Download - Universidade de Aveiro-RMN
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 1/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Elucidação Estrutural e
Metabolómica na IndústriaFarmacêutica
Brian J Goodfellow
1
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 2/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
The Pharmaceutical Industry
We have a challenging and inspiring mission: toimprove the quality of human life by enablingpeople to do more, feel better and live longer.
GlaxoSmithKline
As a global biopharmaceuticalcompany, our activities touch many
people’s lives
DreamHealth is our dream. Because we believe thatdreams should be pursued, we are researching anddeveloping new drugs to offer a longer and betterlife. In Portugal and worldwide. Caring for yourHealth. And there are dreams that come true...
2
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 3/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
The Pharmaceutical Industry
$
2
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 4/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
How do we get new drugs ?
What is a drug molecule?
! A drug molecule has one or more functional groups positioned in 3D spaceon a structural framework that enables the molecule to bind to a targetedbiological macromolecule, the receptor
biological response
+“druggable” target“drug-like” molecule
Properties: low Mwt, not toolipophilic, not too hydrophilic, needsfunctional groups
Properties: usually protein, leads tobiological response, does not causetoxicity
3
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 5/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
New Drugs - recent trends
Reasons:! fundamentally difficult
! high failure rate (4% of compounds become a drug and of these 89 % failclinical trials)
! expensive > 800 million dollars and can take 15 years
! cost minimization in industry leads to fewer drugs
! Mainly based on high throughput screening (HTS) or structure based drugdesign (SBDD)
4
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 6/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
http://www.kubinyi.de/lectures.html
New Drugs - SBDD and HTS
! Requires 3D structure of protein target to design drugs to bind to activesite, therefore need rapid methods for structure determination of targets
Chemical Microarrays (Graffinity, Heidelberg)
! ca. 100,000 fragments linked onto chip
! parallel detection of weak, specific interactions
! confirmation of hits by functional testing and X-ray/NMR
! Corporate databases (100,000s of compounds) are screened automaticallyby robots - identify high-affinity binders but produce false leads - need toidentify lead compounds that interact with the target in a biologicallyrelevant mechanism
5
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 7/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
The diversity of NMR enables the techniqueto be applied at multiple stages along the
drug discovery pathway
New Drugs - How can NMR help ?
NMR is being used for:
! protein structure determination
! lead discovery and optimization
! evaluating in vivo selectivity andefficacy
! analyzing drug toxicity profiles andidentifying new drug discoverytargets
6
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 8/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
NMR can:
! rapidly determine protein and protein-ligand structures
! efficiently screen fragment-based libraries to identify biological relevantligand interactions and identify new therapeutic targets
! monitor changes in the metabolome from biofluids and cell lysates toexplore in vivo drug activity
New Drugs - How can NMR help ?
7
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 9/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Protein structure determination - NMR
C
H
C
H5Å
NOE
1 Å = 1x10-10 m
Input: intercity distance table Output: map of USA
9
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 10/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Protein structure determination - NMR
C
H
C
H5Å
NOE
1 Å = 1x10-10 m
Input: 1H-1H distance table Output: structure of protein
100 VAL H 100 VAL HB 3.32 #peak 288 #SUP 0.96 #QF 0.96 21 LEU H 21 LEU HG 3.97 #peak 289 #SUP 0.98 #QF 0.93189 LYS H 189 LYS HB2 3.71 #peak 290 #SUP 0.86 #QF 0.86100 VAL H 100 VAL QG2 3.97 #peak 292 #SUP 1.00188 VAL QG1 189 LYS H 4.32 #peak 293 #SUP 0.84 #QF 0.84 45 GLU H 46 VAL H 4.60 #peak 297 #SUP 0.96 #QF 0.96131 TYR QD 189 LYS H 4.55 #peak 300 #SUP 0.79 #QF 0.79 45 GLU H 45 GLU QG 3.96 #peak 321 #SUP 0.98 #QF 0.98 45 GLU H 45 GLU HB2 3.30 #peak 323 #SUP 0.91 #QF 0.91 45 GLU H 45 GLU HB3 3.30 #peak 324 #SUP 0.95 #QF 0.95 71 ASP H 114 ALA H 3.83 #peak 330 #SUP 0.99 #QF 0.99 72 LYS H 114 ALA H 4.37 #peak 337 #SUP 0.82 #QF 0.82138 TYR QD 139 ALA H 4.10 #peak 339 #SUP 0.96 #QF 0.96 32 GLU HB2 33 GLU H 4.52 #peak 467 #SUP 0.94 #QF 0.94 23 THR QG2 33 GLU H 3.83 #peak 470 #SUP 0.31 #QF 0.31 99 LYS HB3 101 TRP HE1 5.13 #peak 482 #SUP 0.44 #QF 0.44 84 PHE HB2 85 ALA H 4.01 #peak 493 #SUP 0.98 #QF 0.98 85 ALA H 101 TRP HD1 4.05 #peak 497 #SUP 0.99 #QF 0.99 85 ALA H 168 TYR QD 4.95 #peak 499 #SUP 0.94 #QF 0.94………..
9
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 11/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
amide
aromatic
methyl
Lehninger, Nelson & Cox, 3rd ed.
aliphatic
! But to obtain a structure we need toidentify every peak
! get a ca. peak from each 1H in theprotein
! but 150 residue protein has ~1000 1Hsignals
! incomplete dispersion of frequencies
! need to separate resonances inanother frequency dimension
Protein structure determination - NMR
many peaks overlapped
10
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 12/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
2D
t 1
!M
1D
FourierTransform
2D FourierTransform
Protein structure determination - 2D NMR
11
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 13/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Protein structure by NMR - going to nD
! faster structure determination needs higher dimensions (and isotopicenrichment of the protein)
! But a 5D experiment for a 30 sec FID would take (128x32x16x8x4).......around 2 years !
12
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 14/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
a mathematical calculation that converts a table of
distances into a map or structure
Backbone atoms of the final
family of 20 conformers 173-180
Dias et al. J Biol Chem (2006) vol. 281 (42) pp. 31553-31561
Dias et al. J Biomol NMR (2005) vol. 32 (4) pp. 338-338
Protein structure by NMR - example using up to 3D
! p22HBP a 22 kDa protein
! Used 1851 distances and took ca. 1 year fromstart to finish
13
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 15/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! Robotic can be used for automated production of protein samples for NMRand x-ray
! New NMR pulse sequences shorten the time required to collect a set ofNMR spectra: automated projection spectroscopy (APSY); G-matrix Fouriertransform NMR (GFT-NMR) are two examples
! Dimensionality of traditional multi-dimensional triple resonance experimentsis reduced = shorter acquisition time. Works by joint sampling of multiple
chemical shifts in a single indirect dimension
Protein structure by NMR - speed up data collection
14
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 16/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! Robotic can be used for automated production of protein samples for NMRand x-ray
! New NMR pulse sequences shorten the time required to collect a set ofNMR spectra: automated projection spectroscopy (APSY); G-matrix Fouriertransform NMR (GFT-NMR) are two examples
! Dimensionality of traditional multi-dimensional triple resonance experimentsis reduced = shorter acquisition time. Works by joint sampling of multiple
chemical shifts in a single indirect dimension
Protein structure by NMR - speed up data collection
14
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 17/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Protein structure by NMR - Structural genomics
15
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 18/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
NMR can:
! efficiently screen fragment-based libraries to identify biological relevantligand interactions and identify new therapeutic targets
New Drugs - How can NMR help ?
16
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 19/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Ligand binding by NMR
! where/how does the ligand bind ?
! which ligand of a series binds ?
! NMR techniques for ligand bindinguse the fact that the ligand, whenbound, has properties similar to the
large target or use magnetizationtransfer between target and ligand
17
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 20/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! Saturation Transfer Difference isb a s e d o n a t r a n s f e r o fmagnetization from the protein tothe bound molecule
[ligand] = 1 mM
[protein] = 35!M
expt time 1-4 hours
Ligand binding by NMR - which ligand(s) binds ?
STD experiment for p38 kinase domain
saturate baseline
saturate protein
difference
18
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 21/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Ligand binding by NMR - which ligand(s) binds ?
[ligand] = 0.1-0.5 mM
[protein] = 25 !M
expt time 20 hours
INPHARMA
! LA binds so that HA to HT transferoccurs
! LA dissociates and LB enters! HT to HB transfer now occurs! Result is a NOE peak between HA and
HB mediated by HT
19
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 22/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! Diffusion Ordered SpectroscopY (DOSY)
DOSY
" 1Hrápido
lento
Desvio químico
Difusão
Cromatografia em camada fina TLC
Polaridade
Edição espectral por coeficiente de difusão
Ligand binding by NMR - which ligand(s) binds ?
Chromatograpy, TLC
P o l a r i t y
D i f f u s i o
n
fast
slow
chemical shift
Spectra editing by diffusion constantAdapted from EJ Cabrita, Presentation, UA 2008
20
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 23/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
D i f u s ã o
Desvio químico
lento
rápido
D i f u s ã o
lento
rápido
Host (H)
Guest (G)
H + G HG
Ligand binding by NMR - which ligand(s) binds ?
D i f f u s i o n
D i f f u s i o n
slow
slow
fast
fast
chemical shift Adapted from EJ Cabrita, Presentation, UA 2008
21
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 24/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! HSQC = Heteronuclear SingleQuantum Correlation
! one peak for each NH gives a“fingerprint” of the protein
! need to label the protein with 15Nand or 13C
! fast experiment 5-20 mins
Ileal bile acid binding protein, 14 kDa
Ligand binding by NMR - where/how does the ligand bind ?
! NMR chemical shift mappingidentifies ligand binding sites
! binding site obtained rapidly andthe process can be automated(robotic sample-changers and flow-probes)
22
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 25/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
[ligand] = 0.5-1 mM
[protein] = 0.5-1 mM
expt time 1-4 hours
Ligand binding by NMR - where/how does the ligand bind ?
23
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 26/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
PPIX-induced shift perturbations
small negative shiftslarge negative shifts
small positive shifts (0.05-0.1ppm)
large positive shifts (>0.1ppm)
Ligand binding by NMR - where/how does the ligand bind ?
hydrophobic interaction
between p22HBP and heme
24
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 27/39
Eluciadçao Estrutural e Metabolomica na Indústria Farmacêutica
Summary of some NMR methods for binding studies
Approach Observe Use Description
Shift mapping target1º screeningHit validationBinding site
Perturbs chem shifts oftarget near binding site
STD ligand1º screeningHit validation
Identifies compounds thatbind weakly.
Bulid up curves identifyFGs
waterLOGSY ligand 1º screeningIdentifies compounds thatbind via water mediated
NOEs
DOSY ligand 1º screeningHit validation
Mesures differences indiffusion rates for ligandsin bound versus free state
SAR by ILOE ligand-to-ligand Fragment based DDLead optimization
Detects protein mediatedligand-ligand interactions(occupying adjacent sites)
INPHARMA ligand-to-ligand Lead characterizationDetects protein mediatedligand-ligand interactions
(competition for samesite)
25
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 28/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
NMR can:
! monitor changes in the metabolome from biofluids and cell lysates toexplore in vivo drug activity
New Drugs - How can NMR help ?
26
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 29/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! Basically, a 1D 1H NMR spectrum of a lysed cell or biofluid captures itsmetabolic state (NMR only observes the most abundant metabolites)
Metabolomics by NMR
Proteins
Genomics (DNA)25,000 genesTranscriptomics (RNA)100,000 mRNAs
Proteomics (proteins)
1,000,000 proteins
The metabolome reflects the
general cellular state
Metabolomics (metabolites)
2,500 metabolites (small
molecules)
27
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 30/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Metabolomics by NMR
Metabolomics definition
“the quantitative measurement of the dynamic multiparametric metabolicresponse of living systems to pathophysiological stimuli or geneticmodification” (Imperial College, London, 1999)
Perturbedsystem
- Disease- Xenobiotics- Genetic modification- Diet- Environmental effects
Fingerprint ofbiochemical perturbation
Metabolic adjustmentsto mantain homeostasis
! Changes in metabolic profiles
NMR of:Biofluids (plasma, urine, etc.)TissuesCells
Non-perturbedsystem
28
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 31/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Metabolomics by NMR - multivariate analysis
! Comparing 1 or 2 spectra by eye is easy - 40 is harder…..! Use multivariate analysis (PCA) to identify differences between spectra
! Always need many spectra to filter out external/experimental variables
expt time = 5 mins x Nº samples
some samples have a lot moretrehaolse than others
other differences harder to see!
29
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 32/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
!"#%"#&"#'"#("##"#)"#*"#+"#,"#
!"#$%&' )* +
! Scores plot identifies grouping of samples - similar spectra group together! Loadings plot identifies differences between grouped spectra
expt time = 5 mins x Nº samples
Metabolomics by NMR - multivariate analysis
!"#"$
!"#""&
!"#""'
!"#""(
!"#"")
"
"#"")
"#""(
"#""'
"#""&
"#"$
!"#"( !"#"* !"#") !"#"$ " "#"$ "#") "#"* "#"(
!"# %&'(
!" ' )*&+(
+,-./,0 123,-4-5670
8** 123,-4-5670
+,-./,0 9.7:,-7/;
8** 9.7:,-7/;
cells frozen in N2(l) then extractedusing chlorofrm/methanol/water,freeze dried and buffer added
30
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 33/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! The effect of the drug 8-azaxanthine on the target urateoxidase can be seen
! The drug specifically inhibitsurate oxidase
Metabolomics by NMR - in vivo models
cells frozen in N2(l) thenground and buffer added
31
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 34/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Rat urine
Metabolomics by NMR - in vivo, biofluids
phosphate buffer added to urine
32
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 35/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
The COMET project: 5 pharmaceutical companies + Imperial College (UK)
! Generation of a comprehensive database of NMR spectra of biofluids
treated with various toxicants (147 studies; >30000 samples)
Achievements
! Predictive screening methodologies that both detect abnormal metabolicstates and classify target organ toxicity (mainly liver and kidney)
! Animals (rodents) subjected to treatments generating typical toxicologicallesions and their biofluids analysed by NMR.
Approach
Metabolomics by NMR - in vivo, toxicity
33
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 36/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
! Differences in metaboliteprofiles for Aito Town,Chicago, and Guangxip o p u la t i o n re ve a le ddifferences related togenetic, dietary, and gutmicrobial factors
Metabolomics by NMR - in vivo, know your subject
34
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 37/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
Metabolomics by NMR - disease
! Can distinguish malformations from controls in diffusion edited spectra of AF
! 22 metabolites were found to changesignificantly in the AF of malformed fetuses,compared to controls, together with fourunassigned resonances
!
Results confirmed that malformed fetusessuffer altered energy metabolism and kidneyunderdevelopment
35
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 38/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
MetabolicPhenotype
Nutritionalstatus
Gut microbiota
Disease Co-/pre-administration of
other drugs
P h a r m a c o - m
e t a b o n o m i c s
P h a r m a c
o - g e
n o m i c
s
Genotype
Metabolomics by NMR - the future
! Personalized medicine
36
7/23/2019 Universidade de Aveiro-RMN
http://slidepdf.com/reader/full/universidade-de-aveiro-rmn 39/39
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica
The diversity of NMR enables the technique to be applied at multiple stagesalong the drug discovery pathway
! lead discovery and optimization helped:
! by rapidly determining protein and protein-ligand structures
! efficiently screening fragment-based libraries to identify biologicalrelevant ligand interactions and new therapeutic targets
! Evaluation of in vivo selectivity and efficacy helped by:
! monitoring changes in the metabolome from biofluids and cell lysates toexplore in vivo drug activity and by analyzing drug toxicity profiles andidentifying new drug discovery targets
Conclusion
! Protein structure (UAveiro, ITQB, FCT-UNL, UCoimbra)
! Ligand binding (UAveiro, ITQB, FCT-UNL, UCoimbra)
! Metabonomics (UAveiro - diseases)
37