Antimalarial drug development
through product development
partnerships
Tomo Nozaki, MD, PhD
Graduate School of Medicine
The University of Tokyo
GHIT-PDPs Webinar Series:
Advancing innovations for neglected diseases
during and beyond the pandemic
September 3, 2021

Screening platform: 2018-2020"High throughput screening of DDI compound library against theasexual blood stage of Plasmodium falciparum"
Hit-to-Lead platform: 2021-2023"Hit-to-Lead development of DDI series for antimalarialdevelopment"
Jeremy Burrows

Norio Shibata
Nagoya Instituteof TechnologyPaul Willis

James Duffy

Tomo NOZAKI
Who am I?
Professor
Department of Biomedical Chemistry
Vice Dean
Graduate School of Medicine,
The University of Tokyo
M.D., Ph.D. (Keio University School of Medicine)Specialty: Molecular parasitology (Entamoeba, Plasmodia, Trypanosoma,and Leishmania)
Major interests: Metabolism, Pathogenesis, Evolution, and Drug development
My dream when I was a medical student: a doctor in Amazon

Tomo NOZAKI
Who am I?
Major achievements
Metabolism
Cysteine/methionine, FeS, NADH, CoA

Pathogenesis

Adv Parasitol, 60, 1-99, 2005; Clin Microbiol
Rev, 20, 164-187, 2007; Adv Parasitol, 83, 192, 2013; Biochimie 95, 309-319, 2013; CurrOpin Microbiol 20C:118-124, 2014; FrontMicrobiol 9:2902, 2018; Front Cell Inf Microbiol
11:639065, 2021

Vesicular traffic, proteases, phago(& trogo)cytosis, lipid transport

Evolution

Nature 433, 865-868; Nat Commun 8,101, 2017; PLoS Pathog, 14:e1006882,2018.; PLoS Pathog, 17:e1009551, 2021. PLoS Pathog., 17:e1008909, 2021

Mitochondria under anaerobic environment

Drug development

Proc Natl Acad Sci USA, 106, 21731–21736; Proc Natl Acad Sci USA
112:E2884-90, 2015; Trends Parasitol. 34:1038-1055, 2018.

Target identification and anti-amebic drug lead identification
FEBS J 275, 548-560, 2008; IUBMB Life, 61, 1019-1028, 2009; J Antimicrob Chemother 66,2045-2052, 2011; Front Microbiol 6, 962, 2015.; Parasitol Int 102432, 2021

How we started MMV-UTokyo collaboration: background

When we wanted to start our drug discovery
campaign,....

• A majority of screening projects were conducted bypharmaceutical companies, using their own
libraries.
• There were very few projects from Japanese
academia. Only a limited set of their institutionowned natural compounds were tested against
malaria and other parasitic diseases.

Why not use academia-owned chemical libraries
for malaria drug discovery.

How we started MMV-UTokyo collaboration
We have expertise in molecular parasitology, and we are keen todevelop medicines for malaria and other parasitic diseases.
üWe have excellent target (enzyme)-based and cell-based(phenotypic) assays.
üStructurally-defined chemical and natural microbial culture brothlibraries are readily available.
So, ........
üWe need external help for chemistry, preclinical development
including safety testing, PK/PD, and ADME.
üWe need a compass for directions and good collaborative
network for the above.
üThen, ........
üWe started searching potential collaborators and fundingsources.

Our MMV-UTokyo collaboration

We aim at:
Discovery and optimization of new compounds that are
efficacious against Plasmodium falciparum and have newscaffold and novel mechanism of action
Desired properties include:
••
•
•
•
•
•
•

Fast acting for treatment
Long duration for prophylaxis
Oral dose
Efficacious against drug resistant field strains
Targeting multiple lifecycle stages
Low propensity of raising drug resistance
Cheap
Applicable for vulnerable populations (children and women of childbearing potential)
Burrows, J. N. et al. Malar. J. 16, 26 (2017)from a Japan-made structurally defined chemical libraryObjective
Discover new compounds (with novel scaffold andmechanism of action) against Plasmodium falciparumfrom a Japan-made structurally defined library

Compound library

THE UNIVERSITY OF TOKYOThe compounds are obtained from Drug Discovery Initiative (DDI),The University of Tokyo- 210,150 compounds

Assay
P. falciparum erythrocytic stage
3D7 386 well plates
LDH/SYBR Green assaysPfLDH

Yulia Rahmawati
diaphorase

Haruka Ohno
NatsukiWatanabeAssay evaluation and validation
S/B Profile

1.000

6.00

0.800

5.00
S/B Value

Z' ValueZ' Value Profile

0.600
0.400

4.00
3.00
2.00

0.200

1.00

0.000

0.00
0100

200

300

400

Number of Plate

Z’ value : 0.76 ± 0.07
Excellent assay

500600

700

0100

200

300

400500600

Number of Plate

S/B value : mostly > 3700

Inhibition profile and hit rate
Number of actives% Inhibition

Number of(2 μM)

compounds

≧30

5,831

≧502,505

≧70

1,580

≧801,307≧90974

Total number

210,150

Active rate (>90% inhibition at 2 µM): 0.5%(>50%): 1.2%

Screening cascade
210,150 compounds (DDI)
THE UNIVERSITY OF TOKYO

Primary screening

P. falciparum

3D7 asexual stage (phenotypic) assay(LDH assay)
Cut off: 90% inhibition at 2 µM at 72 h974
Tertiary assay

Secondary (confirmation) assay

3D7

P. falciparum
asexual stage
2 µM; 72 h, 50% cut-off
LDHAssay

648

SYBR GreenAssay

yeast DHODHexpressing 3D7 strain
2 µM; 72 h, 70% cut-off

Quaternary assay

482 actives

Dd2 chloroquine
resistant strain
2 µM; 72 h, 70% cut-offScreening cascade
Quaternary assay

482 actives

Dd2 chloroquine
resistant strain
2 µM; 72 h, 70% cut-off

End of screening projectExclusion by:• No malaria drug fragment (overlaps with known antimalarials excluded)• Structural novelty (Similarity < 0.7 with compounds in MMV databases)

106'Chemist's eye'selection based on
chemical attractiveness8 actives(4 commercial available)

IC50 / EC50 determination
Cytotoxicity against humancells (HepG2) (Safety index > 10)

44(11 commercial available &33 non-commercialavailable)

3 CA + 1 NCA
actives selected82

Prioritization by:
Potency, physicochemical
properties, safety index,structural alerts(StarDrop multiparameter
optimization > 0.1)

Screening cascade (after active discovery)Three hit-to-lead candidates
Series 1

T-117040 (MMV1804220)

Series 2

T-132522 (MMV1804245)

Series 3

T-210671 (MMV1804223)• Good lipophilicity (eLogD: 2.72-3.53) as starting actives• Reasonable kinetic solubility (46~190 µM)• Series 1-3 inhibit none of the following known targets: DHODH, electrontransport chain including bc1 complex, ACS, CARL, PI4K, and ATP4

Profiling of three hit-to-lead candidates
Series 2

Series 1

IC50 3D7 asexual stages (LDH72 hr)

Series 3

T- 117040 (MMV1804220) T-132522 (MMV1804245) T-210671 (MMV1804223)
0.75 µM
1.3 µM
0.23 µM
Slow

Fast

Fast

1.58 µM0.52 µM

0.16 µM

IC50 P. berghei liver activity

0.11 µM

9.77 µM

0.041 µM

% inhibition @ 1 µM
Pf male and female gamete
formation

NA43-96%0-3.9%

IC50 human primary
hepatocytes 96 hr>10 µM>10 µM>10 µMhERG K+ CHO (patch clamp)>30 µM

1.26 µM>30 µM

17 µL/min/mg

27011644.5 µL/min/106 cells

95.6

93D7 rate of kill
IC50 drug resistant Dd2(SYBR Green 72 hr)

Metabolism by humanmicrosomes CLint
Metabolism by rat hepatocytes

Advantages and issues of three hit-to-lead candidates
Series 1

T-117040 (MMV1804220)

Series 2

T-132522 (MMV1804245)

Series 3

T-210671 (MMV1804223)

Strengths ••Liver stage activity
Metabolic stability• Fast kill• Sexual stage activity• De-risked potential
toxicity issue• Fast kill
• Liver stage activityIssues

Improve potency
Define SAR• Need to identify minimal
pharmacophore

203• Peptide like (Oral absorption?)• Complex synthesis(3 chiral centers)• Address liabilities in new
molecules
13••

Number ofderivatives
synthesizedSummaryü

U Tokyo/MMV screened DDI library, Japanese
academia-owned chemical library of 210k compounds,
and identified 3-4 candidates to advance to "hit-to-lead".

üStructural optimization (based on potency,
physicochemical properties, stability, and SAR) andprofiling of the improved actives (the rate of kill, multiple
life cycle stages, mechanism of action, and ADME) are
under way.

Future perspective and requests from academiaüüüü

We have directly gained and will gain knowledge and expertise on antimalarial(& other IDs) drug development from MMV and also indirectly via liaison withexperts in pharmaceutical industries and other international partners.
We, parasitologists in academia, need to create a broader network covering
natural and synthetic chemistry, safety, and even regulatory sciences.
MMV bridges a gap between academia and industries (also within academia)
by promoting networking and helping us be connected with experts withnecessary disciplines.
MMV also provides young researchers an opportunity for exposure to theoutside of the lab, and lots of experience, encouragement, and excitement forrealization of their research.

üWe are looking forward to good progress of our structurally optimized initialleads toward Lead Optimization in 1.5 years, and Preclinical/Clinical platforms
in 3-4 years.

üIt would be nice if GHIT Fund also aids in connecting us with pharmaceuticalindustries and even contract research organizations (CROs).

MMVPaul Willis
James Duffy
Jeremy Burrows

Acknowledgements
Acknowledgements
Drug Discovery Initiative
Hirotatsu Kojima
Riyo Imamura

U Tokyo
Yulia Rahmawati
Haruka Ohno
Natsuki Watanabe
Ghulam Jeelani
Arif Nurkanto

Tomoo Shiba

Nagasaki U

Nagoya Institute Tokyo Medical Dental Uof Technology
Tomoko Ishino
Norio Shibata
Yuji Sumii

Kyoto Inst Technol
Daniel Inaoka
Takaya Sakura
Kiyoshi Kita

NITE
Mihoko Mori

Naoki Shinzawa

Keio UBikaken

Juntendo UKiyotake Suenaga
Naoaki Kurisawa
Arihiro Iwasaki

Masayuki IgarashiHideyuki Muramatsu
Kazuro Shiomi

Makoto Hirai

National Institute of
Infectious Diseases
Yumiko Saito-Nakano
Takeshi Annoura

Shizuoka Pref U
Yuta Tsunematsu
Ryota Shizu