Very Deadly Disease Called Malaria Biology Essay

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The very deadly disease called Malaria caused by a parasite called plasmodium spp. is solely responsible for millions of deaths every year in many tropical countries and most especially the third world developing countries. Dihydrofolate Reductase - Thymidylate Synthase (DHFR-TS) is a very important enzyme in the metabolism of Folate and thus the biosynthesis of Thymidine which is a key nucleotide in DNA. An Inhibition of DHFR-TS enzyme affects plasmodium falciparum DNA synthesis and hence other important cell biological functions that is dependent on DNA replication hence causing the eventual death of the parasitic cell.

This report discusses the key structural and properties of a novel DHFR-TS inhibitor for plasmodium falciparum called Pyrimethamine, quantitative structure-activity relationship (QSAR) studies on its mechanism and mode of action and a brief insight to the problem of antimalarial drug-resistance. It also slightly looks at attempts using quantum cache, a computer-aided drug design (CADD) software to research modelling and docking simulations of Pyrimethamine into plasmodium falciparum Dihydofolate Reductase - thymidylate synthase enzyme active site and other DHFR-TS inhibitors including Proguanil, Cyclouanil Chloroquine and Methotrexate. With the calculation of the binding energy at the active site, the biological activity of the inhibitor can be hopefully ascertained and thus new inhibitors designed.


B.1 Malaria Disease

Malaria is an infectious disease contracted after being bitten by a vector in most cases an Anopheles mosquito carrying the protozoan plasmodium parasite. There are four different species of the plasmodium parasite which include plasmodium vivax, plasmodium ovale and plasmodium malariae but the World Health Organization (WHO) recognises the deadliest specie accounting for more than 80% of malaria-related deaths as the plasmodium falciparum spp. Initial symptoms of an infection include a loss of appetite, fatigue, fever and vomiting and if infection is untreated, it can lead to more complicated cases including anaemia, kidney failure, convulsions and eventual fatality.

(Copyright of World Health Organization. '')[1]

B.2 Plasmodium Falciparum Spp.

An Anopheles mosquito takes up the plasmodium falciparum parasite through biting an infected individual whilst sucking the infected blood. The parasite develops in the gut wall of the mosquito from oocysts to sporozites that then migrate and stay in the salivary glands of the mosquito. This newly infected mosquito, then goes on to bite a new individual transmitting the infection by releasing the plasmodium falciparum sporozites into their blood stream.

Plasmodium falciparum sporozites once in the blood make way to the liver where they develop into full grown merozoites and multiply. Then they return back into the main bloodstream and attack the human red blood cells infecting them and preventing them from binding to oxygen. This results in depleted oxygen levels and eventual cell death.[1]


C.1 Role of Dihydrofolate Reductase (DHFR) in Plasmodium falciparum folate synthesis

Unlike human cells that have DHFR and Thymidylate synthesis individually, in the plasmodium falciparum parasite DHFR-TS exits as a bi-functional enzyme. Plasmodium falciparum parasite uses Dihydrofolate Reductase-Thymidylate (DHFR-TS) enzyme for its folate synthesis and hence Thymidine production.

Fig 1: A 3D model structure of malaria Dihydrofolate reductase-thymydilate synthase (DFHR-TS) enzyme

(Copyright of the Protein Data Bank ( '')[2]

Fig 2: Mode of action of dihydrofolate reductase-thymidylate synthase (DHFR) enzyme

(Copyright of the Nature Cancer Gene Therapy.[3]

Dihydrofolate reductase (DHFR) first reduces dihydrofolate to tetrahydrofolate using the cofactor NADPH. Then tetrahydrofolate is coupled with methylene to form methylene-tetrahydrofolate which then methylates deoxyuridine monophosphate (dUMP) to give thymidine monophosphate (dTMP)using the enzyme Thymidylate synthase (TS) while itself is converted back to dihydrofolate (DHFR) to complete the process. An Inbibition of Dihydrofolate reductase - thymidylate synthase (DHFR-TS) by DHFR-TS inhibitors would prevent the biosynthesis of thymidine and hence DNA synthesis. Examples of a few potent DHFR-TS inhibitors include chloroquine, mefloquine and atovaquone.

C.2 Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (pfDHFR-TS) active site

A full three dimensional structure of PfDHFR-TS has not yet been found experimentally by X-ray diffraction but QSAR studies have been done using high resolution homology-modelling methods to develop structural templates based on the three dimensional Human and E.Coli DHFR-TS active site structures in the Protein Database Bank (PDB). These finished structural templates were then tested for validity using PROCHECK and validation proved over 95% of the active site amino acid residues modelled where favourable. [5]

The homology-model of pfDHFR-TS showed the DHFR domain with 228 residues is linked with the thymidylate synthase domain of 286 residues by a 94 amino acid sequence.

(Details of the full homology modelling and validation procedure are not included in this report but referenced properly at the end)

C.3 Quantitative Structure Activity Relationship (QSAR) studies of Pyrimethamine as a known potent inhibitor of plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase (pfDHFR-TS) enzyme

Characterization of Pyrimethamine

Fig 3: Structure of Pyrimethamine Fig 4: A 3D structure of Pyrimethamine

Pyrimethamine commonly marketed as Daraprim is a bacteriostatic antibiotic that treats malaria infection by inhibiting bacterial and protozoan DHFR-TS enzyme and hence their DNA synthesis. It is a very potent antimalarial drug with 87% protein binding affinity and its high selectivity to pfDHFR-TS. It has also been used in combination with sulphadoxine marketed as Fansidar to treat malaria resistant to chloroquine.

Fig 4: A three dimensional structure showing pyrimethamine docked in the active site of pfDHFR-TS

(copyright of Faculty of Science, Mahidol University.[4]

A three dimensional quantitative structure activity relationship (QSAR) study into the docking and interaction of pyrimethamine in the active site of plasmodium falciparum dihydrofolate reductase enzyme (pfDHFR) was researched. The QSAR study focused on the free binding energies to estimate interaction and inhibition constants. Also other biological activities like the EC50 of pyrimethamine was measured in vitro and taken into account to estimate the ligand binding interactions and thus potency of the antimalarial drug compound.

Binding and interactions of pyrimethamine at pfDHFR active site using molecular dynamics simulations (MDS) by Tokarski and Hopfinger

Fig 5: The 10A pruned binding site model of PfDHFR with key residues of the binding site indicated by ball-stick models in dark shading, NADPH is in light shaded ball-stick representation and the PYR inhibitor is shown in a space-filling shaded representation.

(copyright of Journal of Computer-Aided Molecular Design, 15: 787-810, 2001. KLUWER/ESCOM© 2001)

Model evaluation of conformational stability of the pfDHFR - pyrimethamine complex done by 'Tokarski and Hopfinger' was used in my research. This showed that a pfDHFR homology model on its own contained 3827 atoms including protons and thus had to be pruned to show main compounds involved in the enzyme inhibition and thus avoid conformational explosion during computational molecular dynamic calculations. [5] A summary of the results of the study detailed below showing varying radii (R), root - mean - square values (R.M.S) and corresponding non scaled ligand-enzyme model binding energies for pruned enzyme-ligand complex:

(R=8, r.m.s.=1.80 Å, ELR(LR) = −36.7 kcal/mole)

(R=10, r.m.s.=1.60 Å, ELRs(LR) = −37.3 kcal/mole)

(R=12, r.m.s.=1.94 Å, R = 12, ELR(LR) = −37.6 kcal/mole).

With a very low EC50 value pyrimethamine drug showed its biological activity as a very potent inhibitor. However like every other malaria drug, there is a problem of side effects and resistance.


Investigation into inhibitors of pfDHFR-TS involved understanding the Computer Aided Drug Design software used, 'Quantum Cache' and its uses in the construction and optimization of chemical compounds. A series of practical exercises involving the design of compounds and geometry optimization where done previously in another module to fully understand the computer aided drug design software 'Quantum Cache' and details of this very early -on practical exercises are not included in this finally report but are recorded in the Log book. [7]

Once a better understanding of the software had been achieved, a research was then done into pfDHFR-TS structures available in the Protein Data Bank (PDB). Part of the findings suggested that there were two types of data files; Homology models which involved the design of drug structures using computer aided representations based on very theoretical research and X-ray diffracted structures which involved the actual screening of proteins to get a full and accurate representation of the protein structure needed and then further modified using scientific computational methods and validated by molecular mechanics calculations. The better and more accurate structures are the X-ray diffracted structures and thus are used throughout this research experiment.

A search was run on the PDB website ( for plasmodium falciparum DHFR-TS in complex with an inhibitor. From the returned results, they were a few homology model and relevant X-ray diffracted structures found. This experimental methodology focuses on the analysis of these relevant X-ray diffracted PDB files modified slightly to fit personal intended research; calculations done on them using molecular mechanics; results achieved from calculations done and interpretation of these results.

D.1 PDB File 2BL9:

The first file downloaded was the 2BL9 file showing the X-ray diffracted crystal structure of plasmodium Vivax DHFR-TS in complex with Pyrimethamine and one of its derivatives.

Fig 6: Protein Database (PDB) File 2BL9 showing the 'X-ray crystal structure of plasmodium Vivax DHFR in complex with pyrimethamine and its derivative'

(copyright of Protein Data Bank;[6]

This structure was then saved as '2BL9.pdb' and opened in the 'Quantum Cache' computer aided drug design software.

Fig 7: 2BL9 structure opened in Quantum Cache software showing circled errors-in-translation from PDB which include; wrong hybridization, free oxygen atoms from surrounding water molecules and wrong bond lengths between residues.

Before a structure is deemed suitable for any work to be done on it, the errors-of-translation from downloading it from the Protein Database Bank (PDB) on must first be corrected. This is an intensive process as it involves making sure that every single atom present in the overall structure is properly represented in terms of charge, bonding, bond lengths and hybridization. This is referred to as structure 'cleaning' through out this report.

'Cleaning' of downloaded 2BL9 PDB structure:

After deleting a free oxygen atom (water molecules) surrounding the structure and correcting the hybridization of a wrongly charged atom, an error message would appear every time before I tried to save and warn me of the next incorrectly charge atom (highlighted), then I would correct the error and attempt to save again.

Fig 8: Showing a part of the 2BL9 structure showing highlighted incorrectly represented atoms

This was done repeatedly until all atoms had been corrected and the structure was 'clean' and suitable for work to be carried out on it. The file saved as '2BL9-DHFR+pyrimethamine.csf' as a reference standard for the 2BL9 structure. Hydrogen atoms where then added using the "Beautify Valence" command and the file saved as '2BL9-DHFR+pyrimethamine+H.csf' (Ref. Method 1).

Fig 9: Showing a 'clean' 2BL9 structure

The plasmodium DHFR-TS structure was locked except for the pyrimethamine ligand structure docked in the active site of the structure, which was unlocked by selecting the ligand in the sequence (1240 X); using the adjust command, then Unlock.

Fig 10: A picture showing the protein sequence of plasmodium DHFR-TS with the pyrimethamine molecule highlighted in the active site

Fig 11: A maximised picture showing the pyrimethamine molecule highlighted within the active site of the DHFR-TS structure

Initial Molecular Mechanics Calculations and Results for 2BL9 structure:

Initial geometry optimization of pfDHFR-TS protein structure and docked pyrimethamine ligand; Calculation run using:

Property of: Chemical Sample

Property: Optimized geometry

Using: CONFLEX with MM3

Final Steric energy value = 5930.2797 kcal/mol (ETOTAL)

Pyrimethamine ligand selected and cut into a new Quantum Cache worksheet; Calculation run using:

Property of: Chemical Sample

Property: Current Energy

Using: CONFLEX with MM3

Final Steric energy value = 621.5964 kcal/mol(ELIGAND)

Active site of pf-DHFR without the docked pyrimethamine ligand; Calculation run using:

Property of: Chemical Sample

Property: Current Energy

Using: CONFLEX with MM3

Final Steric energy value = 5346.6482 kcal/mol(EPROTEIN)



EBINDING = 5930.2797 - (621.5964 + 5346.6482)


Second Molecular Mechanics Calculations and Results for 2BL9 structure:

Into a new worksheet, the reference standard for the 2BL9 structure saved as '2BL9-DHFR+pyrimethamine.csf' was opened. Entire protein structure was locked before before applying the "beautify valence" command and "H-atom" commands to the structure. Then the pyrimethamine ligand in the protein structure was selected from the protein sequence(1240 X), unlocked, bonding corrected, the valence and H-atoms of entire structure was beautified again and the new structure saved as 2 - 2BL9-DHFR+pyrimethamine.csf (Ref method 2)

Initial geometry optimization of pfDHFR-TS protein structure and docked pyrimethamine ligand; Calculation run using;

Property of: Chemical Sample

Property: Optimized geometry

Using: CONFLEX with MM3

Final Steric energy value = 3419.7090 kcal/mol (ETOTAL)

Pyrimethamine ligand selected and cut into a new Quantum Cache worksheet; Calculation run using:

Property of: Chemical Sample

Property: Current Energy

Using: CONFLEX with MM3

Final Steric energy value = -66.7277 kcal/mol (ELIGAND)

Active site of pf-DHFR without the docked pyrimethamine ligand; Calculation run using:

Property of: Chemical Sample

Property: Current Energy

Using: CONFLEX with MM3

Final Steric energy value = 3532.0896 kcal/mol (EPROTEIN)


EBINDING = 3419.7090 - (-66.7277 + 3532.0896)


Reference method 2 of saving and calculating binding energies yielded a slightly more negative value than reference method 1 meaning '2' was a more accurate method of energy calculation than '1'. This method was used for all subsequent energy calculations for other DHFR inhibitors designed.

Construction of a new pyrimethamine structure and an attempt to dock relevant structure into 2BL9-DHFR-TS active site.

Into a new worksheet, the structure saved as '2 - 2BL9-DHFR+pyrimethamine.csf designed using reference method '2' explained above was opened. The pyrimethamine ligand present in active site was selected, copied and pasted into a new worksheet with care taken not to in anyway, alter the structure of the ligand.

On a separate worksheet, a new pyrimethamine molecule was constructed, geometry optimized, 4 dihedral angles defined as shown below:

Dihedral angles A, B and C defined using 30 steps of 12degrees between 180 to -180 angles

And conformational analysis ran using;

Property of: Conformations of a small [< ~100 atoms] molecule

Property: Global minimum search

Using: CONFLEX with MM3

And then:

Property of: Chemical Sample

Property: Rigid Map

Using: MM energies (MM3)

This was an extensive search that ran for hours and yielded 42 conformations with the lowest energy conformer having a final steric energy value = -45.8865kcal/mol. This conformer was the closest structure resembling the original pyrimethamine docked in the 2BL9 DHFR-TS protein structure.

Original Pyrimethamine Molecule New pyrimethamine molecule (Lowest conformer)

Both structures were alligned together with parts of the original structure (above left) and the new structure (above right - pink), supperimposed as shown below;

RMS value: 0.15476

Both structures did not perfectly superimpose of each other and this could have been as a result of more dihedral angles needing to have been defined. However this would have been an ever longer and most exhuastive search. Once this superimposing was done, the pyrimethamine structure from the original 2BL9 DHFR-TS structure was deleted and the New pyrimethamine lowest confomer structure was ready to be docked into an active site.

In a new work space on quantum cache, the DHFR-TS active site without the original pyrimethamine structure was opened, locked, geometry optimized and the structure defined as the active site using: 'Edit - Group Atoms - Active site - Name:DHFR-TS - Group' command. Then the New pyrimethamine structure was copied onto the same work space and defined as the ligand-to-be-docked using the similar command: 'Edit - Group Atoms - Ligand - Name:DHFR-TS - Group.'

Since the ligand was pink, the active site was coloured grey to see the ligand in the active site of the DHFR-TS once the docking had been done.

Then the ligand was docked into the 2BL9 DHFR-TS active site using the command:

Docking options: Run in active site window

Calculation type: Dock

Ligand: Flexible

Active site: Rigid

Final docking score = -76.688690 kcal/mol

Calculation of EBINDING energy:

Energy of New pyrimethamine ligand (lowest conformer): -45.8665kcal/mol.

Energy of 2BL9 DHFR-TS active site: 3550.8204 kcal/mol

Ligand docked in active site: 3454.5058 kcal/mol.


EBINDING = 3454.5058 - (-45.8665 + 3550.8204)

EBINDING = -50.4481kcal/mol


An attempt was made at docking other clinically used antimalarial drugs whose mode of action based on extensive QSAR studies are thought to be of a DHFR-TS inhibiting nature, into the DHFR-TS active site using the exact same software and method as seen above with Pyrimethamine . Comprehensive details of the process of individual docking are available in the appendices and log book attached with this report but see a summarised table of results below.

DHFR-TS Inhibitor

(Name and structure)


(Best superimposable/ lowest energy conformer)



(2BL9 DHFR -TS Active Site energy) kcal/mol


(Ligand docked into DHFR-TS active site) kcal/mol



































A comparison of the binding energies of all pfDHFR-TS inhibitors docked in active site of pfDHFR-TS shown above indicates largely that Pyrimethamine compound had the most negative value of -50.4481kcal/mol. QSAR studies show that the more negative the EBINDING energy of an enzyme-ligand complex is, the stronger the attraction of the ligand to the enzyme and therefore the more potent the drug compound is.

This means therefore that from the table above in terms of the EBINDING energies and hence potency of the compounds as anti-malarial drugs;

Pyrimethamine> Trimethoprim >Artemisinin > Cycloguanil > Methotrexate > Proguanil

This largely reflects present QSAR studies of anti-malarial antifolates which indicate that, pyrimethamine is the strongest and most potent pfDHFR-TS inhibitor followed by Trimethoprim.

Arteminisin is one of the newer anti-malarial antifolates derived originally from a plant called Artemisia annua .It had been used largely in the past in Chinese herbal medicine before it was found in 2003 to be a very reliable inhibitor of DHFR-TS with very few side effects. Presently, Arteminisin and its analogues are synthesised as potent anti-malarial drugs presently used in Arteminisin Combination Therapies (e.g. Mefloquine and Airplus) for the treatment of chloroquine resistant Malaria.

Even though Methotrexate is a very potent antifolate drug with high potency against plasmodium falciparum recorded in QSAR studies in vitro, it is mainly used for the short-term treatment of malaria in combination therapies. This is because it has a higher affinity for human DHFR than pfDHFR and hence can cause toxicity, adverse side-effects and eventually encourages malaria resistance if administered on its own. Methotrexate is instead used more widely as a chemotherapy drug for the treatment of cancer.

Finally, Proguanil with the highest EBINDING energy of 155.662503kcal/mol indicates it is the least potent of the group of antifolate drugs. This is very correct and due to the fact that Proguanil is not directly a pfDHFR-TS inhibitor but only a prodrug of Cycloguanil which is the actual DHFR inhibitor showing a much better potency with an EBINDING energy value of 61.3896kcal/mol which is less than half of the EBINDING energy value of Proguanil. Proguanil can also be used in combination therapies with chloroquine for chloroquine-resistant strains of plasmodium falciparum malaria


Using the Quantum cache software for the designing and optimization of molecules was brilliant and fairly straight forward. However, the conformational analysis and ligand docking exercises were a real challenge as results can be very sensitive to the different conformational positions of ligands in an enzyme's active site. This might have affected the accuracy of the above EBINDING energies in comparison with published literature binding energy values for all 6 pfDHFR inhibitors.

Also the difference in values can be attributed to a variation in the different types of computer modelling software available today. The Protein Data Bank makes use of a combination of quicker, precise and more expensive and complexly run software on a larger scale whilst Quantum Cache (Scigress) available to Kingston University, even though still accurate on a smaller scale, can be tedious to analyse very large complex molecules and drug targets.

A future scope for this project could be:

An investigation into identifying pharmacophores and key structural parts of a potent inhibitor involved in the enzyme-ligand interaction at the active site and an attempt at designing an analogue of a potential novel inhibitor for the pfDHFR-TS active site paying close attention to the key binding interactions at the active sites involved in binding.

Also an investigation in using extensive QSAR studies of a new research which involves the availability of non active site pockets in pfDHFR-TS enzyme, their function and how a drug can potential interact with the non active site pockets of pfDHFR-TS and to fuction as a pfDHFR-TS inhibitor.