Claire Wallace Presentation - Soci

Inhibitors of Plasmodium Falciparum Calcium‐Inhibitors of Plasmodium Falciparum CalciumDependent Protein Kinase 1 (PfCDPK1), a Novel Target

for the Potential Treatment of Malaria

Claire WallaceYoung Chemist in IndustryYoung Chemist in Industry

8th May 2013

ContentsContents

Why Malaria?

PfCDPK1: A Novel Target

Hit Identification and Early Lead

O i i i d S h i f I id id iOptimisation and Synthesis of Imidazopyridazines

In Vivo Results

Parasite Rate of Reduction Assay

Biotinylation Experimentsy p

Conclusion

Why Malaria?Why Malaria?

A major public health problem in more than 90 countries with combined

population 3.3 billion people (50% of the world’s population)

~220 million new infections and an estimated 660,000 deaths per year, p y

Mainly in Africa but incidence is growing in Asia and Latin America

Estimated incidence of malaria per 1000 population (2006)

Why Malaria?Why Malaria?

Emergence of drug resistance has compromised the therapeutic efficacyf t ti l i l dof most anti-malarial drugs

Artemisinin and derivatives, used incombination therapy, are currently the mainline of defence against drug-resistant malaria

Humans can be infected by 11 species, but two account for vastmajority of cases: Falciparum (~70%) & Vivax (~25%)j y p ( ) ( )

Life CyclePfCDPK1: A Novel TargetLife Cycle

Calcium-dependent protein kinases (CDPKs) are directly regulated by Ca2+ and are found in plants

PfCDPK1: A Novel Target

and organisms in the alveolate lineage, but they are absent in humans

PfCDPK1 is encoded by an essential gene expressed primarily in the merozoite

Responsible for phosphorylation of components of the molecular motor that drive parasite invasion

of red blood cells

Bl ki it i i t th it t ld i t f ith th lif l d t ti ll Blocking parasite invasion at the merozoite stage could interfere with the life cycle and potentially

allow the infection to be cleared

Hit IdentificationHit Identification

NN

NN

NH

NHO

NH2

IC50 = 60 nM

Hit series of Imidazopyridazines showed sub 100 nM IC50 values against CDPK1 and

strong inhibition of parasite growth

Concerns over high log D values, poor microsome stability and selectivity over

human kinases

Kinase Selectivity Panel:

10 µMKey: % inhibition >90: red, 70-90: yellow, <70: green

Hit to Early LeadHit to Early Lead

PfCDPK1 IC50 = 13 nM

P falciparum EC = 400 nM NNP. falciparum EC50 = 400 nM

Cytotox IC50 = 9 µM

Log D = 3.4

NN

NH

N

N

MLM 85 % rem

HLM 63 % rem

PAMPA = 81 nm/s

NH

PAMPA 81 nm/s

In vivo = 46 % reduction in parasitaemia (50 mg/kg dose orally)

Kinase Selectivity Panel:

Left hand side less important to binding affinityKey: % inhibition >90: red, 70-90: yellow, <70: green 1 µM

Introduction of a more basic side chain to improve ADME properties

Phenyl amide replaced with pyridyl isopentylamine which improved binding affinity Phenyl amide replaced with pyridyl isopentylamine which improved binding affinity

and kinase selectivityBioorg. Med. Chem. Lett., 2013, 23, 3064

Optimisation of ImidazopyridazinesOptimisation of Imidazopyridazines

A structure-based design approach using the homology model of PfCDPK1 was used

to attempt to gain increased binding affinity against the target and correspondingly

increase the cellular potencyp y

NN

N

NH

N

N

The model suggested that the binding pocket occupied by N

NH

the alkyl chain was not optimally filled

NN

N

N

N

Virtual libraries were enumerated with a range of groups

at this position and examined through dockingNN

H

N

NH

Model revealed 2-amino pyridines to have superior H

Nbinding energies

Optimisation of ImidazopyridazinesOptimisation of ImidazopyridazinesN

NN

N

N

NN

NN F

NH N

HN

Additional VDW interactions and fewer clashes

Improved electrostatic complementarity and additional H bondImproved electrostatic complementarity and additional H-bond

Reduced entropic penalty due to conformational focussing

Synthesis of ImidazopyridazinesSynthesis of Imidazopyridazines

O O

NN

N

ClB

N

NH2

O

N

NH

O

O

NN

N

Br

N

NH

O

O

NN

NN

NB

NH2

O

DIPEAPd(dppf)Cl2

Br Br

NN

NH2

DIPEANMP130 °C

57%

Cs2CO3THF/H2O

48%

NH

NH

NN

NN

ClF O

H HN

NH

NN

Ni)

H

NN

NH

N

F

H

NN

NH

N

FPd(OAc)2XantphosCs2CO3Dioxane

ii) HCl i Di

AcOHNa(OAc)3BHTHF

39%ii) HCl in Dioxane 24%

<5 steps A prerequisite for MMV collaboration <5 steps – A prerequisite for MMV collaboration

Testing the ModelTesting the Model

NNH2 NNH2 NNH2

NH

NN

N

NN

2

NH

NN

N

NN

2

NH

NN

N

NN

2

F

IC50 < 9 nM

EC50 = 465 nM

NH

NH

N

IC50 < 8 nM

EC50 = 83 nM

NH

N

IC50 < 8 nM

EC50 = 12 nMC50 65

Cytotox IC50 = 6.6 µM

Log D = 1.6

MLM 92 % rem

C50 83


Log D = 1.1

MLM 93 % rem

C50

Cytotox IC50 = >20 µM

Log D = 0.2

MLM 85 % rem

Introduction of a hydrogen bond acceptor ortho to amine showed a significant cellular

HLM 89 % rem

Pampa = 1.6 nm/s

HLM 89 % rem

Pampa = 0.3 nm/s

HLM 93 % rem

Pampa = 3.9 nm/s

efficacy boost

A further boost obtained by the introduction of a fluorine at the 3 position

Poor permeability, could be due to diaminocyclohexane?

Optimisation of Left Hand SideOptimisation of Left Hand Side

N

NNNH2

N

NN

NN

NN

NH

F

NH

NN

NN

NH

F

NH

NN

NN

NH

F

HN

IC50 = 44 nM

EC50 = Inactive

C t t IC 6 7 M

HN

IC50 = < 8 nM

EC50 = 13 nM

C toto IC >20 M

HN

IC50 = 13 nM

EC50 = 40 nM

C t t IC 5 9 M Cytotox IC50 = 6.7 µM

Log D = 2.5

MLM 92 % rem

HLM 82 % rem

Cytotox IC50 = >20 µM

Log D = 0.2

MLM 85 % rem

HLM 93 % rem


Log D = 1.4

MLM 85 % rem

HLM 98 % rem

Variation of left hand side to improve ADME properties

B i id h i i d th l ft h d id

Pampa = 137 nm/sPampa = 3.9 nm/s Pampa = 1.0 nm/s

Basic side chain required on the left hand side

Attempts to remove resulted in loss of cellular efficacy

A range of basic groups with varying pKa explored (lower pKa improved permeability)A range of basic groups with varying pKa explored (lower pKa improved permeability)

Trade off between good ADME and good efficacy!

Optimisation of LinkerOptimisation of Linker

N NN NN N N

NH

NN

N

F

N

NH

NN

N

N

F

N

NH

NN

NN

N

F

IC50 = 16 nM

NH

N

NH

N

IC50 = 9 nMIC50 = 13 nM

NH

N

EC50 = 406 nM


Log D = 3.2

MLM 47 %

EC50 = 388 nM


Log D = 3.1

MLM 57 %

EC50 = 40 nM


Log D = 1.4

MLM 85 % MLM 47 % rem

HLM 80 % rem

Pampa = 170 nm/s

MLM 57 % rem

HLM 77 % rem

Pampa = 63 nm/s

MLM 85 % rem

HLM 98 % rem

Pampa = 1.0 nm/s

Increasing Permeability

Decreasing Efficacy

In Vivo ResultsIn Vivo Results

IC50 (µM)

Cytotox v-50 (µM)

EC50 (µM)

HLM (%

rem)

MLM (%

rem)LogD

PAMPA Papp

(nm/s)

In vivo reduction in parasitaemia

(%)(µM) rem) rem) (nm/s) (%)

0.013 9.0 0.400 63 85 3.4 81 46N

N

NNN

H

N

NN

N

N

N

NH

0.016 3.2 0.390 80 47 3.2 171 44

NNH

NH

N

F

0.012 2.0 0.078 86 61 3.7 48 51

NN

N

NH

N

NH

F

NH

N Cl

N

NNN

H

N2H

<0.008 >20 0.013 93 85 0.2 4 4NN

NH

N

F

Parasite Rate of Reduction AssayParasite Rate of Reduction Assay

Quantifies number of parasites that remain viable after drug treatment

Parasites are treated with the selected drug at a concentration

corresponding to 10 x IC50 over 48 hoursp g 50

Dilutions are made and cultured for 21 days

Samples taken at 21 and 28 days to confirm number of viable parasites

Can group according to control compounds

Fast killers (chloroquine)

Moderate killers (pyrimethamine) Moderate killers (pyrimethamine)

Slow killers (atovaquone)

Parasite Rate of Reduction AssayParasite Rate of Reduction Assay

Controls

ites

+1)

able

par

asi

Slow/Moderate Killers

log

(via

F t KillFast Killer

Biotinylation ExperimentsBiotinylation Experiments

Used to determine other targets that a compound may interact with

Biotin is tethered to compound by a short linker and exposed to potential target

proteins

(Strept)Avidin has a high affinity for the biotin portion and can be used to “pull-out”

the proteins for which the compound has an affinity.

AVIDIN BEADAVIDIN BEAD

PRO

NH

NH

SO

H

H

TEIN

NH

COMPOUNDO

S

Biotinylation ExperimentsBiotinylation Experiments

NH

NH

SO

H

H

NHON

N

N

NH

O N

NH

NN

N

NHN

S

H

H

NH

NH

NN

N

H

NNH

NH

H

O

Assuming a similar binding mode biotin was tethered to the basic side chainAssuming a similar binding mode biotin was tethered to the basic side chain

However, a new target could mean a new binding mode

ConclusionConclusion

A series of PfCDPK1 inhibitors was identified from a high throughput

screen

Subsequent optimisation yielded potent, selective inhibitors with suitable

ADME and pharmacokinetic profiles

Testing in an in vivo efficacy model showed insufficient reduction in

parasitaemia for the project to progress further

On going investigation into off-target effects through biotinylation

experiments

Acknowledgements

MRCT NIMR

Acknowledgements

Simon OsborneTim ChapmanJon Large

Tony HolderJudith GreenBarbara Clough

Nathalie BoulocEla Smiljanic-Hurley

Munira Grainger

MMVKris Birchall

David Tickle

Didier LeroyPaul WillisSimon Campbell

Sadhia Mahmood

Keith AnsellSwiss TPH InstituteSergio Wittlin

Hayley JonesAlison LevyMichelle Raynor

Monash UniversitySue Charman

Debbie TaylorDavid Whalley

Karen White

Figure 5. Classical antimalarial killing rate profiles.

Sanz LM, Crespo B, De-Cózar C, Ding XC, et al. (2012) P. falciparum In Vitro Killing Rates Allow to Discriminate between Different A ti l i l M d f A ti PL S ONE 7(2) 30949 d i 10 1371/j l 0030949Antimalarial Mode-of-Action. PLoS ONE 7(2): e30949. doi:10.1371/journal.pone.0030949http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030949

Claire Wallace Presentation - Soci

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