Studies On New Proluciferin Substrates For Human CYP4 Family Enzymes And Prodrugs For CYP4Z1

The cytochromes P450 comprise a family of enzymes that are integral to metabolism.In general,CYP families 1,2 and 3 include major xenobioticmetabolizing enzymes,which play a major role in pharmacogenomic risk,while CYP4 enzymes are mainly involved in the metabolism of fatty acids and other endogenous substances.CYP4 enzymes,such as the promising anti-breast cancer target CYP4Z1,have roles in human metabolism yet their functions remain largely uncharacterized.Limited knowledge of their physiological substrates and activities hinders their deorphanization and prevents a complete understanding of their cellular roles.It is,therefore,of particular interest to elucidate structure/activity profiles for CYP4 family enzymes in an effort to better understand the structural requirements for their metabolic activities.CYP4Z1 is capable of catalyzing oxidative cleavages of ethers,a reaction distinct from its previously known fatty acid hydroxylase activity.Since CYP4Z1 is also overexpressed in human breast cancer,we reasoned that it might be possible to design and synthesize promising,ether-containing prodrug molecules that would only be activated by the catalytic activity of CYP4Z1.This study represents our first step toward this aim.This thesis describes the synthesis and characterization of seven new proluciferins and their subsequent reactions with a panel of CYP4 enzymes.Biotransformation of these probe substrates was monitored using each of the human CYP4 family members.Eight out of twelve CYP4 enzymes were able to act on at least one of the substrates,whereas four of the enzymes(CYP4A22,CYP4B1,CYP4F3,and CYP4F22)did not convert any of the new substrates.For all substrates,activity of CYP4Z1 was always highest,while that of CYP4F8 was always second highest,and no other CYP4 enzyme was able to metabolize all eight substrates.These data suggest that for substrates with a different general structure,the catalytic properties of these two enzymes might also resemble each other.Moreover,addition of a single fluorine to the phenyl moiety of Luciferin-BE had pronounced consequences on its metabolism by human CYP4 enzymes.Site of metabolism(So M)predictions involving SMARTCyp and docking experiments helped rationalize activity trends linked to substrate accessibility and reactivity.We further report the first homology model of CYP4F8,with predicted substrate recognition residues in a catalytically competent conformation accessed by REST simulations.This model predicts that the catalytic domain of CYP4F8 is narrower than that of CYP4Z1.Thus,with respect to a CYP4Z1-dependent prodrug strategy for the treatment of breast cancer,unwanted activation of candidate compounds by other CYP4 enzymes,especially CYP4F8 must be taken into consideration.Collectively,the findings described herein will inform future efforts to design prodrug therapeutic agents that are efficiently activated by the metabolism of CYP4Z1.