DHFR ENZYME AND ITS INHIBITORS .

DRUG -RECEPTOR BINDING

1. DHFR ENZYME ( CADD) BY: MANSI PANWAR M.PHARMACY(PHARMACEUTICAL CHEMISTRY) PUP PATIALA

2. DIHYDROFOLATE REDUCTASE (DHFR) It is the reductase enzyme which catalyzes the reduction of 7,8 Dihydrofolate through a stereospecific hydride transfer from NADPH (reduced form)cofactor to produced 5,6,7,8 Tetrahydrofolate product. It is found ubiquitously in all dividing cells of prokaryotes and eukaryotes. MECHANISM OF ACTION : Tetrahydrofolate(FH4) ,which is produced from the vitamin folate (B9) is primary one carbon carrier in the body . While one carbons units are attached to FH4 at N5 OR N10 Or form bridge between N5 and N10 by donation of amino acids (majorly serine), (histidine etc.) they can be oxidized or reduced depends upon the requirement by biochemical reaction .

3. One of the major acceptor of this is deoxy - uridine monophosphate which accepts N5,N10 –methylene from FH4 to yield deoxy -thymidine monophosphate(dTMP) , purine precursor. This is essential for purine and thymidylate synthesis ,which are important for cell growth and cell proliferation . DHFR STRUCTURE : It is a cytosolic protein( 186 amino acids ) of 21.3 kDa and uses NADPH as cofactor. The DHFR sequence in humans is 30% similar to that of E.coli and 70% similar to Other mammalian. DHFR . The standard source of this enzyme is mammalian and avian liver. In humans this enzyme is encoded by DHFR gene . It is found in the q11-q22 region of chromosome 5. Four alpha helix connects successive beta strands . A central eight standard beta-pleated sheet makes up the main feature of the polypeptide backbone folding of DHFR. .

4. Seven of these strands are parallel and the eighth is antiparallel . The protein is divided into two subdomains , the adenosine binding sub – domain and the loop domain . The adenosine binding sub -domain is larger of two and binds adenoside moiety of NADPH . The loop subdomains consist of three loops, the Met-20 loop , the F-G (phenylalanine –glycine) loop , and the G-H (glycine –histidine)loop . Between these two loops there is active site . The active site is situated in N-terminal half of the sequence,which includes a conserved Pro- trp dipeptide ; the tryptophan has been shown to be involved in the binding of substrate by the enzyme. The size of the active site is regulated by movements of the two subdomains. E.COLI (ESCHERICHIA COLI ) dihydrofolate reductase is an important target of antibacterial medications . E.coli DHFR is small ( 18kb) protein with an α / ꞵ structure consist of a central eight stranded ꞵ -she et and four flanking α -helices . The active site cleft of divides the protein into two structural domains: 1).adenosine binding subdomain ( light blue ) and 2.)the major subdomain : MET20( green ),dark blue(F-G), and red ( G-H ). The ternary complex of folate and NADP + illustrates the substrate and cofactor binding site .

5. There are five hydrogen bonds between folate and DHFR active site : 1) Two H-bonds between ARG 70 and folate ( carboxylate group of glutamate ) 2) Two H-bonds between GLU 30 and folate ( NH2 and NH moiety of pyrimidine ring of pteridine ring) The GLU 30 is responsible for the protonation of substrate and holds the substrate during the catalytic activity of enzyme. 3) the other residues which interact with the folate are: 1) One H- bond between ASN 64 and folate ( C=O of p-amino benzoic acid ) 2) PHE 34 (through pi –pi interaction ) with folate ( phenyl ring of p-amino benzoic acid FOLLOWING PICTURE ILLUSTRATES HOW THE COMPLEX OF DHFR AND FOLATE FORM :

6. LIST OF VARIOUS INHIBITORS ACT ON THIS DHFR ENZYME : 1. TRIMETHOPRIM –BLOCKS BACTERIAL ENZYME 2 . PYRIMETHAMINE ---PROTOZOAL ENZYME 3. METHOTRAXATE ---MAMMALIAN ENZYME 1.TRIMETHOPRIM : 5-(3,4,5-TRIMETHOXYBENZYL) -2,4-PYRIMIDINE -DIAMINE i s a anti – bacterial drug used to treat various infections like urinary tract infections, respiratory tract infections and g.i.t. tract infections. It is 50,000 TIMES more active against bacterial DHFR enzyme than mammalian DHFR so, no harm to human folate metabolism . It is used with sulphamethoxazole due to their complementarity and synergistic action together to achieve bactericidal action .

7. 1.FIG. DEPICTING THE INTERACTION OF TRIMETHOPRIM WITH THE DHFR AND NADPH( DHRF OF LACTOBACILLUS CASEI) 2. FIG. : H-BOND INTERACTION B/W DHFR OF E.COLI AND TRIMETHOPRIM LIKE THE NATURAL SUBSTRATE , TRIMETHOPRIM BINDS TO THE ENZYME ALONG THE NADPH IN A TERNARY COMPLEX, IN WHICH THE REDUCING END OF THE CO-FACTOR IS SITED NEAR TO PYRIMIDINE RING OF TRIMETHOPRIM . TRIMETHOPRIM ,METHOTRAXATE ,PYRIMETHAMINE HAVE 2,4-AMINOPYRIMIDINE RING WHICH RESEMBLES THE PTERIDINE NUCLEUS OF DIHYDROFOLATE. BY NMR AND X-RAY IT IS FOUND THAT N1 OF TRIMETHOPRIM IS PROTONATED IN BOUND STATE . AND X-RAY CRYSTALLOGRAPHY ALSO SHOWS THAT TRIMETHOPRIM LOCATE IN A DEEP CLEFT OF THE PROTEIN , WITH FAVOURABLE NON-COVALENT INTERACTIONS HELPING TO MAINTAIN ITS POSSITION . THIS BINDING MODE ENABLES IONIC INTERACTIONS BETWEEN THE PROTONATED N1 AND CARBOXYLATE GROUP OF THE CONSERVED AMINO ACID RESIDUE, AND HYDROGEN BONDING INTERATIONS AS SHOWN IN THE PICTURE. THESE COMPLEX INTERACTIONS HOLDING THE TRIMETHOPRIM AND DHFR TOGETHER , RESULTS IN POTENT INHIBITORY EFFECT OF TRIMETHOPRIM UPON BACTERIAL DHFR.

8. METHOTRAXATE : IT is a dihydrofolate reductase inhibitor that is used in the treatment of autoimmune inflammatory disease such as rheumatid arithritis and also as a antineoplastic drug .

9. Interaction of methotrexate with DHFR :

10. Pyrimethamine : It is a directly acting inhibitor of plasdmodial DHFRase . It gradually reduces the schizogony of malarial parasite in blood . It is slowly acting erythrocytic schizontocide . STRUCTURE: Moa: Interactions between DHFR and Pyrimethamine :

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