Molecular Recognition in Chemistry and Biology: Francois Diederich
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Molecular Recognition in Chemistry and Biology François Diederich Department of Chemistry and Applied Biosciences ETH Zurich (Switzerland) Creator Space Science Symposium “Sustainable Food Chain: From Field to Table” 150 th Anniversary BASF Chicago June 22-24, 2015
Transcript of Molecular Recognition in Chemistry and Biology: Francois Diederich
Molecular Recognition in Chemistry and Biology
François Diederich!Department of Chemistry and Applied Biosciences!
ETH Zurich (Switzerland)!!
Creator Space Science Symposium ! “Sustainable Food Chain: From Field to Table”!
150th Anniversary BASF!Chicago!
June 22-24, 2015!
Protein-Ligand Interactions: Their Understanding Guides Structure-Based Design and Optimization of Drugs and Agrochemicals !
OHN
NH
BrN
O
SO2
Cl
NO H
S
N
HN
O
O
Gly61
Reversible!covalent binding!
hydrogen bonding!
hydrophobic !interactions!
halogen bonding!
hydrogen bonding!
And very important in molecular recognition: changes in water solvation!
A Multi-Dimensional Approach for Deciphering and Quantifying Molecular Recognition Principles and Solvent Effects!
Protein-ligand interactions !!Synthetic host-guest complexation!!Dynamic processes in unimolecular systems (torsion balances)!!Small-molecule X-ray crystallography !!Protein-ligand co-crystal structures!!Data base mining in the Cambridge Crystallographic Database (CSD) and the Protein Databank (PDB)!!Structure-based ligand design!!Theoretical calculations!!=> Collaborative networks required!
Content of the Lecture!
Part 1 Energetically Favorable Water Replacements in Protein-Ligand Complexes!!Part 2 !Weak Intermolecular Interactions in Protein-Ligand Complexes !
!- Orthogonal Dipolar Interactions!
!- Cation-π Interactions!
!- Heteroarene Stacking on Peptide Bonds!
!- Halogen Bonding !!!
Part 3 !From Herbicidal Targets to New Antimalarials !
!- IspD: An Enzyme from the Non-Mevalonate (DXP) Pathway of ! Isoprenoid Biosynthesis!
!- Serine Hydroxymethyltransferase (SHMT), a Neglected ! ! Enzyme from the Folate Cycle!
Part I: Challenges in Water Replacements tRNA-Guanine Transglycosylase in Prokaryotes
Zymomonas mobilis!
W. Xie, X. Liu, R. H. Huang, Nature Struct. Biol. 2003, 10, 781-788.!First X-ray: C. Romier, K. Reuter, D. Suck, R. Ficner, EMBO J. 1996, 15, 2850-2857.!
Two homodimers!in complex with tRNA!
Project in collaboration with the group of Prof. Gerhard Klebe (Univ. Marburg)!
tRNA-Guanine Transglycosylase in Prokaryotes!
! tRNA modification: guanine " queuine (Q)!! Introduced in the wobble position (G34) in the anticodon of
tRNA of all organisms (except yeast, archaebacteria) coding for the amino acids Asn, Asp, His, and Tyr!
! Eukaryotes need Q as nutrient but procaryote TGT introduces preQ1 instead of Q!
! If TGT is blocked, bacteria are apathogenic; translation of virulence factor virF mRNA inefficient !
N. Okada, S. Noguchi, H. Kasai, N. Shindo-Okada, T. Ohgi, T. Goto, S. Nishimura, J. Biol. Chem. 1979, 254, 3067–3073. Z. D. Miles, R. M. McCarty, G. Molnar, V. Bandarian, Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 7368-7372. R. M. McCarty, V. Bandarian, Bioorg. Chem. 2012, 43, 15-25. !
queuine!
! Ping-pong mechanism!! A covalent TGT–tRNA intermediate is formed!
! Asp280: attacks ribose C(1‘)!! Asp102: general acid/base via H-bonded water!
W. Xie, X. Liu, R. H. Huang, Nat. Struct. Biol. 2003, 10, 781–788.!
tRNA Guanine Transglycosylase (TGT): Asp280, Asp102- Catalyzed Base Exchange at the Wobble Position 34 of t-RNA!
Schematics:!
Active Site in TGT Bound to tRNA after Base Exchange (X-ray)!
! tRNA phosphate backbone is accommodated in a wide channel!! recognition of U33G34U35!
Active site in Z. mobilis TGT (PDB code: 1Q2S).!
W. Xie, X. Liu, R. H. Huang, Nature Struct. Biol. 2003, 10, 781-788.!
1.28 Å resolution PDB code: 2Z7K
Ribose 34 Pocket is Filled With a Conserved Water Cluster!
• A well conserved five- water cluster, different from Walrafen’s pentamer is located in the ribose 34 pocket and solvates Asn70 and Asp280!• Free energy calculations show that only two of these water molecules can be replaced without penalty !
Ki 77 nM!
Riniker, S.; Barandun, L. J.; Diederich, F.; Krämer, O.; Steffen, A.; van Gunsteren, W. F., J. Comput. Aided Mol. Des. 2012, 26, 1293-1309.!
P. C. Kohler, T. Ritschel, W. B. Schweizer, G. Klebe, F. Diederich, Chem. Eur. J. 2009, 15, 10809-10817!
lin-Benzoguanines with an Amino Linker in Position 4 Display !Single-Digit Nanomolar Inhibition: Binding Efficiency in Two Steps!
cyan! pink!
yellow! green!
Residual water in R34 pocket!
Ki = 55 ± 3 nM!!
(core without C(4) side chain: Ki = 58 nM) !
1.4 Å resolution!
X-ray Crystal Structures: Primary Ammonium Ion Replaces !Water in an Energetically Neutral Way!
Ki = 4 ± 2 nM!3EOS (1.78 Å resolution)!
(core without side chain: Ki = 58 nM)!
X-ray of Cyclohexyl Inhibitor: Substantial Gain in Binding !Affinity by Occupation of a Shallow Pocket!
P. Kohler, T. Ritschel, W. B. Schweizer, G. Klebe, F. Diederich, Chem. Eur. J. 2009, 15, 10809-10817.!
1.3 Å resolution
Ribose-based lin-Benzoguanine Fills Polar Pocket!
Kd = 288 ± 55 (n = 6) nM (from ITC)!Ki = 248 ± 82 (n = 5) nM (from radiochemical assay)!!
OO
OH OH
HN
N N
NH
O
HNNH2
L. J. Barandun, F. R. Ehrmann, D. Zimmerli, F. Immekus, M. Giroud, C. Grünenfelder, W. B. Schweizer, B. Bernet, M. Betz, A. Heine, G. Klebe, F. Diederich, Chem. Eur. J. 2015, 21, 126-135.!
Displacements of Conserved Water Molecules Seen in Co-Crystal Structures by Ligand Parts
From: E. Persch, O. Dumele, F. Diederich, Angew. Chem. Int. Ed. 2015, 54, 3290-3327.
As a first-approximation rule of thumb, ordered water molecules that undergo four bonding interactions to the protein (H-bonds, dipolar contacts), cannot be replaced by ligand parts. In the modeling, they are best treated similar to the protein surface.
The replacement of water molecules with three close contacts to the protein is also very difficult and not recommended.
Water molecules with two directional contacts to the protein are replaceable, but usually with only little or no gain in binding free enthalpy.
On the other hand, water molecules featuring only one polar interaction to the protein are replaceable, often with a substantial gain in free enthalpy.
Part II: Weak Interactions in Protein Ligand Complexes!
C F C
O......
C
O......C O
orthogonal interactions
N
C OHN
......
amide···π stacking
R X O NH......
halogen bonding
Review in the Special Issue of Angew. Chem. for 150 Years BASF !!E. Persch, O. Dumele, F. Diederich, Angew. Chem. Int. Ed.!2015, 53, 3290-3337.!!
CSD Search for C–F...C=O Interactions Observed in a Fluorine Scan of Thrombin Inhibitors!
CSD version 5.29, November 2007!
Left:!# 1848 occurrences of close C–F...C=O contacts (2.3 Å < d1 < 3.8 Å; 1087 hits)!# Q = C, N, O!
Right:!
# Superposition of 50 intermolecular C–F...C=O contacts !(2.77 Å < d1 < 3.0 Å)!
Although weaker than antiparallel dipolar interactions, orthogonal contacts are preferred at short distances for steric reasons!
M. Zürcher, F. Diederich, J. Org. Chem. 2008, 73, 4345-4361, R. Paulini, K. Müller, F. Diederich, Angew. Chem. Int. Ed. 2005, 44, 1788-1805.!
Binding Mode of Nilotinib (Tasigna®) to Abl Tyrosine Kinase!
DFG loop out!
hinge region!
2.21 Å resolution!PDB ID: 3CS9 !
Gly383!Phe382!
Asp381!
Ala380!
Two orthogonal interactions, which make the difference in 2nd generation Abl kinase inhibitors!Short orthogonal contacts:!C=O···C=O!C–F···C=O(amide)!
Therapy of Chronic Myelogenous Leukemia (CML)!
E. Weisberg, P. W. Manley, W. Breitenstein, J. Brüggen, S. W. Cowan-Jacob, A. Ray, B. Huntly, D. Fabbro, G. Fendrich, E. Hall-Meyers, A. L. Kung, J. Mestan, G. Q. Daley, L. Callahan, L. Catley, C. Cavazza, A. Mohammed, D. Neuberg, R. D. Wright, D. G. Gilliland, J. D. Griffin, Cancer Cell 2005, 7, 129–141. !
Efficient Stacking on Protein Amide Fragments!
Michael Harder, Bernd Kuhn, François Diederich!ChemMedChem 2013, 8, 397-404!
NN
N
O
ON
O
S
Cl
Br
H
H
(±)-19
Resolution 1.25 Å.!
L. M. Salonen, C. Bucher, D. W. Banner, W. Haap, J.-L. Mary, J. Benz, O. Kuster, P. Seiler, W. B. Schweizer, F. Diederich, Angew. Chem. Int. Ed. 2009, 48, 811-814.!
Pioneering work in cation-π interactions: D. A. Dougherty, D. A. Stauffer, Science, 1990, 250, 1558-1560.!
(+)!X-Ray Co-crystal Structure in Factor Xa!
Strongly Enhanced Potency through Cation-π Interactions!
In factor Xa: !!
!Ki(NMe3+) = 9 nM! Ki(CMe3) = 550 nM!
!Ki(CMe3)/Ki(NMe3
+) = 60!!
ΔΔG = 2.5 kcal mol-1 !
Average value approx. 0.8 to 0.9 kcal mol-1 per aromatic ring!
NN
N
O
ON
O
S
Cl
H
H
NN
N
O
O
O
S
Cl
H
HBr
(±)-19 (±)-37
(±)! (±)!
(+)-Enantiomer: !Ki(FXa) = 6 nM !!
!(–)-Enantiomer:!
Ki(FXa) = 271 nM !!
!
!
L. M. Salonen, C. Bucher, D. W. Banner, W. Haap, J.-L. Mary, J. Benz, O. Kuster, P. Seiler, !W. B. Schweizer, F. Diederich, Angew. Chem. Int. Ed. 2009, 48, 811–814.!
Factor Xa: Amide/Heteroaromatic Stacking!
L. M. Salonen, M. C. Holland, Philip S. J. Kaib, W. Haap, J. Benz, J.-L. Mary, O. Kuster, W. B. Schweizer, D. W. Banner, F. Diederich, Chem. Eur. J. 2012, 18, 213–222.!
2Y5H!2Y5G!
N
N
O
O
HH
N+
HetSCl
Br –
Ki = 146 nM! Ki = 1620 nM!
N
O
O
N
Parallel!Parallel displaced!
Amide/π-Stacking Interactions: SCS-MP2/aug-cc-pVDZ//LMP2/cc-pVDZ!
Interaction energy between N-methylacetamide (NMAC, model system for protein backbone, large
dipole moment 3.7 D) and various 5- and 6-
membered heterocycles was calculated using spin
component scaled Møller-Plesset 2 (SCS-MP2)!
NN
N
NNN N
N
NN
HN O
O S NH
N
O
N
SN
O
NN ON
N
NH
N
NH
HN
NH
NNH
O
O
NH
M. Harder, B. Kuhn, F. Diederich, ChemMedChem 2013, 8, 397-404.!
Correlation between Interaction Energy and Dipole Strength!
∆E (SCS-MP2) vs. Dipole Moment !
Interaction energy increases with stronger dipole moments.!
–2.50 0.0 –
–3.30 2.5 161
–4.13 kcal mol–1 4.4 D 173°
ΔE = µ = α =
–1.32 0.0
–2.50 0.0
–3.39 kcal mol–1 0.0 D
ΔE = µ =
Comparison of SCS-MP2 Binding Energies!
The interaction energy is getting more favorable with decreasing π-electron density of the heterocycle. !
The interaction energy increases with stronger dipole moments.!
Rotational Scan to Evaluate Dipole Impact!
ΔE = !r = !d =!x = !α =!
–2.45 !3.55!3.89!1.58!179!
–2.47 !3.52!3.98!1.87!240!
–1.26 !3.61!3.99!1.70!298!
–0.89 !3.70!4.07!1.70!
2!
–1.09 !3.63!4.05!1.80!60!
–2.03 !3.48!4.10!2.16!122!
kcal mol–1 !Å!Å!Å!°!
Ideal relative dipole alignment is antiparallel!!
Best interaction energy in the range from 3.2 – 3.8 Å !
Systematic Investigation of Halogen Bonding in Protein–Ligand Interactions!
L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm, R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez, J. Benz, J.-M. Plancher, G. Hartmann, D. W. Banner, W. Haap, F. Diederich, Angew. Chem. 2011, 123, 329-334; Angew. Chem. Int. Ed. 2011, 50, 314-318.!!L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm, R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez, J. Benz, J.-M. Plancher, G. Hartmann, Y. Isshiki, K.Morikami, N. Shimma, W. Haap, D. W. Banner, F. Diederich, ChemMedChem 2011, 6, 2048-2054.!
Introduction to Halogen Bonding (XB)!
Definition of Halogen Bonding (XB)!
! XB is the non-covalent interaction of a halogen atom X in one molecule with a negative site, such as the lone pair electrons of a Lewis base, in the other.!
! X can be part of a dihalogen, e.g. Br2, or it may be a substituent on some other, often organic, molecule.!
! X can be iodine, bromine, or chlorine, but not fluorine!!
! D is called the halogen bonding (XB) donor.!! A is the halogen bonding (XB) acceptor.!
! Why would halogen atoms, which are generally viewed as being negative, undergo such a reaction? ⇒ σ-hole!!
Halogen Bonding: Size of σ-Hole Increases with Lower Hybridization of XB Donor !
! The size of the σ-hole on iodine increases with decreasing hybridization state of the C atom of the XB donor (sp > sp2 > sp3)!
! In parallel, the electronegative area decreases from C(sp3) to C(sp2) and changes to an electroneutral surface potential for C(sp)!
!Electrostatic potential surface at the electron density of 0.001 electrons Bohr–3!
O. Dumele, D. Wu, N. Trapp, N. Goroff, F. Diederich, Org. Lett. 2014, 18, 4722-4725.!
Theoretical Calculations and Solid-State Structures: Rigorous Geometrical Requirements for XB
C X O C! Angle C–X–O = 160–180°!!! Angle X–O=C = 90–180°!
! d(X–O) ≤ sum (vdW radii)!
T. Clark, M. Hennemann, J. S. Murray, P. Politzer, J. Mol. Model. 2007, 13, 291–296. ! K. E. Riley, J. S. Murray, P. Politzer, M. C. Concha, P Hobza, J. Chem. Theory Comput. 2009, 5, 155–163.!!P. L. Wash, S. Ma, U. Obst, J. Rebek, Jr., J. Am. Chem. Soc. 1999, 121, 7973-7974. P. Metrangolo and G. Resnati, Chem. Eur. J. 2001, 7, 2511–2519. ! ! ! A. C. B. Lucassen, A. Karton, G. Leitus, L. J. W. Shimon, J. M. L. Martin, M. E. van der Boom, Cryst. Growth. Des. 2007, 7, 386-392.!P. Metrangolo, F. Meyer, T. Pilati, G. Resnati, G. Terraneo, Angew. Chem. Int. Ed. 2008, 47, 6114 – 6127.!R. Cabot, C. A. Hunter, Chem. Commun. 2009, 2005-2007.!M. G. Sarwar, B. Dragisic, L. J. Salsberg, C. Gouliaris, M. S. Taylor, J. Am. Chem. Soc. 2010, 132, 1646-1653; !
XB Capsules: A New Protocol to Form Molecular Containers!
donor hemisphere!
acceptor hemisphere!
R = Solubilizing groups!A = Halogen bonding acceptor atom!X = Cl, Br, I, F!!The capsules contain 2 independent!guest binding sites, one in each !resorcine[4]arene hemisphere.!Examples for guests: 1,4-dioxane, !1,4.dithiane!!!!
O. Dumele, N. Trapp, F. Diederich, Angew. Chem. Int. Ed., DOI: 10.1002/anie.201502960!
hCathepsin L (hCatL): C=O from Gly61 Converges into S3 Pocket: Target for XB Investigations in a Biological Receptor!
S.F. Chowdhury et al., J. Med. Chem. 2008, 51,1361–1368!
Resolution: 1.45 Å!
Unbound hCatL: Cys25Ala mutation: M. A. Adams-Cioaba, J. C. Krupa, C. Xu, J. S. Mort, J. R. Min, Nat. Commun. 2011, 2, 197. !!PDB code: 3iv2, 2.2 Å resolution.!
Overlay Bound (orange) and Unbound (grey) hCatL:!
X IC50 [µM] logD PDB ID Res. [Å]
F 0.34 2.36 2XU4 1.12
H 0.29 2.11 – – CH3 0.13 2.57 2XU5 1.60
Cl 0.022 2.73 2YJC 1.14
Br 0.012 2.96 2YJ2 1.15
I 0.0065 3.23 2YJ8 1.30
XN
O
SO2
Cl
NHO
CN
Inhibition of Human Cathepsin L (hCatL)!
L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm, R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez, J. Benz, J.-M. Plancher, G. Hartmann, D. W. Banner, W. Haap, F. Diederich, Angew. Chem. Int. Ed. 2011, 50, 314–318;!L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm, R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez, J. Benz, J.-M. Plancher, G. Hartmann, Y. Isshiki, K. Morikami, N. Shimma, W. Haap, D. W. Banner, F. Diederich, ChemMedChem 2011, 6, 2048–2054. !
SAR evidenced by inhibition of hCatL and X-ray co-crystal structures!
H to I mutation worth up to ∆∆G = –2.6 kcal/mol!
X-Ray Co-Crystal Structures!
PDB ID: 2YJ8!res. 1.30 Å!!IC50 = 0.0065 µM!
P212121 !
S3 pocket of hCat with iodinated inhibitor (green)!
L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm, R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez, J. Benz, J.-M. Plancher, G. Hartmann, Y. Isshiki, K. Morikami, N. Shimma, W. Haap, D. W. Banner, F. Diederich, ChemMedChem 2011, 6, 2048–2054. !
Halogen bond highlighted red, distances in Å!
Σ (v.d.W. O + I) = 3.5 Å!
X-ray Co-Crystal Structures of Br, Cl, and F Inhibitors!
All in space group!P212121 !
L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm, R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez, J. Benz, J.-M. Plancher, G. Hartmann, Y. Isshiki, K. Morikami, N. Shimma, W. Haap, D. W. Banner, F. Diederich, ChemMedChem 2011, 6, 2048–2054. !
2YJ2!1.15 Å!!0.012 µM!
2YJC!1.14 Å!!0.022 µM!
2XU4!1.12 Å!!0.34 µM!
at C=O: HB ⟂ XB!
C=O and F-inhibitor bridged by H2O!
PDB ID, resolution, IC50:!
Σ (v.d.W. O + Cl) = 3.3 Å!
Σ (v.d.W. O + Br) = 3.4 Å!
Overlay Shows Slight Pyrrolidine Pucker Changes Allowing Fluorine to Avoid the C=O Dipole!
Adjustments through slight changes in the 5-ring pucker!
Part IIIa Pseudilins – Allosteric Inhibitors of the Non–Mevalonate Pathway Enzyme IspD !
A. Kunfermann, M. Witschel, B. Illarionov, R. Martin, M. Rottmann, W. Ho ̈ffken, M. Seet, W. Eisenreich,!H. Kno ̈lker, M. Fischer, A. Bacher, M. Groll, F. Diederich, Angew. Chem. Int. Ed. 2014, 53, 2235-2239.!!Matthias C. Witschel, Wolfgang Höffken, Michael Seet, Liliana Parra, Thomas Mietzner, Frank Thater, !Ricarda Niggeweg, Franz Röhl, Boris Illarionov, Markus Fischer, Adelbert Bacher, François Diederich, !Angew. Chem. Int. Ed. 2011, 50, 7931-7935.!!
Biosynthesis of Isoprenoids!
!! Two different pathways: mevalonate and DXP (mevalonate independent)!! Animals exclusively use the mevalonate pathway!! DXP pathway found in bacteria, algae, and protozoa (e.g. P. falciparum)!! In higher plants both pathways are present in a compartmentalized way:
Cytoplasm: mevalonate; Chloroplast: non-mevalonate.!
!
O
SCoA
O
CO2
O
OH
O P O
O
O
H
OPP OPP
IPP-isomerase
3 +
mevalonatedependent DXP
H H
H
HO
H
isoprenoids
IPP DMAPP
beta-carotenecholesterol
K. Bloch, Steroids 1992, 57, 378–383.!W. Eisenreich, M. Schwarz, A. Cartayrade, D. Arigoni, M. H. Zenk, A. Bacher, Chem. Biol. 1998, 5, R221–R233.!
=> Synergisms between anti-parasite and herbicide chemistry <=!!M. Witschel, M. Rottmann, M. Kaiser, R. Brun, PLoS Negl. Trop. Dis. 2012, 6, e1805.!M. Witschel, F. Stelzer, J. Hutzler, T. Qu, T. Miezner, K. Kreuz, K. Grossmann, R. Aponte, H. W. Höffken, ! F. Carlo, T. Ehrhardt, A. Simon, R. L. Parra, PCT Int. Appl. 2013, WO 2013182472 A1.!
Enzymes of the DXP (“Non-Mevalonate”) Pathway of Isoprenoid Biosynthesis: IspD as Drug Target!
CO2
O
H O
O
OH
P
O
O
O
O
OH
P
O
O
OOH
O
O
OH
P
O
O
O
OH
HO
O
OH
P
O
O
O
OH
HO
P O ON
N
NH2
OO
O
HO OH
O
OH
P
O
O
O
OH
O
P O ON
N
NH2
OO
O
HO OH
PO
O
O
OP
OH
O
OH
P OOO
O
O
OH
O P
O
O
O
P O
O
O
OPP
OPP
+dxs
IspCdxr
IspD
IspE IspF
IspG IspHIPP-Isomerase
CO2 NADPH NADP+
CTP PPi
ATP ADP CMP
DXP
MEP CDP–ME
CDP–ME2P
G3Ppyruvate
MECDP
HMBPP
IPP
DMAPP
Fosmidomycin!(H. Jooma et al., Science 1999, 285, 1573)!
IspD is a Homo-Dimer: Complex with CDP-ME and Overlay with Apo-Enzyme!
Apo enzyme: PDB Code 4NAI, 1.5 Å resolution, A. Kunfermann, M. Witschel, B. Illarionov, R. Martin, M. Rottmann, W. Ho ̈ffken, M. Seet, W. Eisenreich, H. Kno ̈lker, M. Fischer, A. Bacher, M. Groll, F. Diederich, Angew. Chem. Int. Ed. 2014, 53, 2235–2239.!Complex with CDP-ME (4-diphosphocytidyl-2C-methyl-D-erythritol): PDB code 1INI, 1.8 Å resolution, E. coli IspD; S. B. Richard, M. E. Bowman, W. Kwiatkowski, I. Kang, C. Chow, A. M. Lillo, D. E. Cane, J. P. Noel, Nat. Struct. Biol. 2001, 8, 641-648.!
4NAI: 1.5 Å, AtIspD (apo)!1INI: 1.8 Å, EcIspD (CDP-ME bound)!
Arabidopsis thaliana (At)!
N
N
N
N
OH
Cl
7
IC50 (AtIspD) = 140 nM
M. C. Witschel, H. W. Höffken, M. Seet, L. Parra, T. Mietzner, F. Thater, R. Niggeweg, F. Röhl, B. Illarionov, F. Rohdich, J. Kaiser, M. Fischer, A. Bacher, F. Diederich Angew. Chem. Int. Ed. 2011, 50, 7931-7935.!
BASF HTS on AtIspD: The Screening Hit, a Triazolopyrimidine, !Binds Into a Hitherto Unknown Allosteric Pocket!
• 1.4 Å resolution, PDB code: 2YC3 • Very tight and hydrophobic benzyl pocket • Not much space for further substituents • pKa = 3.9 → binds as anion • bridging water molecule
NN
NN
O
ClHN
NH2
NH2
Arg157 HN
Ile265
OHN
Val266
NHO
Ile240
HN
OIle177
HN O
Val263
O
H
H
NH
O
Ala202
N
N
N
OH
Cl
15NC
14: IC50 = 274 ± 15 µM!!pKa1 = 2.32, pKa2 = 5.22 → !binds as di-anion, desolvation decreases activity
N
N
N
OH
Cl
14OHO
15: IC50 = 35 ± 7 nM!
PDB code: 2YC5!
1.6 Å resolution!
PDB code: 2YMC!
1.8 Å resolution!
Replacing the Bridging Water!
Pseudilins: a New Lead!
! Identified as AtIspD inhibitors via HTS!! Binding of allosteric pocket by different interactions!! Marine bacterium Pseudomonas bromoutilis!! Pseudilins already known for their antibiotic properties!
P. R. Burkholder, R. M. Pfister, F. H. Leitz, Appl Microbiol. 1966, 14, 649–653.!A. Kunfermann, M. Witschel, B. Illarionov, R. Martin, M. Rottmann, W. Ho ̈ffken, M. Seet, W. Eisenreich,!H. Kno ̈lker, M. Fischer, A. Bacher, M. Groll, F. Diederich, Angew. Chem. Int. Ed. 2014, 53, 2235–2239.!
Cl
Cl
OH
HN
BrBr
Br
Br
Br
OH
HN
BrBr
Br
4NAK: 1.8 Å !
Pseudilin Binding is Facilitated by Metal Ions (X-ray)!
IC50 AthIspD [µM]! IC50 PvIspD [µM]! EC50 !Pf cell [µM]!
R1 !R2 !R3! w/o metal! 40 µM Cd2+! w/o metal! 40 µM Cd2+!Br !Br !Br! 13 ± 2! 1.4 ± 0.2! 48 ± 9_! 57 ± 12! 1.27!Cl !Cl !Br! 12 ± 1! 2.2 ± 0.2! 56 ± 8_! 41 ± 7_! 1.07!Cl !Cl !Cl! 19 ± 2! 4.3 ± 0.8! 46 ± 6_! 40 ± 7_! n.d.!F !F !Br! 79 ± 6! 13 ± 2_! 64 ± 15! 51 ± 15! n.d.!H !Br !Br! 52 ± 6! 19 ± 2_! 36 ± 10! 24 ± 6_! 11.72!
A. Kunfermann, M. Witschel, B. Illarionov, R. Martin, M. Rottmann, W. Ho ̈ffken, M. Seet, W. Eisenreich,!H. Kno ̈lker, M. Fischer, A. Bacher, M. Groll, F. Diederich, Angew. Chem. Int. Ed. 2014, 53, 2235–2239.!
R2
R1
OH
HN
R3R3
R3
Other divalent cations, such as Zn(II), give similar results!
Pseudilin Binding: Halogen Bonding Involved !
! Cd2+ coordinated by phenol OH, pyrrole N,Gln238, and a water molecule!
! Halogen bonding to backbone carbonyl of Val239!
A. Kunfermann, M. Witschel, B. Illarionov, R. Martin, M. Rottmann, W. Ho ̈ffken, M. Seet, W. Eisenreich,!H. Kno ̈lker, M. Fischer, A. Bacher, M. Groll, F. Diederich, Angew. Chem. Int. Ed. 2014, 53, 2235–2239.!
4NAK: 1.8 Å !R2
R1
OH
HN
BrBr
Br
A: R1 = R2 = Br!B: R1 = R2 = Cl!
4NAL: 1.8 Å!
A! B!
Pseudilin Binding: Halogen Bonding Competes with Ion Chelation!
!! The pseudilin derivative missing a halogen para to the phenolic OH
changes binding conformation to regain halogen bonding thereby abolishing ion coordination (70% syn, 30% anti)!
4NAK: 1.8 Å ! 4NAL: 1.8 Å!
A. Kunfermann, M. Witschel, B. Illarionov, R. Martin, M. Rottmann, W. Ho ̈ffken, M. Seet, W. Eisenreich,!H. Kno ̈lker, M. Fischer, A. Bacher, M. Groll, F. Diederich, Angew. Chem. Int. Ed. 2014, 53, 2235–2239.!
4NAN: 1.8 Å!
Br
H
OH
HN
BrBr
Br
Part IIIb. !Pyrazolopyrans – Inhibitors of Serine Hydroxymethyltransferase (SHMT)!
M. C. Witschel, M. Rottmann, A. Schwab, U. Leartsakulpanich, P. Chitnumsub, M. Seet, S. Tonazzi, G. Schwertz F. Stelzer, T. Mietzner, C. MacNamara, F. Thater, C. Freymond, A. Jaruwat,
C. Pinthong, P. Riangrungroj, Y. Yuthavong, P. Mäser, P. Chaiyen, F. Diederich, !J. Med. Chem. 2015, 58, 3117–3130.!
Folate Metabolism!
N
N NH
HN
H2N
OH HN
O
NH
CO2H
CO2H
HN
NH
HN
N
NH
N
N
NH
HN
N
NH
O
OO
HO3PO
OH
N
NH
O
OO
HO3PO
OH
NADPH
NADP+Ser
Gly
SHMT DHFR
TSdUMP dTMP
DHF
5,10-CH2-THF
THF
DHPDHFSDHPS
Pteridinediphosphate pABA+
! Folate cycle proven drug target!! Folate synthesized de novo in
plants and pathogens, uptake from nutrition in humans!
! Crucial for DNA precursor synthesis!! No inhibitors for SHMT so far!
!
Tetrahydrofolate!
C. K. T. Pang, J. H. Hunter, R. Gujjar, R. Podutoori, J. Bowman, D. G. Mudeppa, P. K. Rathod,!Mol. Biochem. Parasitol. 2009, 168, 74–83.!
PABA!
• Catalyzes the one-carbon transfer to THF!• Co-factor: pyridoxal-5’-pyridine (PLP)!• Two redox states (unique cysteine pair)!
Serine Hydroxymethyltransferase (SHMT): A Homo-Dimer!
X-ray: V. Trivedi, A. Gupta, V. R. Jala, P. Saravanan, G. S. Rao, N. A. Rao, H. S. Savithri, H. S. Subramanya, J. Biol. Chem. 2002, 277, 17161–17169.!P. Chitnumsub, W. Ittarat, A. Jaruwat, K. Noytanom, W. Amornwatcharapong, W. Pornthanakasem, P. Chaiyen, Y. Yuthavong, U. Leartsakulpanich, Acta Crystallogr. D 2014, 70, 1517–1527.!
SHMT from Bacillus stearothermophilus (PDB code: 1KL2; 2.7 Å)!
N
HO
H O
OPO32-
PLP
Pyrazolopyrans: From Herbicide to Antimalarial!
! BASF 2004: HTS on Arabidopsis thaliana SHMT (AtSHMT) reveals pyrazolopyrans with μM (IC50) activity. Structure based design improves activity on AtSHMT to < nM rang.!
! Cell-based screening of herbicidal plant SHMT inhibitors on Plasmodium falciparum (Pf) identifies compounds with EC50 values in the nM range!
!
!!!!! Enantioselectivity in both target and cell-based assay!
(±) EC50 Pf NF54 = 2.80 nM; IC50 Pf SHMT = 370 nM!
(+) EC50 Pf NF54 = 1.85 nM; IC50 Pf SHMT = 60 nM!
(–) EC50 Pf NF54 = 48.9 nM; IC50 Pf SHMT = 5420 nM!
SO
O
CN
O
CN
HN N
NH2*
Thienyl Ester: Binding Mode of Pyrazolopyrans to P. Vivax SHMT!
Plasmodium Vivax SHMT!PDB: 4TMR, 2.7Å!
SO
O
CN
O
CN
HN N
NH2*
PABA channel!
Pyrazolopyrans: Profiling of Lead Compound!
! Undiminished activity on drug resistant Pf strains TM90C2B (Chloroquin, Pyrimethamine, Mefloquin, Atovaquone) and K1 (Chloroquin, Sulphadoxine, Pyrimethamine, Cycloguanil) !
!!EC50 TM90C2B = 5 nM!!EC50 K1 = 5 nM!
!! Very low cytotoxicity on mammalian (L6: rat, HepG2: human) cells!
!EC50 L6 = 31100 nM!!EC50 HepG2 > 20000 nM!
!! Excellent activity on P. berghei (liver stage)!
!EC50 P. berghei = 9 nM!
! In vivo (mouse model) activity proven but yet unsufficient metabolic stability!! t1/2 = 2 – 13 min!
! Low selectivity against human SHMT!
SO
O
CN
O
CN
HN N
NH2*
(±) !
Conclusions!
Protein-ligand structural work increasingly reveals the rules for favorable replacements of water seen in cryo-X-ray co-crystal structures. !!!Orthogonal dipolar interactions, in particular those involving organofluorine, have become essential keys in the toolbox of molecular recognition. Dipolar interactions also control favorable stacking of heteroarenes on peptide bonds.!!!Halogen bonds rival strong neutral hydrogen bonds as energetically favorable contacts in protein-ligand complexation.!!!Structure-based design of new drugs and crop protecting agents greatly benefits from in-depth insight into molecular recognition forces.!!!There exist substantial synergisms between herbicide development and the invention of new anti-parasite drugs. These synergisms have been successfully exploited in the development of new anti-malarials with new targets, IspD from the non-mevalonate pathway of isoprenoid biosynthesis and SHMT from the folate cycle. !
