drug work?”

•  100-500 patients •  Most drugs fail in

Ph II due to lack of sufficient efficacy

•  Confirm findings in large patient population

•  1000-5000 patients

•  Greater chance to find rare side effects

•  Compare to “gold standard” treatment

•  Monitor long-term safety

•  Patient population more heterogeneous

•  Adverse reactions may lead to withdrawal of drug


Commercialization

•  Overall R&D process takes 10-‐17 years •  Overall average cost of a new drug ~$1B

~10,000 compounds synthesized ~100 tested in animals ~10 reach Phase I clinical trials ~1 reaches the market

4



Development Investigational New Drug (IND)

Application



Translational Drug Discovery

Selec6ng a Drug Target

•  Understanding the biology relevant to the disease of interest is criKcal to success •  This is not always well understood at the beginning of a drug discovery project •  Not all targets will produce the desired biological effect or to a sufficient degree to

be efficacious •  Historically, many drugs have been developed where the drug target or biological

mechanism was not known

5

Lead Discovery

•  Bioassays must be developed in order to idenKfy and prioriKze “hits” for medicinal chemistry opKmizaKon

•  Some assays may be used to eliminate certain compounds for further consideraKon –  Off-‐target selecKvity, cytotoxicity, etc.

DefiniKons: •  Hit – a compound that has some acKvity against the chosen biological

target •  Lead – a compound that has some acKvity against the chosen biological

target, belongs to a class of compounds that show promise to lead to a drug, but is not good enough to be the drug itself

•  TesKng Funnel – a set of assays used to filter compounds to idenKfy promising leads

6

Finding a Hit or Lead Compound

7

High Throughput Screening (HTS)

8

100’s to 1,000,000’s of compounds

HTS Assay

Hits

Screening hits are potential leads that need to be validated!

Hit Triage: Valida6ng the hit

•  Is the hit real? Need to validate!

•  Confirm idenKty –  Resynthesize or purchase pure compound and retest

•  Dose response curve (IC50 determinaKon) •  Orthogonal tesKng •  Secondary tesKng •  Confirm mechanism of acKon

9

Selec6ng the Lead

•  This is a process—mulKple hits/leads may be selected iniKally for followup (Hit-‐to-‐Lead phase) –  Don’t know which hit/lead will be result in a drug

•  Is the hit drug-‐like? •  Is there good potenKal for opKmizaKon? •  SelecKvity, solubility, toxicity, etc. •  Is it patentable? •  Ease of synthesis? •  Are there related compounds with SAR that makes sense?

10

Drug design is the applica6on of medicinal chemistry towards the op6miza6on of PD and PK

•  Pharmacodynamics (PD) refers to what the drug does to the body –  E.g., opKmizaKon of interacKons of a drug with its target

•  PharmacokineKcs (PK) refers to what the body does to the drug –  E.g., opKmizaKon of the ability of a drug to reach its target

Aim -‐ To op6mize binding interac6ons with target

•  To increase ac6vity and reduce dose levels •  To increase selec6vity and reduce side effects Reasons

Op6mizing Pharmacodynamics

Types of Binding Interac6ons

•  ElectrostaKc or ionic bonds (20-‐40 kJ/mol) •  Hydrogen bonds (16-‐60 kJ/mol)

–  Hydrogen bond acceptor (HBA) –  Hydrogen bond donor (HBD)

•  Van der Waals interacKons (2-‐4 kJ/mol) •  Dipole-‐dipole/ion-‐dipole interacKons •  Repulsive interacKons •  Hydrophobic interacKons

13

Structure-‐Ac6vity Rela6onships (SAR)

•  RelaKonship between the chemical structure of a molecule and its biological acKvity

•  Determining the SAR allows for opKmizaKon of potency and reducKon of off-‐target toxicity

•  Typically specific chemical groups are modified, replaced or added to determine the SAR

14

OH

CWHM-0263D7 = 1100 nM

CWHM-1233D7 = 222 nM

O

O

CWHM-0253D7 = 7600 nM

ClCWHM-122

3D7 = 14000 nM

OH

O

CWHM-0273D7 = 560 nM

O

CWHM-1213D7 = 16000 nM 1x

-8x

2x

2x

-28x

-60x 5x

OH

CWHM-0283D7 = 6000 nM

O

-6x

N

NN

O

OR3

OH

TCMDC-124587CWHM-023

3D7 IC50 = 940 nMO

OH

Cl

H-bond donor or acceptor

aromatic center

basic or positive center

H-bond donor or acceptor

aromatic center

basic or positive center

Pharmacophore

O H

C O 2 A S P

SER

PHE

Binding site

Developing a Pharmacophore

A pharmacophore is a model which describes important characteristics for binding to a target

Pharmacokine6cs: Access to the target

•  Aqueous Solubility – drug needs to dissolve

•  AbsorpKon – drug needs to reach blood stream

•  DistribuKon – drug needs to reach the target

•  Metabolism – need drug stability to maintain sufficient concentraKon to affect target over Kme (half-‐life)

•  ExcreKon – eliminaKon of drug in a reasonable Kme period

AD

ME

Pharmacokine6cs & Drug Design

•  Drugs must be sufficiently polar to be soluble in aqueous condiKons •  Drugs must be sufficiently polar to interact with molecular targets •  Drugs must be sufficiently ‘greasy’ to cross cell membranes •  Drugs must be sufficiently ‘greasy’ to avoid rapid excreKon •  Drugs must have a balance of both hydrophilic and lipophilic

characteris?cs

Pharmacokine6c (PK) Parameters

1

10

100

1000

10000

0 2 4 6 8 10 12

Plasma Co

ncen

tra6

ons

(ng/mL)

Time (hr)

IV (1 mg/kg)

PO (2.5 mg/kg)

PK Parameters (rat) CL 18 mL/min/kg T½ 0.4 hour Vdss 0.6 L/kg BA 20%

•  Clearance (CL) – rate at which a compound is cleared from the body •  Half-‐life (t1/2) – period of Kme in which half of the compound has been

eliminated from the plasma •  Volume (V) – represents how widely the compound is distributed to the

Kssues •  Bioavailability (BA or %F) – % of compound that reaches the blood stream

from the oral dose

Many Hurdles for Preclinical Drug Discovery

19

•  Find right target •  Develop assays •  Find a lead •  OpKmize potency •  Increase selecKvity •  Needs to be absorbed •  Reduce metabolism •  Geung to the target

Kssue •  Low toxicity •  Efficacious in animal

model •  Not too difficult to

synthesize •  Stable to long-‐term

storage •  Crystalline compound •  Aqueous solubility •  Etc…

Alonso & Tanner, Nature Medicine 2013, 19, 150-155

•  ~225 million cases/yr •  ~1 million deaths/yr •  In Africa:

–  $1.8B healthcare costs –  Loss of $12B GDP

•  Resistance to most exisKng drugs is widespread

“Malaria dispropor+onately affects poor people who cannot afford treatment or have limited access to health care, trapping families and communi+es in a downward spiral of poverty.”

– World Health Organiza6on website

Malaria – The Need for New An6malarials

Plasmodium Life Cycle

Four Species P. falciparum P. vivax P. ovale P. malariae

Malaria Medicine Cabinet

N

OH

HN

NCl

amodiaquine

NHN

NCl

chloroquine

HO

O

N

N

quinine

4-Aminoquinolines Aryl-amino alcohols 8-Aminoquinolines

Antifolates Peroxides

NH

N CF3CF3

HOH

mefloquine

NH2HNN

Oprimaquine

N

N

Cl NN

N

ClN

piperaquine

Napthoquinone

H2N

S NH

O O NN

OO

N

N NH2

ClNH2 Cl

HN

HN

NH

HN

NH

Cl

OHO

O

H

H

atovaquoneproguanilpyrimethaminesulfadoxine

Cl

Cl

ClOH

N

lumefantrine halofantrine

OH

N

Cl

Cl

CF3

O

O

O

H OO

H

H

O

O

O

H OO

H

H R

artemisinindihydroartemisinin R = Hartemether R = Meartesunate R = COCH2CH2CO2-

Goal: Iden6fy an6malarial drug with novel chemotype and mechanism of ac6on

Poten6al Targets for New Malaria Drugs

Kappe et al, 2010 Science 328: 862

Russo et al., Nature 2010, 463, 632 Boddey et al., Nature 2010, 463, 627

Plasmodium Aspar6c Proteases

Asp Protease Func6on(s) Poten6al for Drug Disovery

Plasmepsins I-‐IV Degrade hemoglobin in red blood cells for food source

Poor—funcKonally redundant with each other and falcipains

Plasmepsin V Regulates export of hundreds of parasite proteins; potenKal

role in both BS and LS

AxracKve but challenging—essenKal gene, but limited tools/assays

Plasmepsins VI-‐X Unknown Unknown

Signal PepKde PepKdase

Degrade unstable proteins; potenKal role in mulKple life

stages

AxracKve but challenging—essenKal gene, but limited tools/assays

Aspar6c Proteases are Druggable Targets •  Renin for hypertension •  β-‐secretase (BACE) for Alzheimer’s Disease •  HIV-‐1 Protease Inhibitors

–  Ten FDA-‐approved marketed drugs, mostly based on transiKon-‐state mimics –  Structure-‐based drug design (SBDD) played major role in HIV-‐1 protease drug discovery –  Major challenges in protease inhibitor drug development: high MW, poor absorpKon,

cell permeability, poor aqueous solubility, high hepaKc clearance

HN N

OH

PhO

NH

O

NH

ON

H2NO H

H HN N

OH

SO

NH

O

H

H

Ph

HO

OHN N

OH

PhO

S

O

OH

HO

O

NH2

saquinavir nelfinavir

darunavir

1995 1997

2006

O

OH

O

HNS

O ON

CF3

tipranavir2005

Plasmodium falciparum has at least 11 asparKc proteases

Hybrid Approach -‐IdenKfy target of a phenotypic hit

-‐Use tools of target based drug discovery to opKmize

How do we iden6fy a new an6malarial drug lead?

Phenotypic Approach

–  Iden6fies compounds that kill the parasite –  Compounds ac6ve in cells—more “drug-‐like”

–  Source for most an6malarial drugs –  Target is usually not known making op6miza6on emperical

Parasite dies

Target-‐based Approach –  Inhibi6on of a specific protein target will lead to parasite death (presumably) – High throughput tools for op6miza6on: enzyme assays, structure-‐based drug design –  Potent inhibitors may not have good cell/in vivo potency

Inhibition of target or

mechanism

Phenotypic High Throughput Screens

Strategy: Mine phenotypic screening databases for drug-‐like aspar6c protease inhibitors as good lead compounds for

op6miza6on as drugs

Aspartic Protease Inhibitors

Antimalarial Compounds

•  Renin inhibitors •  HIV Protease inhibitors •  BACE inhibitors •  Cathepsin D/E inhibitors

•  GSK TCAMS •  SJCRH collection •  Novartis’ “Malaria Box” •  Scientific literature

Lead compounds for op?miza?on as new an?malarial agents

Pf Asp Protease

inhibitors?

Target-based Approach

Phenotypic Approach

ONH

P2 OHN

P1 ONH

P1' OHN

P2'NH

O

O-H

O

OHO-H

ONH

P2 HN

P1 ONH

P1' OHN

P2'NH

O

O

H-O

O

O OHH

ONH

P2 OHN

P1 OH2N

P1' OHN

P2'NH

O

O-H

O

O

OH

Substrate Protein Transition-State Intermediate Hydrolysis Products

Asp Asp'

Inhibi6on of the Asp Protease Mechanism

Phritonavir

OHN

NH

HN N

NS

O

OOH

O

NS

Ph

Known Inhibitors of Asp Protease β-‐Secretase

Blue structures used in substructure search of the Tres Cantos AnKmalarial Set (TCAMS)

= key catalytic Asp residue-binding moiety

An6malarial Aspar6c Protease Inhibitors in TCAMS

Gamo et al, Nature, 2010, 465, 305-310

209 examples in TCAMS Antimalarial EC50 30-5000 nM

35 examples in TCAMS Antimalarial EC50 140-1500 nM

1 examples in TCAMS Antimalarial EC50 840 nM

Hydroxyethylamines Aminohydantoins Spiropiperidines

Are these plasmepsin inhibitors? Are they good drug-‐like starKng points?

Lipinski’s Rule of Five proper6es of “Drug-‐like” molecules -‐5 or less hydrogen bond donors -‐10 or less hydrogen bond acceptors -‐Molecular weight under 500 -‐ParKKon coefficient log P less than 5 (lipophilicity)

Aminohydantoins – Med Chem Strategy

NHN O

HN

4 (TCMDC-136879)

NHN O

R R

H2N R

Asp

Asp

Aminohydantoin-Aspartic Protease BACE Binding Mode

TCAMSdata mining

35 examples in TCAMSIC50 range 140-1500 nM

NHN O

X R1

R3R2

1. Prepare R1/R2/R3 analogs to develop SAR2. Derivatize X to confirm aspartic protease target

X = NH, O, S

ACS Med Chem Lett 2014, 5, 89-93

“Hit Compound”

Evidence for Asp Protease Mechanism of Ac6on

CWHM-209 MW 411

cLogP 4.75 3D7 IC50 > 5 µM

PM-II IC50 >10 µM

CWHM-117 MW 393

cLogP 3.93 3D7 IC50 = 309 nM PM-II IC50 = 4 nM

PM-IV IC50 = 15 nM

CWHM-208 MW 394

cLogP 3.86 3D7 IC50 > 10 µM PM-II IC50 > 10 µM PM-IV IC50 > 10 µM

3INH. Malamas et al., J. Med. Chem., 2010, 53, 1146

Aminohydantoin/BACE Binding Mode

HNN

O

HN

O O

HNN

O

S

O O

HNN

O

O

O O

SAR and Selec6vity

3INH. Malamas et al., J. Med. Chem., 2010, 53, 1146

Aminohydantoin/BACE Binding Mode

é 3D7 potency é PM2 potency é Selectivity

S3 Pocket?

NHN O

HN

O O

3D7 = 0.463 µMPM2 = 0.004 µMBACE = 12.0 µM [3000x]

NHN O

HN CH3

O O

3D7 = 7.92 µMPM2 = 0.487 µMBACE = 7.92 µM [25x]

NHN O

HN CH3

O3D7 = 6.88 µMPM2 = 1.01 µMBACE = 1.68 µM [2x]

NHN O

HN

O3D7 = 0.750 µMPM2 = 0.013 µMBACE = 18.1 µM [1400x]

N N

Iden6fica6on of a Lead Compound(s)

!

! ! ! !cLogP! 4.0$ 2.1$ 4.3$ 2.6$

Pf!3D7!IC50!(µM,!72h)!

0.463$ 0.459$ 0.383$ 0.463$

Pf!Dd2!IC50!(µM,!72h)!

0.480$ 0.526$ 0.367$ 0.442$

Pf!3D7!IC50!(µM,!48h)!

0.751$ 0.404$ 0.339$ 0.571$

HepG2!IC50!(µM)! 9.4$ >50$ 8.0$ 30$

NHN O

HN

O O

NHN O

HN

O

O O

NHN O

HN

O O

NHN O

HN

O O

O

Pharmacokinetics (PK) in Rat •  Half-life = 2.9 h •  Bioavailability = 16%

Dose−response in the P. chabaudi ASCQ-‐infected murine four-‐day suppressive test

CWHM-117

NHN O

HN

O O

CWHM-117

Orally efficacious in mice infected with Plasmodium falciparum •  89% suppression of parasitemia achieved at 100 mpk (qd) •  Full suppression achieved at 300 mpk (qd) Goal is to improve potency another 10-‐fold

“Lead Compound”

37



Development Investigational New Drug (IND)

Application



Hits to Leads

N

NN

O

O

OH

Cl

N

NN

O

O

OH

O Cl

NHN O

HN NHN O

HN

O O

•  Potent and SelecKve •  Orally Efficacious •  Need 10x more potency

Aspar?c protease inhibitors represent novel an?malarial chemotypes and mechanism of ac?on

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