U SING SIGHT TO EXPLOIT MOLECULAR KNOWLEDGE
Andrea Bernini
Strutural Biology Lab – www.sbl.unisi.it andrea.bernini@unisi.it
02/04/2019@Dipartimento Ingegneria dell’Informazione e Scienze Matematiche
Structural biology is a branch of molecular biology, biochemistry,
and biophysics concerned with the molecular three-dimensional structure of biological macromolecules such as proteins, RNA, and DNA.
Structural bioinformatics is the branch of bioinformatics which is related to the analysis and prediction of the structure of biological
macromolecules. It deals with generalizations about macromolecular 3D structure such as comparisons of overall folds and local motifs, principles of molecular folding, evolution, and binding interactions, and
structure/function relationships, working both from experimentally solved structures and from computational models. Structural bioinformatics can be seen as a part of computational structural biology.
B IRTH OF S TRUCTURAL B IOLOGY
1958: John Kendrew and 3D structure of myoglobin.
Oggi: la stessa molecola di mioglobina visualizzata in grafica 3D con un software gratuito
S TRUCTURAL LEVELS OF PROTEINS
P ROTEIN FOLDING AND ENERGY
LANDSCAPE
S IR K ENDREW AND M YOGLOBIN , 1958
This plasticine model of myoglobin, made by Sir John Kendrew, was the first ever model to be made of a protein molecule. In 1958 John
Kendrew (1917-1977) and Max Perutz (1914-2002) were able to produce a model of its 3-dimensional structure, for which they were awarded the Nobel Prize for chemistry in 1962.
The contorted cylindrical shape, showing the track of polypeptide chain, is supported by wooden rods protruding from a pegboard base;
dimensions of base 18" x 1 1/2"; overall height 8 1/2". The forest of rods
obscured the view of the model and made it hard to adjust. Its size made it cumbersome and problematic to move.
1965: Kendrew commissiona ad A. A. Barker i primi modelli della mioglobina in palline di metacrilato (e li vende a 600$, stimabili in 4’300$ alla data odierna) .
F ROM PHYSICAL MODELS TO ABSTRACTION ….
The domain I of CD11a (membrane integrin), wire model decorated with pipecleaner (late 90’s)
The same protein rendered by computer graphics (MolMol)
E ARLIEST C OMPUTER R EPRESENTATIONS
As early as 1964, Cyrus Levinthal and his colleagues at MIT had developed a system that displayed, on an
oscilloscope, rotating "wireframe"
representations of macromolecular structures.
In a similar way, ATARI created the videogame Asteroids in 1979.
Videogame came later than molecular representation but development rate had been different…
E VANS & S UTHERLAND C OMPUTERS : 1980-1990
During the 1980's, the most popular computer system for crystallographers was manufactured by Evans &
Sutherland. These computers, costing about $250,000 in 1985, displayed the electron density map, and enabled an amino acid sequence to be fitted
manually into the map.
M OLECULAR GRAPHICS FOR THE MASSES : R OGER S AYLE ' S R AS M OL , 1993
In 1990, Roger entered graduate school in computer science at the University of Edinburgh. Roger developed his program into a more molecular visualization system, and by 1993, it was being used in
teaching and for images in research publications. Roger generously
made the program available to the world scientific community free of charge when he received his Ph.D.
in June, 1993. In January, 1994, Roger was employed by
GlaxoWellcome, which supported the continued development of
RasMol freeware, including the first version for the Macintosh, for the next two years.
www.openrasmol.org
JMOL, THE MOLECULAR VIEWER OF THE INTERNET AGE
Jmol is a free, open source molecule
viewer in the form of a Java applet. It is
cross-platform,
running on Windows, Mac OS X, and
Linux/Unix systems.
jmol.sourceforge.net
S OURCES OF MOLECULAR STRUCTURES
Protein Data Bank (www.rcsb.org) – macromolecules e.g. 1EY2
PubChem (pubchem.ncbi.nlm.nih.gov) – small molecules
M OLECULAR D YNAMICS S IMULATION OF
P ROTEINS
Protein structure
• Protein Data Bank
• In house determination (NMR)
• In house modelling (by homology,…)
Parametrisation
• Amber all atom force field for proteins
• B3LYP/6-31G charge calculation for drugs (ab initio)
Simulation engine
• GROMACS
• Parallel calculation
• GPU enabled
Molecular dynamics trajectory
• Time evolution of structure
• Snapshot every 1ps
• Microsecond scale; tens of µs coming soon
Two proteins, involved in the genetic disease Alkaptonuria, are actually under investigation:
1,2-homogentisate dioxygenase (HGD) and 4- hydroxyphenyl-pyruvate dioxygenase (HPPD, bound to nitisinone in the above picture).
B INDING SITE DISCOVERY AND
VIRTUAL DRUG REPOSITIONING FOR PHARMACEUTICAL CHAPERONES
TARGETING HOMOGENTISATE 1,2-
DIOXYGENASE FOR THE TREATMENT OF
THE INBORN ERROR OF METABOLISM
DISORDER A LKAPTONURIA .
ruptures of muscles, tendons, ligaments
black spots in eyes
blue-black ears black urine
black cartilage
FOTO: Liverpool
+ aortic valve disease
deficiency of homogentisate 1,2-dioxygenase (HGD) and incapacity to metabolise homogentisic acid (HGA)
(LaDu et al. 1958) -inborn error of metabolism
HGA accumulates in the body at 2,000 times the normal rate, in form ochronotic pigment it is attacking the bones and turning them black and brittle
it causes severely debilitating osteoarthritis, heart disease, and other serious health complications
patients become increasingly disabled as they get older
the first genetic disease to be discovered more than 110 years ago (MIM 203500; Garrod 1908)
ALKAPTONURIA (AKU)
ALKAPTONURIA (AKU)
- world-wide incidence is 1:250 000 – 1: 1 000 000
http://www.indiegogo.com/projects/cure-black-bone-disease
• mapping of the AKU gene to 3q13.33 (Pollak et al. Nat Genet 1993, Janocha et al. Genomics 1994)
• the HGD gene and cDNA described (Granadino et al. Genomics 1997, Fernandez-Canon et al. Nat Genet 1996)
HGD protein -> dimer of trimers
expressed mainly in liver, kidneys and prostate (Fernandez-Canon et al. Nat Genet 1996)
crystal structure described (Titus et al. Nat Struct Biol 2000)
expression also in chondrocytes, synoviocytes, osteoblasts (Lischi et al. J Cell Physiol 2012)
Rodriguez et al. Hum Mol Genet 2000
HGD trimer side view of hexamer – dimer of
trimers
AKU – autosomal recessive inheritance father mother
healthy healthy
AKU healthy healthy healthy child
healthy AKU healthy carrier healthy healthy
carrier carrier
Homogentisate 1,2-dioxigenase (HGD
)• Enzyme involved in the metabolic pathway of tyrosine and phenylalanine
degradation.
Pharmacological chaperones
• Pharmacological chaperones (PC) are small molecules helping the stabilization of a protein thus recovering functionality.
[Muntau AC et al. J Inherit Metab Dis; 2014]
PC
Wild type Mutants
Pharmacological chaperones
• Current PC’s are desiged to target an existing binding site (e.g. cofactor).
Transthyretin and Tafamidis
[Bulawa C. et al. Pnas; 2012]
Epos: Ensemble of Pockets on Protein Surface
Patch file Plas file
PID freq[%] mean vol[Å^3]
min
vol[Å^3] max vol[Å^3] mean pol.
min pol.
max pol.
mean depth [Å]
min depth
[Å]
max depth[Å]
40 40.206 362.585 185.411 718.201 0.354 0.300 0.394 6.336 2.695 10.580
88 83.505 521.479 245.932 911.854 0.356 0.315 0.392 7.607 4.296 11.161
89 83.505 519.932 102.084 787.060 0.363 0.324 0.400 8.691 5.200 10.973
90 98.969 296.350 179.515 933.911 0.297 0.253 0.350 7.247 2.042 13.162
115 53.608 702.886 187.278 1.058.844 0.386 0.344 0.431 3.458 0.954 8.897
139 63.918 405.702 169.256 771.454 0.374 0.317 0.438 6.686 3.302 10.998
150 26.804 401.203 202.646 722.240 0.376 0.333 0.450 6.625 4.540 10.625
162 41.237 316.139 206.822 493.636 0.340 0.300 0.366 8.098 3.877 12.735
3. Drug score > 0,6 evaluated with PockDrug for the prediction of the druggability.
Pocket filtering
Pocket Drug Score
40 0.48
88 0.3
89 0.41
90 0.79
139 0.16
150 0.12
162 0.68
168 0.26
178 0.66
190 0.21
279 0.19
287 0.4
305 0.16
321 0.55
322 0.22
357 0.84
Pocket 357
Pocket 162
Pocket 90
Pocket 178
http://pockdrug.rpbs.univ-paris-diderot.fr/cgi-bin/index.py?page=Home
Top ranked molecules for each pocket
Pemetrexed ∆G= -7,8 Kcal/mol
Dihydroergotamine ∆G= -10,4 Kcal/mol
Olaparib ∆G=-11,6 Kcal/mol
Genz-10850 ∆G= -11,5
Kcal/molBinding site discovery workflow
Virtual screening
A set of molecules are tested in each pocket by molecular docking
simulation
The affinity between pockets and molecules are calculated
The molecules with higher affinities are potential chaperones stabilizing the protein- protein interface
A KNOWLEDGMENTS
Silvia Galderisi
Anna Visibelli Vittoria Cicaloni
