DE NOVO DESIGN OF A THYMIDYLATE KINASE INHIBITOR

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DE NOVO DESIGN OF A THYMIDYLATE KINASE INHIBITOR
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Thymidylate Synthase
Enzyme Reaction

• Notes
• dTMP is one of the building blocks for DNA
  synthesis
• Enzyme inhibition inhibits DNA synthesis and cell
  division
• Enzyme inhibitors are potential anticancer agents
• Inhibitors can be modelled on the substrate or
  cofactor

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Thymidylate Synthase
Cofactor

• Notes
• Provides a 1-C unit for biosynthetic pathways
• Inhibitors can be based on the cofactor structure
• Difficult to gain selectivity between enzymes
  using the same cofactor

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De Novo Design

• Notes
• The design of a novel inhibitor based on the
  structure of the binding site
• Crystallize target enzyme with known inhibitors
• Establish structure by X-ray crystallography
• Molecular modelling studies to carry out
  following
• Identify binding site and binding regions
• Design structure to fit the binding site
• Incorporate functional groups to make binding
  interactions
• Possibility of better selectivity between
  different targets

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De Novo Design
Enzyme inhibitors

• Notes
• 5-Fluorodeoxyuridylate binds to the substrate
  binding site
• CB 3717 binds to the cofactor binding site

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De Novo Design
Binding interactions for CB 3717

• Notes
• Identifies binding interactions of pteridine ring
• Identifies available amino acids and bridging
  water molecule

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De Novo Design

• Notes
• Remove CB 3717 in silico
• Set up a grid in the empty binding site
• Place an aromatic CH probe at each grid point
• Measure hydrophobic interactions
• The binding pocket for the pteridine ring is
  hydrophobic
• Identify a hydrophobic scaffold to fit the pocket
• Scaffold must be smaller than binding pocket to
  allow introduction of functional groups
• Add functional groups to make additional binding
  interactions

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De Novo Design

• Notes
• Hydrophobic naphthalene group forms van der Waals
  interactions with the binding site
• Room for additional binding groups

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De Novo Design

• Notes
• Lactam introduced to allow additional hydrogen
  bond interactions with the binding site
• Naphthostyryl scaffold

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De Novo Design

• Notes
• Amino substituent added to gain access to vacant
  region
• Placed at 5-position for synthetic feasibility
• Can vary N-alkyl groups without introducing an
  asymmetric centre

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De Novo Design
Notes The benzyl group mimics the benzene ring of
the cofactor
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De Novo Design

• Notes
• Phenylsulfonylpiperazine group is added for water
  solubility
• Positioned to protrude from binding site
• Makes contact with surrounding water
• No desolvation penalty

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De Novo Design

• Notes
• De novo designed inhibitor is now synthesized and
  tested
• Active inhibitor
• Co-crystallized with enzyme
• Crystal structure determined

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Binding Interactions
Intended
Actual
Water shifted

• Notes
• Inhibitor binds deeper into pocket than expected
• Ala-263 shifted due to steric clash
• Water molecule displaced to different position

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Structure-based Drug Design

• Notes
• Molecular modelling studies of actual binding
  interactions
• Identifies 4 regions for modification
• Possible analogues are overlaid with lead
  compound to test whether they fit the binding
  site
• Synthesis of analogues

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Structure-based Drug Design

• Region R1
• Substituent fits hydrophobic pocket
• Pocket becomes hydrophilic with depth
• Polar functional group at the end of the alkyl
  chain may be beneficial
• CH2CH2OH has greater binding affinity
• Methyl better than ethyl

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Structure-based Drug Design

• Region R2
• Carbonyl oxygen replaced with amidine group
• Capable of binding to Ala-263 instead of
  repelling it
• More basic and protonated - allows ionic
  interaction and stronger hydrogen bonding
  interaction
• Increased inhibition
• Binding interactions identified from crystal
  structure

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Structure-based Drug Design
Binding interactions
Ionic and stronger H- bonding interactions

• Notes
• Binding interactions as expected
• Ala-263 not displaced
• Bridging water molecule not displaced

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Structure-based Drug Design

• Region R3
• Small hydrophobic pocket available in the region
• Methyl or chloro-substituent both beneficial for
  activity

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Structure-based Drug Design
Region R4 Morpholine ring found to be beneficial
for selectivity and pharmacokinetics
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Structure-based Drug Design

• Notes
• Structures are synthesized containing
  combinations of the optimum groups at each
  position
• Amidine is the most important group for enhanced
  activity
• Inclusion of all the optimum groups is not
  beneficial

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Structure-based Drug Design

• Notes
• Amidine, morpholine and methyl group are
  introduced
• No change at R3
• Potent inhibitor (500 x more active)
• Structure taken forward for clinical trials

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De Novo Design

• Principles
• De novo design is useful in designing lead
  compounds for structure-based drug design
• Designed structure should be loose fitting and
  flexible
• Allows possibility of different binding modes if
  binding does not take place as predicted
• Allows scope for further modification and drug
  optimization
• Compounds should be synthetically feasible
• Compounds should be in a stable conformation
• Desolvation penalties need to be considered