Malaria Libre

Project Meeting

24th June 2021

2

• Status of action items from previous meeting

• Project update

❖ Metabolomics studies – Carlo

❖ Blood stage specificity assays - Shailja

❖ Hemozoin adsorption studies - Katherine

❖ Next set of molecules for aryl piperazine scaffold

❖ Cyclopropyl amides – an update

Agenda

3

Action itemsStatus of action items from March 2021

Action Item Responsible group/ scientist Status

Synthesis of compounds TCGLS,CDRI, Clint TCG- ongoing, CDRI – reinitiated(compounds

submitted for 3D7 activity), Clint-??

Blood stage specificity – dose response in ring &

schizont stage

Shailja Slides 15-18

Establishment of MIR assay Shailja/ Shruthi intends to put the assays on

board(discuss separately) Discuss separately

Mechanism of action studies: metabolomics Carlo

Slides 4 -14

Screening of compounds in Hemozoin

adsorption model

Katherine de Villiers

Slides 19 -31

Follow up meeting to discuss funding

opportunities

Kiran to coordinate Done, Plan for the ICMR grant, not sufficient

data for India alliance grant

Metabolomics

Aim: Use untargeted metabolomics analysis to determine the mode of action of aryl imidazole and aryl piperazine

antimalarials.

Methodological details:

Parasitaemia: 90%

Haematocrit: 0.5%

Parasite stage: 28-34 h trophozoites

Extraction: Cold methanol

Detection: LC-MS

Analysis: Untargeted (IDEOM)

Sample analysed: Cell pellet

Troph 26 h ± 4h

B

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

A

GFP Pf332 Merge

MC

RBCMMC

RBCM

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

Overlay

SLO

-SLO/Sap

+PrK +

SLO

α-PfSBP1 (N)

α-GFP

100

75

50

37

25

kDa

150

50

37

25

SN P P P

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

C

D

Saponin

Proteomics sample

Precipitate proteins Dissolve

proteins

Ring 8 h ± 2h Metabolomics

sample

Peptidomics sample

QuenchMetaboliteExtrac on LCMSanalysis

MetabolomicsDataanalysis

Solubilise Reduce&Alkylate

Digest

Label

Frac onate

ProteomicsDataanalysis

LCMSanalysis

Centrifugal filtration

Collect Flow-through

Desalt

LCMSanalysis

Pep domicsDataanalysis

Troph 26 h ± 4h

B

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

A

GFP Pf332 Merge

MC

RBCMMC

RBCM

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

Overlay

SLO

-SLO/Sap

+PrK +

SLO

α-PfSBP1 (N)

α-GFP

100

75

50

37

25

kDa

150

50

37

25

SN P P P

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

C

D

Saponin

Proteomics sample

Precipitate proteins Dissolve

proteins

Ring 8 h ± 2h Metabolomics

sample

Peptidomics sample

QuenchMetaboliteExtrac on LCMSanalysis

MetabolomicsDataanalysis

Solubilise Reduce&Alkylate

Digest

Label

Frac onate

ProteomicsDataanalysis

LCMSanalysis

Centrifugal filtration

Collect Flow-through

Desalt

LCMSanalysis

Pep domicsDataanalysis

Troph 26 h ± 4h

B

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

A

GFP Pf332 Merge

MC

RBCMMC

RBCM

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

Overlay

SLO

-SLO/Sap

+PrK +

SLO

α-PfSBP1 (N)

α-GFP

100

75

50

37

25

kDa

150

50

37

25

SN P P P

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

C

D

Saponin

Proteomics sample

Precipitate proteins Dissolve

proteins

Ring 8 h ± 2h Metabolomics

sample

Peptidomics sample

QuenchMetaboliteExtrac on LCMSanalysis

MetabolomicsDataanalysis

Solubilise Reduce&Alkylate

Digest

Label

Frac onate

ProteomicsDataanalysis

LCMSanalysis

Centrifugal filtration

Collect Flow-through

Desalt

LCMSanalysis

Pep domicsDataanalysis

100% MeOHTroph 26 h ± 4h

B

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

A

GFP Pf332 Merge

MC

RBCMMC

RBCM

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

Overlay

SLO

-SLO/Sap

+PrK +

SLO

α-PfSBP1 (N)

α-GFP

100

75

50

37

25

kDa

150

50

37

25

SN P P P

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

C

D

Saponin

Proteomics sample

Precipitate proteins Dissolve

proteins

Ring 8 h ± 2h Metabolomics

sample

Peptidomics sample

QuenchMetaboliteExtrac on LCMSanalysis

MetabolomicsDataanalysis

Solubilise Reduce&Alkylate

Digest

Label

Frac onate

ProteomicsDataanalysis

LCMSanalysis

Centrifugal filtration

Collect Flow-through

Desalt

LCMSanalysis

Pep domicsDataanalysis

Metabolomics

sampleQuench

Metabolite

extractionTroph

26 h ± 4h

B

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

A

GFP Pf332 Merge

MC

RBCMMC

RBCM

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

SLO SLO/Sap

RBCM

PVM

PV

MC RBCM

PVM

PV

MC

P P

Overlay

SLO

-SLO/Sap

+PrK +

SLO

α-PfSBP1 (N)

α-GFP

100

75

50

37

25

kDa

150

50

37

25

SN P P P

GFP PfSBP1 Merge Overlay

GFP MAHRP2 Merge Overlay

C

D

Saponin

Proteomics sample

Precipitate proteins Dissolve

proteins

Ring 8 h ± 2h Metabolomics

sample

Peptidomics sample

QuenchMetaboliteExtrac on LCMSanalysis

MetabolomicsDataanalysis

Solubilise Reduce&Alkylate

Digest

Label

Frac onate

ProteomicsDataanalysis

LCMSanalysis

Centrifugal filtration

Collect Flow-through

Desalt

LCMSanalysis

Pep domicsDataanalysis

Ice cold PBS

LCMS analysisMetabolomics

data analysis

+/-Drug

Carlo Giannangelo, Monash University

Aryl imidazole metabolomicsCompound Concentration File name Duration Replicates Notes

MMV1794348 250 nM P_17_250nM 5 h 5 Test compound

MMV1794348 650 nM P_17_650nM 5 h 5 Test compound

MMV000073 1 μM P_73 5 h 4* Positive control

(PfATP4

inhibitor)

Chloroquine 200 nM P_CQ 5 h 5 Positive control

MIPS2222A 1 μM P_2222A 5 h 4 Positive control

(unknown MoA)

MMV690903 650 nM P_69 5 h 5 Negative control

DMSO 0.01% P_DMSO 5 h 5 Negative control

DHA 100 nM P_DHA_3h 3 h 4* Positive control

DMSO 0.01% P_DMSO_3h 3 h 4* Negative control

Data Quality

▪ 4-5 replicates from each treatment were analysed.

▪ 4 pooled quality control samples and solvent blanks were analysed at regular intervals

▪ Relative variability in median peak intensity is ≤ 6% in pooled Quality Control samples (low instrument-induced variability).

▪ Relative variability in median peak intensity is < 10% for replicates within groups (acceptable biological variability).

▪ No normalisation required (several outlier samples were removed).

*one outlier replicate was excluded during analysis.

0

500000

1000000

1500000

2000000

2500000

3000000

Me

dia

n p

ea

k i

nte

ns

ity

Median (positive)

Aryl imidazole metabolomics overview▪ 515 putative metabolites were identified in this study (comparable to other similar studies)

▪ Principal component analysis showed that parasites treated with MMV1794384 (P_17) cluster together with the inactive aryl imidazole (MMV690903, P_69) and DMSO control, with no distinct metabolic profile

▪ Metabolic perturbations caused by DHA and MMV000073 (PfATP4 inhibitor) account for the majority of variance in the dataset

Principle component analysis

MMV1794348

(P_17)

[MMV1794348] 250 nM 650 nM

5 h pulse IC10 IC50

72 h IC50 3.5 x 9 x

Aryl imidazole significantly perturbed metabolites

* Pvalue < 0.05 and fold change > 1.5 vs DMSO

MMV1794348

3-Methyleneoxindole

MMV1794348Quinaldic acid

4-Nitroaniline

6-aminonicotinate

0.00001

0.0001

0.001

0.01

0.1

10.1 1.0 10.0 100.0 1000.0 10000.0100000.0

Pvalu

e (

Tte

st)

Intensity Ratio

P_17_250nM

P_17_650nM

Significance

threshold

▪ 4 metabolites were significantly perturbed by MMV1794348 treatment (pvalue < 0.05 and fold change > 1.5 vs DMSO)

▪ These metabolites are unrelated and not from a specific metabolic pathway and are unlikely to be related to the MoA

Aryl imidazole preliminary metabolomics summary

▪ MMV1794384 (P_17) caused no distinct metabolic profile and antimalarial activity is unlikely to involve perturbation of parasite metabolism

▪ The mechanism of action does not overlap with DHA or PfATP4 inhibitors

▪ The mechanism of action requires further investigation

Aryl piperazine metabolomics

Compound Concentration File name Duration Replicates Notes

MMV024406 1 μM P_1 5 h 3 Phenyl amide

MMV1804508 1 μM P_5 5 h 3 Cyclopropyl

MMV1804743 1 μM P_6 5 h 3 Cyclopropyl

Atovaquone 1 μM P_Atov 5 h 3 Positive control

MMV1803903 1 μM P_7 5 h 3 Inactive phenyl amide

MMV1803901 1 μM P_8 5 h 2 Inactive cyclopropyl

DMSO 0.01% P_DMSO 5 h 3 Negative control

Data Quality

▪ 3 replicates from each treatment were analysed.

▪ 3 pooled quality control samples and solvent blanks were analysed at regular intervals

▪ Relative variability in median peak intensity is < 5% in pooled Quality Control samples (low instrument-induced variability).

▪ Relative variability in median peak intensity is < 12% for replicates within groups (acceptable biological variability).

▪ No normalisation required (several outlier samples were removed).

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

Me

dia

n p

ea

k i

nte

ns

ity

Median (positive)

Aryl piperazine metabolomics overview▪ 584 putative metabolites were identified in this study

▪ Principal component analysis showed that parasites treated with the cyclopropyl, MMV1804503 (P_5), cluster with atovaquone-treated parasites (pink)

▪ The other cyclopropyl tested, MMV1804743 (P_6), and the active and inactive phenyl amides cluster with untreated parasites

Principle component analysis

MMV1804508

(P_5)

MMV1804743

(P_6)

MMV024406

(P_1)

MMV1803903

(P_7)

inactive

Atovaquone-like effect of MMV1804508▪ MMV1804508 (P_5) perturbed pyrimidine biosynthesis, similar to atovaquone

▪ Metabolites upstream of DHODH accumulated in MMV1804508 and atovaquone treated parasites, while downstream pyrimidines were depleted

▪ MMV1804743 (P_6) did not have a significant impact on pyrimidine metabolism under these conditions

• MMV024406 (P_1) also depleted downstream pyrimidines, but did not have the same “atovaquone-like” profile

MMV1804508

(P_5)

MMV1804743

(P_6)

MMV024406

(P_1)

MMV1803903

(P_7) inactive

MMV1803901

(P_8) inactive

Aryl piperazine impact on nucleotide metabolism

N-carbamoyl aspartate -3 3 - 3 -3 -3 -

Dihydroorotate -3 3 -1 4 -3 -3 -

Orotate -2 2 - - -2 -2 -

Adenine - - - - - - -

Inosine - - - - - - -

Guanine - - - 1 - - -

Adenosine - - - - - - -

dGDP - - - - - - -

Inosinic acid - - - 1 - - -

Deoxycytidine - - - - - - -

Dihydrothymine - - - - - - -

Hypoxanthine - - - - - - -

Guanosine monophosphate -1 - -1 - -1 - -

Sulfate - -1 - - - - -

dCDP - -1 - - - - -

Adenosine monophosphate -1 -1 - - -1 - -

Cyclic GMP -1 -1 - - - - -

Uridine 5'-monophosphate -1 -1 - -1 -1 - -

Cytidine - -1 - - - - -

Deoxyadenosine monophosphate -1 -1 - - - - -

dCMP -2 -1 - - -1 - -

Cytidine monophosphate -1 -1 - -1 -1 - -

5-Thymidylic acid -2 -1 - -1 -1 - -

-2 -1 - 1 2

-2 -1 0 1 2

log2 fold change

P_1

P_5

P_6

P_A

tov

P_7

P_8

P_D

MS

O

Nucleotide metabolism

Aryl piperazine preliminary metabolomics summary

Cyclopropyls

▪ MMV1804508 (P_5) displayed an “atovaquone-like” profile, suggesting either inhibition of the bc1 complex or DHODH.

▪ MMV1804743 (P_6) had no pronounced impact on parasite metabolism, possibly due to the concentration and/or duration of compound exposure in this study.

Phenyl amides

▪ MMV024406 (P_1) also perturbed nucleotide metabolism and had a distinct profile to atovaquone.

▪ The relevance of altered nucleotide metabolism to the MoA of phenylamides is unclear, as a similar profile was also seen in the inactive phenylamide control, MMV1803903 (P_7).

MMV1804508

(P_5)

MMV1804743

(P_6)

MMV024406

(P_1)

MMV1803903

(P_7)

MMV1803901

(P_8)

Possible Next steps

14

1. Concentration and/or time dependent changes in metabolite levels

2. Thermal proteomics (CETSA) for target ID

3. Untargeted proteomics could also give an indication of the MoA

Ring stage

activity assay

Trophozoite

stage activity

assay

Schizont stage

activity assay

Asexual blood stage specific assay Shailja

IC 50: 7.685 µM (5 hr)

7.385 µM (10 hr)

mmv 24406 (Ring stage) mmv 24406 (Trophozoite stage stage)

IC 50: 1.478 µM (5 hr)

1.169 µM (10

hr)

IC 50: 991 nM (5 hr)

375.8 nM (10 hr)

mmv 24406 (Schizont stage stage)

mmv 24408 (Ring stage) mmv 24408 (Trophozoite stage stage) mmv 24408 (Schizont stage stage)

mmv 1804508 (Trophozoite stage stage)mmv 1804508 (Schizont stage stage)mmv 1804508 (Ring stage)

IC 50: 729 nM (5 hr)

633 nM (10 hr)

IC 50: 903 nM (5 hr)

759 nM (10 hr) IC 50: 2.954 µM (5 hr)

2.252 µM (10

hr)

IC 50: 7.465 µM (5 hr)

759 nM (10 hr)IC 50: 553 nM (5 hr)

124 nM (10 hr)IC 50: 288 nM (5 hr)

63.78nM (10 hr)

MMV1804508 (Ring stage) MMV024408 (Ring stage) MMV024406(Ring stage)

Duration 5 h 10 h 72 h 5 h 10 h 72 h 5 h 10 h 72 h

IC50 (µM) 7.465 0.759 0.157 0.90 0.739 0.322 7.685 7.385 0.033

MMV1804508 (Trophozoite

stage)

MMV024408 (Trophozoite stage) MMV024406 (Trophozoite stage)

Duration 5 h 10 h 5 h 10 h 5 h 10 h

IC50 (µM) 0.553 0.124 2.954 2.252 1.478 1.169

IC50 (µM) > 20 0.57 ± 0.18 > 20 1.3 ± 0.20 > 20 1.4 ± 0.15

MMV1804508 (Schizont stage) MMV024408 (Schizont stage) MMV024406 (Schizont stage)

Duration 5 h 10 h 5 h 10 h 5 h 10 h

IC50 (µM) 0.288 0.063 0.729 0.633 0.991 0.375

Note : Highlighted section represent data from other group

Compond

ID

IC50 (72 hr) IC50 (72

hr)

24406 157.3 nM 369 nM

24408 322.5 nM 376 nM

1804508 33.30 nM 166 nM

Buller, R. et al. Quinoline binding site on malaria pigment crystal: a rational pathway for antimalaria drug design. Cryst. Growth Des. 2002, 2, 553-562.

BackgroundDr Katherine de Villiers and Dr Tania Oliver

Stellenbosch University, South Africa

In silico β-haematin preparation

(0 0 1)

(0 1 1)

(0 1 0)

(1 0 0)

• Dimer co-ordinates obtained

from crystal structure.

• Only hydrogens were optimized

utilizing :

− DFT

− DNP

− GGA PW91

S. Pangola, P. W. Stephens, D. S. Bohle, A. D. Kosar and S. K. Madsen, Lett. to Nat., 2000, 404, 307–310

R. Buller, M. L. Peterson, Ö. Almarsson and L. Leiserowitz, Cryst. Growth Des., 2002, 2, 553–562.

Optimised inhibitorInhibitor Adsorption

AnnealingAdsorption energy (kcal/mol)

ሻ𝐸𝑎𝑑𝑠 = 𝐸(𝑐𝑟𝑦𝑠𝑡𝑎𝑙 + 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟ሻ − (𝐸𝑐𝑟𝑦𝑠𝑡𝑎𝑙 + 𝐸𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟

Computational parameters

Forcite module used on BIOVIA Materials Studio.

Force field: cvff (parameterised for β-haematin)

Quality: Ultrafine

Charges: Qeq

Adsorption calculation

Crystal faces

(001)

a)

b

c0

b

c0

(011)

b)

(100)

(010)c)

b

a0

(001)

a)

b

c0

b

c0

(011)

b)

(100)

(010)c)

b

a0

(001)

a)

b

c0

b

c0

(011)

b)

(100)

(010)c)

b

a0

(001)

(010) and (100)

(011)

Rotate

about b-axis(001)

1°

2°

A B

C D

E

Summary

• Three repeat calculations on the (001) face to determine the

adsorption energy (Eads) for the five compounds from the MMV data set

are nearing completion.

• All compounds are structurally related and adsorb in similar

fashions.

• A adsorbs solely onto the secondary binding site of the (001) face,

whereas compounds B, C, D, and E adsorb onto both the primary

and secondary binding sites.

• Thus far, there is no clear correlation between the biological IC50 data

provided and the in silico Eads results.

Inhibitor

(IC50

(3D7/Dd2))

Our

referenc

e

Eads

Average

(kcal.mol-1)

Standard

deviation

MMV892566

(0.27 / 0.52)

A -74.7 1.2

MMV1803899

(0.37 /0.68)

B -77.0 5.8

MMV024406

(0.42 /0.82)

C -65.9 2.2

MMV893309

(0.30 / 0. 85)

D -60.1 5.6

MMV1899689

(25 / -)

E -77.7 7.3

Inhibitor

(IC50 (3D7/Dd2))

Our

reference

pKa Protonation state %

present

at pH 4.8

Eads

Avera

ge

Standar

d

deviatio

n

MMV892566

A

1.95 (Pyridine F3) 0 0.01 -73.0 1.03

(0.27 / 0.52) 3.07 (Pyridine

inner)

1+ (Pyridine NH+ (not F3)) 98.16 -74.7 0

8.7 (Pyridine

outer)

2+ (Two outer pyridines) 0.15 -79.7 0.54

2+ (Pyridine NH+ (not F3) and inner

Pyridine)

1.67 -74.9 0

MMV1803899

B

3.03 0 0.01 -67.8 0.15

(0.37 /0.68) 9.06 1+ (Pyridine CH3) 98.32 -77.0 5.67

2+ (Pyridine NH+ and inner Pyridine) 1.68 -72.7 1.37

MMV024406

C

3.03 0 0.01 -66.5 0.56

(0.42 /0.82) 8.7 1+ (Pyridine CH3) 98.31 -65.9 0

2+ (Pyridine NH+ (not Cl outer) and inner

Pyridine)

1.67 -66.2 2.11

MMV893309

D

2.98 1+ (Pyridine next to n-n ring) 13.1 -67.1 3.17

(0.30 / 0. 85) 5.61 2+ (Pyridine next to n-n and inner) 0.21 -70.1 3.83

8.67 2+ outer pyridines 85.37 -59.1 1.84

3+ all pyridines 1.31 -58.5 2.90

MMV1899689

E

-0.44 0 0.01 -65.4 5.46

(25 / -) 3.03 1+ (Pyridine next to n-n ring) 98.31 -77.8 1.19

MMV892566 (A) +1 (98% present)

All protonation states were observed to dock onto the secondary binding site.

View down X-

axis

View down Z-

axis

MMV1803899 (B) +1 (98% present)

- Neutral MMV1803899 molecule was observed to dock onto the secondary binding site.

- The 1+ protonation state docked into the primary with a portion of the molecule on the secondary binding site.

- The 2+ protonation state docked onto the secondary binding site.

View down X-

axis

View down Z-

axis

MMV024406 (C) +1 (98% present)

- The neutral MMV024406 molecule was observed to docked into the primary with a portion of the molecule on the secondary

binding site.

- The 1+ protonation state docked onto the secondary binding site.

- The 2+ protonation state docked onto the secondary binding site.

View down X-

axis

View down Z-

axis

MMV893309 (D) +2 (85% present)

- All the protonation states were observed to dock into the primary with a portion of the molecule on the secondary binding site.

View down X-

axis

View down Z-

axis

MMV1899689 (E) +1 (98% present)

- All the protonation states were observed to dock into the primary with a portion of the molecule on the secondary binding site.

View down X-

axis

View down Z-

axis

Future work

• Complete repeat calculations on the (001) face.

• Consider alternative faces – in particular the (011) face.

All compounds in which the total number of pi-pi

interactions in this distance range was 2 or fewer in

aggregate for both the (001) and (011) faces were virtually

inactive (IC50 > 100 mM) or almost so (IC50 > 80 mM). For

the remaining compounds, a direct proportionality was

found between adsorption energy with the (011) face and

the experimental b-hematin inhibition IC50.

• Determine β-hematin IC50 (NP-40 detergent)

• Cell fractionation assay to confirm MOA (Hz) in cells.

F.P. L'abbate et al. Eur. J. Med. Chem., 159 (2018) 243e254

32

Confidential

Aryl piperazines (series 1c)

No. of compounds synthesised

Focus of current exploration, all

regions for modification are being

explored

MMV1803899

Pf potency (3D7, µM) range Best activity in range of 0.3-0.5 0.36

SAR understandingLimited modifications tolerated(next

slide)-

PRR Fast kill in 2 point FACS and PRR Fast in 2 point FACS

Blood stage specificity To be initiated To be initiated

MoA

Unknown: no finger print match in

pH fingerprinting assay, no cross R

in STPH

No cross R in STPH or fingerprint

match in PH fingerprinting assay

yDHODH screeningNot a bc-1 or DHODH

inhibitor(MMV024406)ND

Potency (liver stages)MMV024406 active in Pb and Pf

assayND

Potency (transmission),DGFA inactive inactive

eLogD generally 3-4 3.6

Solubility (i.e. PBS)Can be modulated, some

compounds have ~ 50uM49

Metabolic stabilityLow to moderate in h mic and r

hepsh mic: 45; r heps: 2.9?

CYP InhibitionCan be modulated through

structural modificationsIC50 > 30uM

hERG (K+CHO) IC50 , µMCompounds with lower liability had

lower potency<1

In vivo PKIdentify potent compounds before

selectionND

MMV1803899

Low Caco2

permeability

• Moderate potency

• hERG liability

• Poor metabolic stability

33

Action itemsModifications in the amide region

MMV024406

Pf 3D7 LDH : 0.42µM

elog D: 3.7

NF54 IC50

3D7(LDH or SYBR)IC50

Identify vectors for improving activity, moving away from hidden aniline, essentiality of amide

• Substituted pyridines are tolerated

• Other heteroaryls or polar sub not tolerated

• Cycloalkyls were detrimental

Essentiality of amide

4.03 4.79

0.36

1.01

Next steps – explore

hydrophobic space?

Next steps – explore into the hydrophobic space?

Other linkers(stability, additional interactions?)

KishoreSanjay ?

TCGFNDR

34

Action itemsInitial SAR investigations and chemistry plans

MMV024406

Pf 3D7 LDH : 0.42µM

3D7 LDH/SYBR IC50

Modification of linker – impact on potency, metabolic stability, hERG

Core hopping – improvement in potency?

New data

Next steps

Sanjay

Sanjay ?

TCG

Possibility of improving activity?

35

Action items Core hopping

MMV024406

Pf 3D7 LDH : 0.42µM

NF54/3D7 IC50(µM)

Core hopping – improvement in potency?

Mimic 4-Aqs (Prem)

Previous data

Modulating the steric bulk and electronics

on the core

Is 3-Cl Py

a linker?

Clint

Kishor

Crizotinib K1 IC50: 0.78uM

Next steps

Ideas from virtual screening hits

37

Action itemsUpdate on cyclopropyl amide series

Recap from last meeting(s):

• MMV1804508 and MMV1804743 are hypersensitive to atovaquone resistant strain,TM90C2B and active in ELQ resistant cell lines

• MMV1804508 screened in barcoded resistant cell lines at Sanger- mechanism of resistance not identified

• MMV1804508 and MMV1899468 are not DHODH inhibitors

Key points of discussion with Akhil Vaidya, Drexel University

• Screen compounds in Pf bc-1 assays and glu-gal assays

• Generate resistant mutants with MMV1804508

• Screen in additional bc-1 mutant strains

Comment : Given the compounds inhibit male gamete formation, they mayn’t be bc-1 inhibitors or may exhibit poly pharmacology

Status: Compounds were shipped, studies are to be initiated

3

8

Profile of front runner

Criterion (Early Lead) MMV1804508

In vitro

3D7/Dd2 IC50 M 0.19/0.07

Pb(sporozoite), Pf (schizont) 0.29/ongoing

DGFA, IC50 MPf FGF(inactive), Pf

MGF(0.87)

FACS/PRR Slow rate of kill(3.82)

MoA Not known

ADME properties

h microsomes CLint, (mL/min/kg) /t1/2(min) 33/42

r hep,Clint, uL/min/106 / t1/2(min) 138/5

CYP inhibition (3/5 > 10, none < 1), M >10

Solubility PBS pH 7.4 (>10), M 7.5

hPPB (% bound) ongoing

Permeability Caco-2, Papp A to B (10-6 cm/s) / efflux ratio

4.2/1.6

Safety profile

Cytotoxicity HepG2, M 9.7

hERG , M 10.33

Discussion point:

Can this compound be used for in vivo PoC?

Issues: Moderate activity in 3D7

Low metabolic stability – can be mitigated by using i.p

route for PoC

39

Next steps

MMVID

3D7/Dd2LDH IC50(uM)

Synthesis of focussed compoundsSwitch to pyrimidine in place of 3-chloropyridine for a few compounds (lower lipophilicity , ~ 2 fold improved activity in MMP)

1899468

0.13/0.34

clogP:4.18

clogP:2.99 1847005

0.42

elogD:3.16

ClogP:4.5

Improved HLM

clogP:3.17

Explore some

polar groups

MMV1804743

0.42

HLM:1.7

rheps:14.2

Improve activity

Lower

lipophilicity

Synthesis ongoing

at TCG

Improve activity

Lower lipophilicity

40

Ways of contribution• Design and synthesis of compounds to achieve the objectives

• Synthesis of compounds

• Carry out experimental in vitro Met ID

• Screen front runners in lab derived Indian strains other than 3D7; asexual intraerythrocytic blood

stage assays, mechanism of action

Details of objectives and plans: https://www.mmv.org/mmv-open/malaria-libre/malaria-

libre-data-repository

Ways of contributing to the project

41

Back up

42

Action items Role of terminal pyridyl

MMV024406

Pf 3D7 LDH : 0.42µM

NF54/3D7 IC50(µM)

Previous data

Next set of compounds?

(Discussion point)

43

Series 1a - Discussion

Criterion (Early Lead) MMV1804508 MMV1804743

In vitro

3D7/Dd2 IC50 M 0.19/0.07 0.42/ND

Pb(sporozoite), Pf (schizont) 0.29/ongoing 0.52/ongoing

DGFA, IC50 MPf FGF(inactive), Pf

MGF(0.87)ND

FACS/PRR Slow rate of kill(3.82) Ongoing at TCG

MoA Not known Not known

ADME properties

h microsomes CLint, (mL/min/kg) /t1/2(min) 33/42 1.7/805

r hep,Clint, uL/min/106 / t1/2(min) 138/5 14/49

CYP inhibition (3/5 > 10, none < 1), M >10 ND

Solubility PBS pH 7.4 (>10), M 7.5 2.5

hPPB (% bound) ongoing ongoing

Permeability Caco-2, Papp A to B (10-6 cm/s) / efflux ratio

4.2/1.6 -

Safety profile

Cytotoxicity HepG2 (>100 fold IC50), M 9.7 25

hERG (SI>100 fold IC50), M 10.33 ND

Pb(sporozoite) IC50~3D7 LDH IC50

DGFA active

Slow rate of kill : generate MIR

Reinitiate work on series with focus of

targeting TCP4

3D7 can be used as surrogate for driving

SAR( generate liver stage data with few

more compounds to confirm this

observation)

Right time to reinitiate:

wait for y DHODH and cross R data?

MMV1804508 MMV1804743

Profiling in resistant strains at STPH(Recap)

44

MMV643121 MMV390048 MMV018912 MMV000130 MMV034055 MMV1803899 MMV690095

DDD107498 MMV390048 DSM265 GNF156 ELQ300

Mutated locus

Mutations (amino

acid changes) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)

Dd2 wt N/A 0.3436 14.97 10.05 8.990 24.51 462.3 385.4

Dd2 DDD107498 PfeEF2 Y186N 1558 NT NT NT NT 379.5 455.5

Dd2 390048 PfPI4K S743T NT 108.6 NT NT NT 439.0 395.0

Dd2 DSM265 Pfdhodh NT NT 277.2 NT NT 481.0 481.8

Dd2 GNF156 Pfcarl Ile1139Lys NT NT NT 1625 NT 397.4 373.6

Dd2 ELQ300 PfcytB Ile22Leu NT NT NT NT 216.7 418.9 423.9

MMV1803899 MMV690095

IC50 (nM)

NF54 186.1 173.3

K1 589.6 503.5

7G8 522.2 423.8

TM90C2B 384.6 202.5

RF12 (PH-1263-C; Ross et al. 2018

Nature Communications) 774.3 592.0

Dd2 462.3 385.4

MMV690095 MMV1803899

Compounds don’t show cross resistance in tested strains

45

MMVID

Pf3D7 LDH IC50 uM

RA: result awaited

Post meeting data

024408

0.43

Series 1a : Snapshot of modifications explored

1848300

2.22

1848186

0.881848511

0.63

1848693

0.30

1848694

0.52

Synthesis

ongoing

Synthesis ongoing 1848596

>25

1848594

>25

1848595

>25

1848597

2.8

Replacement of piperazine

46

Series 1a : modifications to improve metabolic

stability(update)

[O]

[O]

[O]

[O]

MMV ID 024408 1846943 1847005 1848300 1848511 1804743 1848187

Pf3D7(LD

H,IC50

uM)

0.43 0.6 0.42 2.2 0.62 0.42 0.31

elogD 4.3 4.7 3.2 3.3 3.9 5.2 5.4

HLM, Clint 18 12 2.5 65 260 1.7 21.3

r hep,Clint 233 103 93.5 26.5 127 14.2 74.2

MMV1846943

MMV1847005

MMV1848300MMV1804743

MMV1848187

MMV1848511

Confidential

Addressing poor metabolic stability of MMV023327

[O]

[O]

[O] MMV023227

MMV1794034MMV893302MMV892881

MMV893303

Putative metabolites

MMV ID 023227 692840 892881 1794034 893302 893303

Pf3D7(SYBR/

LDH,IC50 µM)

0.46/1.1 0.33/- 0.72/- 0.86/- 0.92/- 0.7/2.4

elogD 3.4 4.4 3.4 4.7 4.8 4.8

HLM,

Clint,mL/min/mg

205 173 54 246 98 70.3

r hep,Clint,

uL/min/106

206 ND ND 163 22.4 13.4

MMV692840

47

• Replacement of dimethyl imidazole with imidazopyridine improved metabolic stability

• Replacement of phenyl with pyridyl as scaffold improved metabolic stability

48

Rate of killing profile

Compound Dose Lag phase(h) Log PRR PCT99.9%

(h)

MMV1804317 10xIC50 24 2.82 62

Pyrimethamine 10xIC50 24 3.55 61

49

Series 1a(cyclo propyl amide)

Mechanisms picked up in a pH finger printing assay:

Pf ATP4, Pf FNT, Cl- transporter, V-ype H+ ATPase, protonophores, possible Pf HT or glycolysis

Compounds profiled:

MMV1804508 MMV1804743

Don’t have any of the mechanisms of action indicated above

MMV1804508 has slow rate of killing

Compound Dose Lag phase(h) Log PRR PCT99.9%

(h)

MMV1804508 10xIC50 72 3.82 94

Pyrimethamine 10xIC50 24 3.55 61

Parasitology

Transmission to

Human

43 – 48 h

15-30 mins

5.4 days

9 days

15 mins

1h

12-36h

9-12 days

Pb (sporozoite): 0.26; 1.03(MMV1794348); 1.58(MMV1804317)

Pf (3D7, NF54, Dd2): 0.24, 0.18, 0.33; No cross

résistance in UCSD panel of resistant strains

Stages (Ring/Troph/ Schizont): ongoing for exemplar along

with metabolomics @ Darren Creek lab

Pf/ Pv ex vivo: ND

aryl imidazole – MMV690095

Pb (sporozoite): 0.17; Pb (schizont): 0.12; Pf(schizont) : 0.19

Aryl piperazine: phenyl amides

Pf (3D7, NF54): 0.42,0.26; No cross resistance in

STPH panel of resistant strainsDGFA: in active

DGFA: in active

Aryl piperazine: cyclopropyl amides

Pb (sporozoite): 0.29 (MMV1804508); 0.53 (MV1804743)

Pf assay ongoing

Screening ongoing at STPH for exemplar

Pf MGF: 0.87uM; Pf FGF: >25uM

Moderate rate of killing

Fast rate of killing

Slow rate of killing

Exemplars sent for screening at Sanger(multiplex cross resistance assay)

Profiling in MMV assays for complete biological characterisation and series prioritisation for early lead

50

MMV024406

51

Action items Aryl imidazole - strategy

Core hopping

Rigidification

Replacement of benzyl

substituents with

(subs)cyclic amines,

fused amines

Objective: identify vectors for improving activity, impact on Dd2/3D7

Heterocycles,

Substitution on aryl ring

52

Action items Aryl imidazole – core hopping

MMV ID

3D7/Dd2 LDH IC50 (µM)

Objective: Identify vectors for improving activity and impact on Dd2/3D7 ratio

1804317

0.67/6.17

• Low Dd2/3D7shift is observed with substituted aryl compounds

• Profile MMV1900172 or MMV1900434 in Sanger multiplex assay

53

Action items Aryl imidazole - rigidification

Rigidification

Replacement of benzyl

substituents with

(subs)cyclic amines

MMV ID

3D7/Dd2 LDH IC50 (µM)

Ongoing/to be initiated at TCG

Objective: identify vectors for improving activity

Way forward: focus on central core rigidification modifications

Profile MMV1900786 in Pf Dd2 assay

CDRI