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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
4
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
6
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
15
• 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.
✔
✔ ✔
✔
✔
✔
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
✔
✔
✔
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