USING 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.

BIRTH OF STRUCTURAL BIOLOGY

1958: John Kendrew and 3D structure of myoglobin.

Oggi: la stessa molecola di

mioglobina visualizzata in

grafica 3D con un software

gratuito

STRUCTURAL LEVELS OF PROTEINS

PROTEIN FOLDING AND ENERGY

LANDSCAPE

SIR KENDREW AND MYOGLOBIN, 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) .

FROM 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)

EARLIEST COMPUTER REPRESENTATIONS

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…

EVANS & SUTHERLAND COMPUTERS:

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.

MOLECULAR GRAPHICS FOR THE MASSES:

ROGER SAYLE'S RASMOL, 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

SOURCES OF MOLECULAR STRUCTURES

Protein Data Bank (www.rcsb.org) – macromolecules

e.g. 1EY2

PubChem (pubchem.ncbi.nlm.nih.gov) – small molecules

MOLECULAR DYNAMICS SIMULATION OF

PROTEINS

Proteinstructure

•Protein Data Bank

• In housedetermination(NMR)

• In housemodelling (by homology,…)

Parametrisation

•Amber all atomforce field for proteins

•B3LYP/6-31G charge calculationfor drugs (ab initio)

Simulationengine

•GROMACS

•Parallel calculation

•GPU enabled

Moleculardynamicstrajectory

•Time evolution of structure

• Snapshot every1ps

•Microsecondscale; 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).

BINDING SITE DISCOVERY AND

VIRTUAL DRUG REPOSITIONING FOR

PHARMACEUTICAL CHAPERONES

TARGETING HOMOGENTISATE 1,2-

DIOXYGENASE FOR THE TREATMENT OF

THE INBORN ERROR OF METABOLISM

DISORDER ALKAPTONURIA.

ruptures of muscles, tendons, ligaments

black spots in eyes

blue-black earsblack 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 oftrimers

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 typeMutants

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/mol

Binding 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

AKNOWLEDGMENTS

Silvia Galderisi

Anna Visibelli

Vittoria Cicaloni