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DHODH: Structure-Guided Design of a New Class of Drugs

Defeating Malaria Together

Meg Phillips, Ph.D. UT Southwestern

Pipeline to development of a DHODH-based anti-malarial

Target

Actual or potential inhibitors

Validated hits

Leads Drug Candidate

Drug

HTS Screen Animal models

Safety Pharmacology

Preclinical and Clinical development

Iterative medicinal chemistry

Optimize efficacy and pharmaceutical properties

Inform with X-ray Structure

2001 Phillips enzymology/ structure/HTS; Rathod

parasitology, med chem

2004 NIH funding Phillips/Rathod

Program: Brewer/ Rogers

2006 Charman: ADME

2007 MMV/NIH funding to Phillips/Rathod/Charman

Global Academic and Industrial Partnership facilitated by MMV and NIH – “virtual” drug company

DSMteamAdvisors: Bathurst,

Burrows, Floyd, Matthews, Craft, Dayan

GSK, Buckner

2009: 3 DHODH teams - DSM/GSK/

Genzyme

2009 Coteron/GSK

Identifying a target - pinpointing vulnerable biology in the cell

Bases (nucleotides) are the building blocks of DNA

Nucleus Chromosome

DNA

Base pairs

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How Parasites Replicate their Genetic Material

UMP

G A

T C

Building blocksfor DNA and RNA

SalvageU

Synthesis

CO2 + amino acids

Host

Host and Malaria Parasite

An essential pathway X

X

DHODH the target of a novel potential therapy

DNA and RNA

U

G A

T C

Building blocksfor DNA and RNA

CO2 + amino acids

DHODH

DHO

orotate

MalariaDHODH

HumanDHODH

Drug binding site

Differences in the shape of the drug binding site Parasite enzyme can be selectively blocked

Identifying malaria parasite selective DHODH inhibitors

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Start with 200,000 drug like chemicals – find the needle in the haystack

High throughput screen (HTS) to identify DHODH inhibitors

•  Plate potential inhibitors on 384 well plates (520) and assay enzyme •  Identified 1350 compounds that inhibited malaria DHODH

Blue to white screen Inhibitor = blue

UT Southwestern screening core – Phillips lab

Baldwin, et al J. Biol. Chem. 2005, 280, 21847-21853

Triazolopyrimidines - The Lead Compound Series

•  HTS identified potent and selective inhibitors of P. falciparum DHODH

•  ~12 structural classes; 60 compounds with IC50 < 500 nM

•  Lead series – triazolopyrimidines

•  PfDHODH ( IC50 = 50 nM); human DHODH (>200,000 nM)

Phillips, et al J. Med. Chem. 2008, 51, 3649-3653

>200,000 nM

Frac

tion

activ

e Typical drug 5 – 10 nM

Mechanism of parasite killing by DSM1

Painter, et al Nature 2007, 446, 88-91.

•  DSM1 potent inhibitor of parasite growth in vitro

University of Washington – Rathod lab

>6,000 nM

Pyrimidine biosynthesis

%pa

rasi

te s

urvi

val

Pyrimidine biosynthesis

Malaria DHODH

Parasites grow Parasites die

Mechanism of parasite killing by DSM1

•  DSM1 potent inhibitor of parasite growth in vitro

•  DHODH is primary target of cell killing

•  Identified a potent DHODH inhibitor, but not effective in animals

•  Next step understand metabolism and plasma exposure

Malaria DHODH

Pyrimidine biosynthesis

Parasites grow

DSM1 Yeast DHODH

%pa

rasi

te s

urvi

val

University of Washington – Rathod lab

Identifying compounds that kill parasites in animals and man

Gujjar, et al J Med Chem. 2009 Apr 9;52(7):1864-72; Gujjar, R., et al. (2011) J. Med. Chem., in press.

•  Library of ~80 analogs with naphthyl replacements synthesized •  Identified compounds that show good stability

ADME: Monash University – Charman lab

Chemistry: University of Washington – Rathod lab DSM1

DSM74

Malaria parasite growth inhibited in P. berghei mouse model

Gujjar, et al J Med Chem. 2009 Apr 9;52(7):1864-72 University of Washington – Buckner/Rathod lab

PfDHODH 270 nM hDHODH > 100,000 nM Pfalcip 3D7 320 nM

•  First proof of concept that DHODH inhibitors can suppress parasites in vivo

•  No evidence of toxicity Oral dosing

Compound lacks potency required of clinical candidate

Another round of lead optimization

X-ray structure provided insight to improve potency

UT Southwestern Med Center – Phillips lab

N

N

N

N

HN

R1

R

R1 = CF3, SF5 or Cl

R = CF2CH3, CF3, OCH2CH3, CH2CH3and many others

A library of 100 molecules were synthesized to find the optimal R-group GSK Tres Cantos – Coteron lab

•  Improved potency 30-60-fold for redesigned molecules (DSMRD) •  PfDHODH and whole cell activity meet development criteria •  Metabolism and plasma exposure?

InvitroP.falciparumassay

WO 2011/041304; PCT/US2010/050532, 4/07/11, 2011

X-ray structure provided insight to improve potency

Candidates pharmacokinetic profile in rats

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•  Good oral bioavailability

•  Prolonged plasma exposure

•  Long half-life supports meeting Target Product Profile of once daily dosing

Monash University – Charman lab

Identified a potent compound with good plasma exposure, what about in vivo activity?

Candidate mouse efficacy study – SCID mouse

•  Single oral dose per day for 4 days

•  As potent as Chloroquine (ED50 = 3.2 mg/kg)

•  works on drug resistant parasites

•  Candidate meets development criteria for efficacy

GSK Tres Cantos - Iñigo Angulo Barturen and Santiago B. Ferrer

Pipeline to development of a DHODH-based anti-malarial

Target

Actual or potential inhibitors

Validated hits

Leads Drug Candidate

Drug

HTS Screen Animal models

Safety Pharmacology

Preclinical and Clinical development

Pink, et al Nature reviews drug discovery (2005) 4, 727 - 740.

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Ames negative CEREP panel clean Rabbit Ventricular Wedge assay clean

Animal Tox - ongoing

Summer 2011

Iterative medicinal chemistry Optimize efficacy and pharmaceutical properties

Inform with X-ray Structure

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The Team – a global partnership

GSK Spain – Med chem support: Jose Coteron, Ph.D. María Marco-Martín and Jorge Esquivias-Provencio, chemists

SCID Mouse models: Iñigo Angulo Barturen and Santiago B. Ferrer

Advisors: Ian Bathurst (MMV), Jeremy Burrows (MMV), John Rogers (NIH), Dave Matthews, David Floyd, Carl Craft

Funding: NIH U01AI075594; Medicines for Malaria Venture

UT Southwestern Meg Phillips, Ph.D.

Farah El_Mazouni Xiaoyi Deng Nick Malmquist Betsy Goldsmith Jeff Baldwin

Monash University Sue Charman, Ph.D. Bill Charman, Ph.D.

Karen White David Shackleford

University of Washington Pradip Rathod, Ph.D.

Ramesh Gujjar John White Rapat Patrapuvich Jenny Gϋler Fred Buckner Sharon Creason Akhil Vaidya

Genzyme – Ted Sybretz and Jeff Klinger