drug%discovery%for%malaria%dbbs.wustl.edu/curstudents/documents/markey/2014-09-09 markey...lead...
TRANSCRIPT
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
Development: 7-10 years
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
Target Discovery Lead Discovery Lead
Optimization Preclinical
Development Investigational New Drug (IND)
Application
Drug Discovery: 3-7 years
Development: 7-10 years
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
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
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
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
Target Discovery Lead Discovery Lead
Optimization Preclinical
Development Investigational New Drug (IND)
Application
Drug Discovery: 3-7 years
Development: 7-10 years
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