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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