1. Improving Gleevec: Insight from the Receptor Structure Gleevec cannot bind to the open (active) form of the Abl kinase - would collide with open conformation of the activation loop

2. Remove portion of molecule causing steric clash with the open (active) conformation of the activation loop Arrived at new class of drug, PD17, predicted to still bind competitively at ATP-binding site of the Abl kinase

3. Gleevec PD17 6 H-bonds2 H-bonds contacts 21 residuescontacts 11 residues IC 50 = 100 nMIC 50 = 5 nM Despite fewer interactions with Abl, the drug PD17 is a better inhibitor

4. Inactive Abl + PD17Active Abl + PD17 Despite making fewer contacts with the target protein, PD17 is a better inhibitor because it binds to both conformations of Abl - Thus, losing an H-bond but removing Gleevec’s steric clash with the open conformation led to an improved drug

5. Improving PD17 PD17 loses the H-bond to threonine 319 that is essential for Gleevec’s activity; however, this residue remains available for H-bonding near the end of PD17 Can PD17 be improved by engineering a new H-bond to Thr319? PD17

6. Hydroxyl group contributes new H-bond even better binding (IC 50 = 0.4 nM) Better binding than Gleevec (IC 50 = 5 nM) PD17 PD166326

7. With further improvements: Dasatinib, first 2 nd -generation kinase inhibitor Gleevec

8. With further improvements: Dasatinib, first 2 nd -generation kinase inhibitor - 325 times more effective than Gleevec against normal CML cancer - effective against tumors expressing 14 out of 15 resistance mutations (all but the dreaded Thr-315  Isoleucine)

9. With further improvements: Dasatinib, first 2 nd -generation kinase inhibitor Tokarski et al. Cancer Res. 2006

10. - dasatinib binds to both active and inactive conformations Phe-382  -stacks with Gleevec pyrimidine ring, locks activation loop in inactive conformation Gleevec occupies a hydrophobic pocket that is otherwise filled by Phe-382 Tokarski et al. Cancer Res. 2006

11. Receptor-Based Design Knowing that BCR/ABL fusion protein is the specific cause of CML... (1) Identify a small molecule that selectively inhibits this kinase (Gleevec) (2) Perform structural studies to understand mechanism of action: - discover new mode of drug action: selective binding to inactive kinase structure (varies from kinase to kinase) (3) Use structural information to make a drug that binds either conformation (PD17) (4) Through a second round of structural studies, add H-bonding interactions to optimize the inhibitor (PD166326)

12. Receptor-Based Design Knowing that BCR/ABL fusion protein is the specific cause of CML... (5) Create 2 nd generation drug – Dasatinib More effective than Gleevec because: a) binds both active and inactive forms.. b) causes few distortions of protein, compared to ATP-bound form.. c) makes fewer interactions with P-loop & other parts of ABL..

13. Receptor-Based Examples 1. Targeting a single protein essential for disease progression Improving Gleevec, a new anti-cancer drug 2. Taking advantage of unique features of a protein target Prophylactic Inhibition of Cholera Toxin

14. Disease: Cholera (caused by bacterium Vibrio cholerae) Traveler’s diarrhea (E. coli) - combined, kill over 1 million people per year Target: pentameric protein toxins The pathogenic bacteria V. cholerae and E. coli affect humans by producing a protein toxin that forms a pentamer - toxin has 5 identical subunits that come together in a star-shape - released in lumen of the intestine - each of the 5 units binds to an oligosaccharide on epithelial cell surfaces, gaining entry into the cell Strategy: design inhibitors to block binding of receptors to natural ligand on cell surface, thus preventing toxin from entering

15. Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure Natural ligand of cholera toxin is an oligosaccharide ending in a terminal galactose sugar Substitutions wouldn’t work at O3, O4; each acts as H-bond donor & acceptor with protein side chains Also, no substitutions at O6, which is bonded to 2 waters Glu Lys Trp Asn H2OH2O H-bond acceptor H-bond donor

16. Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure Substitutions would work at O1, O2 - only lose 1 H-bond, to a displaceable H 2 O 35 galactose analogues purchased + tested to see if they could inhibit binding of natural ligand to the toxin protein 7 had lower IC 50 ’s than galactose itself Glu Lys Trp Asn H2OH2O H-bond acceptor H-bond donor

17. Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure Most potent inhibitor was m-nitrophenyl-  - D -galactoside (MNPG)

18. Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure Most potent inhibitor was m-nitrophenyl-  - D -galactoside (MNPG) - retains favorable binding interactions of the natural ligand - nitrophenyl group displaces a water molecule - structure-based design came up with an inhibitor K d of 10  M, a 100-fold improvement over galactose alone... - however, still much lower than the affinity for the natural ligand

19. Options for designing high affinity protein inhibitors: (1) Make a drug that binds tightly to the binding site - 5 molecules must bind per toxin pentamer, independently (2) Make a penta-valent inhibitor, that is, one molecule with 5 inhibitory “fingers” linked to a central core - 1 molecule binds per toxin pentamer, but fingers bind semi- cooperatively

20. In multivalent binding, binding of 1 finger aligns other fingers with their receptor sites - this increases the overall binding affinity, by decreasing entropic costs associated with multiple ligands binding independently - linkers can also make favorable contacts with the protein surface, further promoting binding allows you to make a potent inhibitor even if the fingers on their own aren’t such good binders each low affinity high affinity strong binding vs.

21. Step 2: Determine whether making a pentavalent ligand improves binding Multi-valent drug design is a strategy to get higher binding affinity by exploiting the presence of multiple, identical binding sites on a target protein - for instance, many proteins are multimeric, meaning composed of several identical subunits - design a single, large molecule which presents multiple copies of an inhibitor, arranged to jam all binding sites on the target

22. Step 2: Determine whether making a pentavalent ligand improves binding to cholera toxin Attach galactose to a scaffold, using flexible linkers to space out 5 sugar residues joined to a central core galactose scaffold flexible linker arm (R 1 ) each one of these arms is the same as the one shown above

23. Step 2: Determine whether making a pentavalent ligand improves binding Attach galactose to a scaffold, using flexible linkers to space out 5 sugar residues joined to a central core IC 50 (  M) Galactose-based finger, alone5,000 Galactose-based pentavalent ligand16

24. Step 3: Combine the 2 ways to improve binding: make a pentavalent ligand using the improved galactose derivative Attach m-nitrophenyl-  - D -galactoside (MNPG) to a scaffold, with linkers to position the fingers over the 5 binding sites of the pentamer

25. Step 3: Combine the 2 ways to improve binding: make a pentavalent ligand using the improved galactose derivative Attach m-nitrophenyl-  - D -galactoside (MNPG) to a scaffold, with linkers to position the fingers over the 5 binding sites of the pentamer IC 50 (  M) Galactose-based finger5,000 Galactose-based 16 pentavalent ligand MNPG finger alone 195 MNPG pentavalent ligand 1 pentavalent ligand shows ~200-fold improvement over the best single-site derivative

26. Yellow = MNPG ligand Green = 1 arm of pentavalent ligand Red = a water molecule that forms hydrogen bonds w/ natural galactose + protein amide; - displaced by an oxygen of the inhibitor’s nitrophenyl ring Pentavalent ligand fills the toxin pocket in similar manner as the free MNPG inhibitor, but with the higher binding affinity that comes with multivalency

27. Step 4: Continue to improve binding affinity: change scaffold (1) Improve fit of linkers - make more rigid: less conformations, binding is more entropically favored - enhance interactions with protein surface - present linker makes van der Waals contacts w/ side chains glu, tyr, his, lys, arg (2) Increase valency: go from penta-valent (5 ligands) to deca-valent (10 ligands)

28. Now design a drug that will bind to 2 toxin pentamers simultaneously

29. green = natural ligand (oligosaccharide w/ terminal galactose) blue = 1 arm of pentavalent (5-armed) ligand brown = 1 arm of decavalent (10-armed) ligand