Computational Pharmacology. Judith Klein-Seetharaman Assistant Professor, Department of Pharmacology. High Points of the Case Study: The Development of Cox-2 Inhibitors. High points regarding the success of the drugs… High points regarding drug discovery principles….
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Assistant Professor, Department of Pharmacology
A Wonder Drug: What is the most commonly-taken drug today?
It is an effective painkiller.
It reduces fever and inflammation when the body gets overzealous in its defenses against infection and damage.
It slows blood clotting, reducing the chance of stroke and heart attack in susceptible individuals.
It may be an effective addition to the fight against cancer.
As you might expect from a drug with such diverse actions, aspirin blocks a central process in the body: Aspirin blocks the production of prostaglandins, key hormones that are used to carry local messages.
Unlike most hormones, which are produced in specialized glands and then delivered throughout the body by the blood, prostaglandins are created by cells and then act only in the surrounding area before they are broken down.
Prostaglandins control many of these neighborhood processes, including the constriction of muscle cells around blood vessels, aggregation of platelets during blood clotting, and constriction of the uterus during labor.
Prostaglandins also deliver and strengthen pain signals and induce inflammation.
These many different processes are all controlled by different prostaglandins, but all created from a common precursor molecule.
COX = Cyclooxygenase (PDB entry Inhibitors1prh) performs the first step in the creation of prostaglandins from a common fatty acid.
It adds two oxygen molecules to arachidonic acid, beginning a set of reactions.
Aspirin blocks the binding of arachidonic acid in the cyclooxygenase active site. The normal messages are not delivered, so we don't feel the pain and don't launch an inflammation response.
http://www.rcsb.org/pdb/molecules/pdb17_1.htmlWhat does COX do?
Two different active sites, collectively prostaglandin synthase: 1, the cyclooxygenase active site discussed; 2, is has an entirely separate peroxidase site, which is needed to activate the heme groups that participate in the cyclooxygenase reaction.
Dimer of identical subunits (two cyclooxygenase active sites and two peroxidase active sites in close proximity)
Each subunit has a small carbon-rich knob, pointing downward anchoring the complex to the membrane of the endoplasmic reticulum, shown in light blue.
The cyclooxygenase active site is buried deep within the protein, and is reachable by a tunnel that opens out in the middle of the knob. This acts like a funnel, guiding arachidonic acid out of the membrane and into the enzyme for processing.
PDB entry 4cox
COX-1 and COX-2 are made for different purposes.
COX-1 is built in many different cells to create prostaglandins used for basic housekeeping messages throughout the body.
COX-2 is built only in special cells and is used for signaling pain and inflammation.
Aspirin attacks both. Since COX-1 is targeted, aspirin can lead to unpleasant complications, such as stomach bleeding.
Needed: specific compounds that block just COX-2, leaving COX-1 to perform its essential jobs. These drugs are selective pain-killers and fever reducers, without the unpleasant side-effects.
Val523 (Ile in COX-1)
=> piece together all available information
Linking variety of databases
Linking the different layers
Understanding drug action
BLAST is a heuristic search method that seeks words of length W (default = 3 in blastp) that score at least T when aligned with the query and scored with a substitution matrix. Words in the database that score T or greater are extended in both directions in an attempt to fina a locally optimal ungapped alignment or HSP (high scoring pair) with a score of at least S or an E value lower than the specified threshold. HSPs that meet these criteria will be reported by BLAST, provided they do not exceed the cutoff value specified for number of descriptions and/or alignments to report.
BLOSUM62 Substitution Scoring Matrix Drug Discovery. The BLOSUM 62 matrix shown here is a 20 x 20 matrix of which a section is shown here in which every possible identity and substitution is assigned a score based on the observed frequencies of such occurences in alignments of related proteins. Identities are assigned the most positive scores. Frequently observed substitutions also receive positive scores and seldom observed substitutions are given negative scores.
The PAM family PAM matrices are based on global alignments of closely related proteins. The PAM1 is the matrix calculated from comparisons of sequences with no more than 1% divergence. Other PAM matrices are extrapolated from PAM1.
The BLOSUM family BLOSUM matrices are based on local alignments. BLOSUM 62 is a matrix calculated from comparisons of sequences with no less than 62% divergence. All BLOSUM matrices are based on observed alignments; they are not extrapolated from comparisons of closely related proteins. BLOSUM 62 is the default matrix in BLAST 2.0. Though it is tailored for comparisons of moderately distant proteins, it performs well in detecting closer relationships. A search for distant relatives may be more sensitive with a different matrix.
The relationship between BLOSUM and PAM substitution matrices. BLOSUM matrices with higher numbers and PAM matrices with low numbers are both designed for comparisons of closely related sequences. BLOSUM matrices with low numbers and PAM matrices with high numbers are designed for comparisons of distantly related proteins. If distant relatives of the query sequence are specifically being sought, the matrix can be tailored to that type of search.Scoring matrices
A. When no information but sequence and physical principles are used
= ab initio structure prediction (Blue Gene IBM )
B. When other information is used (Survey of "ab initio" methods that use pdb information and their relation to protein folding)
requires a method for evaluating the compatibility of a given sequence with a given folding pattern
B0. 3D profiles
B1. Rosetta: conformations from short segments in pdb
B2. Including experimental structural constraints
B3. Threading (=sequence-structure alignment),
B4. Inverse threading and folding experiments Reference Ivet
B4a. using short-range information
B4b. using short- and long-range information
B4. Predicting structural class only Reference Ivet
B5. Predicting active site only?
B6. Predicting protein-protein interaction sites?
B7. Predicting surface shape?
C. When a template with known structure must be available
D. Modeling structures based on experimental data
Both NMR and X-ray underdetermine the protein structure. To solve a structure one must minimize a combination of the deviation from the experimental data and the conformational energy:
D1. NMR (set of constraints on distances and angles)
D2. X-ray crystallography (Fourier transform of the electron density)
Basic Steps in Homology Modeling Drug Discovery
( Blast the query sequence towards the pdb database )
Model Building Drug Discovery
Model Refinement and Evaluation Drug Discoveryhttp://cgat.ukm.my/spores/Predictory/evaluation.html
Class A: Rhodopsin Drug Discovery
, Odorants, Monoamines, Lipid messengers,
hormones (e.g. platelet activating factor,
releasing hormone &
, parathyroid hormone,
Class C: Metabotropic glutamate and
Calcium sensors, GABA
Class D: Fungal pheromone Family
Class E: c
AMP receptor (
Class F: Frizzled/Smoothened family
Ocular albinism proteins
Putative/ unclassified orphansGPCR Family and Their Ligands
The Disulfide Bond is highly conserved across families, but not in putative and orphan receptors
COX 2 Modelling :
Template structure : 1PTH.pdb (cox1 in ovis aries)
query seq:sequence of 1PXX.pdb (cox2 in mus musculus)
model generated using modeller: 2cox.pdb
COX 1 Modelling:
Template structure : 1PXX.pdb (cox2 in mus musculus)
query seq:sequence of 1PTH.pdb (cox1 in ovis aries)
model generated using modeller: 1cox.pdb
A protein conformation has to satisfy three conditions: Drug Discovery
1. Only stereochemically allowed conformations of all residues are acceptable (=avoid steric clashes).
Model system: dialanine peptide
Rotation of the polypeptide chain is permitted around the N-Calpha (angle Phi) and Calpha-C (angle Psi) bonds (except Proline) and the peptide bond (angle omega), which is either trans in most cases (omega=180o) or cis (omega=0o) in rare cases, i.e. at Proline residues. These angles define the backbone conformation, and specific conformations are allowed, as described by the Ramachandran plot.
2. The folded state must be energetically favorable
The native state of globular protein is only 20-60 kJ mol-1 (5-15kcal/mol) more stable than the denatured state. This is the equivalent of one or two water-water hydrogen bonds. It is unclear why this is the case, because the stability of proteins can be increased by adding stabilizing contacts. The main problem in achieving the native state is the loss of conformational freedom (entropy reduction), when going from many unfolded to a single folded conformation. This process is therefore thermodynamically unfavorable. Why does it still occur? Because the loss in entropy arising from conformational restriction is compensated by an increase in entropy arising from the hydrophobic effect. The fact that native protein structures are more stable than unfolded protein by 1-2 H-bonds, means that 1-2 unsatisfied H-bonds in a protein can make the native state unstable.
3. The folded state must be tightly packed.
How tightly packed is the interior of a protein? In theory, relatively loose packing would ensure exclusion of water, since a <1.4Å radius (=size of water molecule) hole is acceptable. However, attraction between atoms (van der Waals forces) cause closer packing than theoretically required by the hydrophobic effect alone. Thus, a protein is like a jigsaw puzzle, except that the pieces in a jigsaw puzzle are rigid, while the side chains in proteins are dynamic and can adopt many conformations.
More details on requirements 2 and 3: The folded state must be energetically favorable and the folded state must be tightly packed.
Terms used in the evaluation of the energy of a conformation (see page 253 in chapter 5, Ref_Lesk for equations):
1. Bond stretching
2. Bond angle bend
3. deviations from planarity and enforcement of correct chirality
4. Torsion angle
5. van der Waals interactions
6. Hydrogen bonds
=> set of conformational energy potentials that fine tune these parameter sets "Potential functions"
The potential functions satisfy necessary but not sufficient conditions for successful structure prediction. Multiple local minima cannot be distinguished reliably from the correct one on the basis of calculated conformational energies. (What does this mean in practice? How many possible structures are there as opposed to the real structures? How different are the structures?)