Mode of Action of Destruxins StructureActivity Relationships

1
Mode of Action of DestruxinsStructure/Activity
Relationships

• Martyn Ford
• Centre for Molecular Design
• University of Portsmouth

2
Search for Novel Drugs Insecticides

• Development of resistant strains unsolved
  problems in healthcare crop protection require
• novel biologically active materials to act as
  leads for the development of drugs and crop
  protectants

3
Destruxins - novel lead compounds

• Destruxins are cyclic peptides
• extracted from fungi
• eliciting a range of biological responses
• including cytotoxicity inhibition of gene
  expression
• These natural products have potential
• as insecticides and control agents of disease

4
Structure of Destruxins

• Cyclic hexadepsipeptides produced by
  entomopathogenic and phytopathogenic fungi
• Five amino acids and one hydroxyl acid
• The family contains over 32 destruxins

5
Genomics proteomics

• Because the destruxins are small cyclic peptides
• there is scope for genomic proteomic database
  searching to identify further leads
• but first, the mode of action must be established
  
• in order to identify appropriate clinical crop
  protectant targets

6
Effect of Destruxinsand mode of action

• Destruxins are nerve poisons and general
  cytotoxins
• inhibit effects of the proliferation of leukemic
  cells and the expression of hepatitis B
• modify the concentration of calcium, provoking
  depolarization of the muscles???

7
Potent Destruxins

• Destruxin A
• Destruxin E

8
Structure/Activity Relationships

• Increased potency is associated with
• the absence of a hydrophilic function
• the presence of a side chain with a double bond
  or an epoxy ring

9
Enniatin a cylic peptide similar to Destruxins

• Enniatins are produced by phytopathogenic fungi
• These compounds belong to a class of ionophoric
  antibiotics
• which induce the transport of group I cations

10
Structure of Enniatins
11
Mode of Action of Enniatin

• Enniatins chelate sodium and potassium
• Ester groups play a role in ligand binding by
  interacting with the cations.
• The aliphatic side chain and the N-methyl group
  are located on the surface of the chelation
  complex to form a hydrophobic environment around
  the cation

12
Mode of Actionof Enniatins (2)

• There are two ways to chelate group I cations
• 11 internal complex

13
Mode of Actionof Enniatins (2)

• There are two ways to chelate group I cations
• 21 external complex
• where the ions are binds by two molecules of
  Enniatins

14
Computational Studiesof Destruxin A

• The X-Ray conformations of Destruxin A and
  Enniatin B have been superimposed in Quanta
• using the match atoms molecular similarity
  procedures
• the carbonyl and N-methyl groups have been
  matched using a Cartesian flexibility fitting
  procedure

15
Superimposition of Destruxin A Enniatin B
16
Molecular Dynamics (MD)

• MD simulations were used to identify a possible
  ionophoric structures for destruxin
• in chelation complexes with Ca2

17
Parameterisation of the MM Force Field

• Standard CHARMm v.21.3 as implemented in MSI
  software
• CHARMm used stand-alone for MD
• CHARMm scripts written to take into account an
  appropriate dielectric for a H2O environment
• http//www.cmd.port.ac.uk/webdocs/destruxin.html

18
Molecular Dynamics of Destruxin A - CHARMm

• Minimisation steepest descent 9000
• Heating from O to 300?K during 10ps.
• Equilibration during 10ps
• Simulation during 1ns
• 3 possible chelation complexes were identified
• and confirmed by cluster analysis!

19
Destruxin Complexes1 2 with Ca2
20
Destruxin complexeswith Ca2 (3)
21
Distance O-Ca2 forDestruxin A
22
Energy Comparison of Destruxin A

• The energies of the four conformations are
  similar
• with low interconversion energy barriers
• Destruxin A may adopt the same conformation as
  Enniatin B

23
Studies of Destruxin D (a less toxic material)
24
Distance O-Ca2 forDestruxin D
25
Predicted Log KOW of Destruxins A D
26
Destruxin Complexes with Na (1 2)
27
Destruxin Complex with Na (3)
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Destruxin Complexes with K (1 2)
29
Destruxin Complex with K (3)
30
Selective binding

• The modelling studies suggest that Destruxin A
  can chelate Ca2
• but not Na or K
• Evidence in support of this hypothesis has been
  obtained using a competition reaction
• using a Ca2 sensitive dye - Antipyrylazo III

31
Experimentation using colourimetric dye

• Competition reaction between the colourimetric
  dye Antipyrylazo III (AA) and Destruxin A
• a- 12.5 ?g/ml of AA
• b- 12.5 ?g/ml of AA 400 ?M of CaCl2
• c- 12.5 ?g/ml of AA 400 ?M of CaCl2 2 mM of
  Dtx A

32
Comparison between Enniatin Destruxin A
33
Biological Significance

• The binding constant of Destruxin A for Ca2 (1.6
  mM) matches the extracellular concentration of
  Ca2 (1-10 mM)
• this is consistent with a putative role for
  Destruxin A as a Ca2 ionophore

34
Ionophoric properties of Destruxin A

• Experiment using the fluorescent dye fura-2
• Placed inside the liposome (Kd for calcium125nM)
• Calcium (800 ?M) Destruxin A (100 ?M) added
  outside the liposome

35
Ionophoric properties of Destruxin A

• Thus addition of Ca2 and Destruxin A to the
  external medium surrounding liposomes containing
  a Ca2 sensitive dye
• results in an increase in dye intensity at 380 nm
  and a decrease at 340 nm

36
Ionophoric properties of Destruxin A

• This result suggests that in the presence of
  destruxin, Ca2 is able to cross the liposome
  membrane to form a complex with the fluorescent
  dye fura-2

37
Proposed Mode of Actionof Destruxin
38
Kinetics of Ca2 transport across liposome
bilayers

• The transport kinetics of Ca2 across membranes
  has been investigated further
• with fura2 dye (5 ?M) inside the liposome
  compartment
• with Ca2 (800 ?M) on the outside
• and destruxin A (100 ?M) added to the external
  bathing medium to initiate the Ca2 transport
  process

39
Kinetic profile after addition of destruxin A

• The time course of the increase in chelated dye
  within the liposome is characterised by
• an initial sigmoidal phase followed by
• a linear increase in dye intensity

40
Interpretation

• The initial sigmoidal phase reflects
• the build up of destruxin (Dtx) in the bilayer
  and
• an associated increase in the transport of Ca2
  ions across the membrane

• This early phase is characterised by
• an initial lag time
• attainment of a steady state accumulation of
  destruxin in the membrane
• approach to a limiting Dtx concentration

41
Interpretation

• Once the Dtx has attained an equilibrium
  concentration in the bilayer
• Ca2 ions will continue to be transported across
  the bilayer, but at a constant rate (33?0.2
  pM/second)

• On reaching the internal bilayer interface
• Ca2 ions will be released into the centre of the
  liposome
• Dtx will diffuse back to the external surface,
  bind more Ca2 repeat the process
• until equilibrium has been established across the
  bilayer

42
Derivative of the Ca2 transport curve

• The transport process can be investigated in
  terms of the 1st derivative of the kinetic
  profile
• which rapidly peaks at 130 pM s-1
• then falls exponentially to a limit of 33 pM s-1

43
Conclusions

• Experiments using liposomes and cation binding
  show that Destruxin is able to behave as an
  ionophoric molecule
• The Kd of value (1.6 mM) for Ca2 is such that
  the peptide will chelate the calcium outside the
  cell (extracellular calcium concentration 1 -
  10 mM)

44
Conclusions

• The chelation complex crosses the membrane to
  unload Ca2
• with an intracellular concentration (ca. 0.1?M)
  substantially below the Kd value (1.6 mM)
• it is proposed that the destruxin molecules
  shuttle across the membrane
• loading unloading Ca2
• causing muscle paralysis and cytotoxicity

45
Future studies

• To search for other destruxin and enniatin-like
  peptides using genomic proteomic database
  sequence searches
• Using these lead structures, identify and
  synthesise peptoid mimics with crop protectant
  and medicinal properties

46
Acknowledgements

• Maria Hinaje - (UoP)
• Dr Lee Banting - (UoP)
• Dr David Salt - (UoP)
• Dr David Livingstone - (ChemQuest)
• Dr Steve Arckle - (UoP)
• Dr Paul Cox - (UoP)
• Dr Bhupinder Khambay - (BBSRC IACR)