Proteins adopt beautiful shapes that enable them to perform an incredible array of tasks. But these wiggly little creatures cannot stay still. Is this a nuisance or a blessing? Proteins are the gooey stuff that makes life possible. They live in a state that is neither solid nor liquid, and their biological functions depend crucially on keeping a balance between order and disorder. However, solid entities are far simpler to understand and we tend to use (and abuse) the rigid body approximation until it breaks. In this colloquium Xavier Barril and Xavier Salvatella will present the issue of protein flexibility from different perspectives, discussing structure and dynamics, and exploring how far we must/can go into the order-disorder spectrum. After a basic introduction to proteins, Xavier Barril will focus on the implications of protein flexibility for drug discovery. Showing that a rigid representation has been, and continues to be, extremely useful. He will present some of the failures and challenges in introducing a more realistic view, but also how the dynamic perspective is gaining ground thanks to the advances in structural biology and computational chemistry.

1. The unbearable lightness of being
(a protein)
Xavier Barril
ICREA Research Professor, Barcelona University
71st ICREA Colloquium
Barcelona, 14th June 2016

3. Protein?

4. Proteins come in all shapes and forms…
…because form follows function

6. Structure Determination: X-ray Crystallography
PDB today
Total: 119480
X-ray: 106777 (89.3%)

7. Structure Determination: X-ray Crystallography

8. Structure-Based Drug Design

9. A New Hope
Progress in science depends on new techniques, new
discoveries and new ideas, probably in that order.
— Sydney Brenner

15. Score
Score
Score
A
B
C
Score
Score
Score
A B C
!Ei = -kBT·ln(Pi) Ei = -kBT·ln(Pi)
NATURE CHEMISTRY | VOL 6 | JULY 2014
SBDD: More is less

16. Finding Hot Spots: MDmix

17. High-Throughput Docking
http://rdock.sourceforge.net
ü Fast (~10k cpds x CPUday)
ü Free & Open Source
ü Advanced features:
• Limited Protein flexibility
• Interfacial water molecules
ü User control:
• Pharmacophoric restraints
• Tethered scaffold docking

18. Dynamic Undocking
• Different number of dynamic undocking runs
were tested (2, 8 or 22). As shown in the picture
for CDK2, increasing the number of runs
improved the results. However, the difference
between 8 and 22 runs is really small.
the
• The GPU time required for each ligand and stage is
20 min for minimisation and equilibration, 15 min fo
each ns of MD simulation and 10 min for each SMD
run.
abase and they are automatically
ameterized using forcefield PFROSST
h tleap.
Sampling
• As the number of runs
profiles increased their s
favourable dissociation p
• Concept of minimum o
replicas.

19. CO-0001
Conc. (µM)
ΔTm
0 500 1000 1500
0
2
4
6
Virtual
Library
Virtual
Hits

20. Nucleus
ER
Correct
Folding
Ribosome
Protein
Function
Normal
gene
Target
Organelle
Rare Diseases
HEALTHY STATE

21. Nucleus
ER
Ribosome
Protein
Deficiency
Mutated
gene
Target
Organelle
Proteasomal
degradation
Incorrect
Folding
Rare Diseases
DISEASE STATE

22. Nucleus
ER
Ribosome
Protein
Inhibited
Mutated
gene
Target
Organelle
Pharmacological Chaperones
Correct
Folding
Proteasomal
degradation
Substrate-compeLLve
pharmacological chaperone
PC THERAPY

23. GLB1 Binding Sites
§ Through SEE-Tx, Minoryx
idenLfied a druggable binding
site at the interdomain region.
§ Such region is surrounded by
mutaLons associated to infanLle
form.
§ MutaLons responsive to
chaperones targeLng such site
may differ from those responsive
to binding-site chaperones.
§ CombinaLon of chaperones
targeLng differenLated binding
sites may provide superior
efficacy and broader
applicability.
Ohto et al, JBC 2012
AcLve site
Allosteric site

24. Virtual
Library
~1.5M cpds
Allosteric
Site
Virtual
Hits
140 tested
CatalyLc
Site
Virtual
Hits
50 tested
DSF
1 AcLve
Series
2 AcLve
Series
Hit IdenLficaLon & Primary Screening
Mel,ng curve
ΔTm
CO-0001
Conc. (µM)
ΔTm
0 500 1000 1500
0
2
4
6
DGJ
KD = 242±40 µM
KD ~ 240µM
KD ~ 2µM
DSF
KD ~ 15µM
DGJ
ΔTm @ 100µM = 2.3ºC
Compound @ 30µM
§ Hit series: Very small molecules (av. Mw = 250Da) with excellent
ligand efficiency (potency in mid microM range).
§ TargeLng of non-exploited binding site.
§ >150 patentable non-sugar like NCE’s already synthesized.
§ IP filed on main series.
§ Back-up scaffolds idenLfied.

25. Series OpLmizaLon
Minoryx’s cpds.
§ Enzyme enhancement >300% (at 10µM)
§ Highly efficient (MW = 270Da)
§ Drug-like profile
§ High in-vivo BBB penetraLon (Brain/plasma >> 1)
§ More that 150 compounds have been
synthesized.
§ This allowed idenLfying several acLve series
and subseries.
§ Most promising series are series A1 (which
includes cpd #3 from previous slide) and
series C1 (cpd # 8).
§ Series C1, show increased cell-based
potency as well opLmized properLes for
BBB penetraLon.
Enzyme enhancement upon treatment of COS cells transiently
transfected with mutant hGLB1 (DGJ: substrate compeLLve PCT with
microM affinity; NN-DGJ: Substrate compeLLve with nM affinity; #3, 4
and 10: Minoryx’s non-compeLLve PCTs; Series A1; Series C1)
T420K
GM1 type 3

26. Non-compeLLve Hit series: InhibiLon of the lysosomal enzyme in lysates from normal
fibroblasts
ü Control 1 and Control 2 exhibited dose-response
inhibitory acLvity on the enzyme with lysates from
normal human fibroblasts, as previously described.
ü In contrast, Minoryx s compounds do not exhibit any
inhibitory acLvity on the enzyme.
Allosteric chaperones do not inhibit
Control 1
Control 2
IC50 = 17µM
IC50 = 0.1µM
Non-compeLLve chaperones

28. Acknowledgements
Group Members
• Sergio Ruiz – rDock; DUck
• Carles Galdeano – Fbw7, BRD4
• Serena Piticchio – Fragments
• Guillermo Gutiérrez – Automaton, Flex
• Míriam Martínez – BRD4, Fbw7
Former Group Members
• Daniel Álvarez – MDmix
• Peter Schmidtke – DUck
Collaborators
• F. Javier Luque
• Minoryx
• Vernalis