Project Grant R21AI182823

NONCOMPETITIVE INHIBITION OF CYP121 DIMERS FROM MYCOBACTERIUM TUBERCULOSIS - PROJECT SUMMARY AND ABSTRACT TUBERCULOSIS (TB) CONTINUES AS A GLOBAL PANDEMIC AND REMAINS THE LEADING CAUSE OF DEATH BY INFECTION WORLDWIDE. THE RISE OF MULTI-DRUG RESISTANT FORMS OF MYCOBACTERIUM TUBERCULOSIS (MTB) HIGHLIGHTS THE NEED TO DEVELOP NOVEL ANTI-TB THERAPEUTICS. A PROMISING DRUG TARGET IS THE ESSENTIAL ENZYME CYTOCHROME P450 21A1 (CYP121) OF MTB, WHICH CATALYZES A PHENOL-COUPLING REACTION TO CONVERT DICYCLOTYROSINE (CYY) INTO THE CYCLICAL COMPOUND MYCOCYCLOSIN. SINCE ATTEMPTS TO DELETE THE CYP121 GENE RESULT IN NON-VIABLE BACTERIA, VARIOUS DRUG DESIGN EFFORTS HAVE FOCUSED ON THE DEVELOPMENT OF COMPETITIVE ACTIVE SITE INHIBITORS OF THE ENZYME. CYP INHIBITION TYPICALLY INVOLVES THE USE OF A NITROGEN HETEROCYCLE THAT FORMS A COORDINATE COVALENT BOND WITH THE HEME IRON. HOWEVER, GIVEN THE HIGH DEGREE OF STRUCTURAL HOMOLOGY WITHIN THE CYP SUPERFAMILY, THEORETICAL COMPETITIVE INHIBITION OF CYP121 CARRIES SIGNIFICANT RISKS DUE TO THE POTENTIAL FOR NON-SPECIFIC INHIBITION OF DRUG-METABOLIZING CYPS LIKE CYP3A4. PATIENTS UNDERGOING TREATMENT FOR TB ARE SUBJECTED TO A RIGOROUS REGIMENT OF MEDICATIONS LIKE RIFAMPICIN AND ISONIAZID, AMONG OTHERS, SOME OF WHICH ARE ALREADY COMPETITIVE INHIBITORS OF DRUG METABOLIZING CYPS. IN THE CURRENT PROPOSAL, THE DEVELOPMENT OF NON-COMPETITIVE INHIBITION OF CYP121 IS PROPOSED. THE ENZYME DISPLAYS THE UNUSUAL FEATURE OF RELYING ON A DISTAL-TO-DISTAL HOMODIMER INTERFACE FOR ITS FUNCTION. NOTABLY, THIS PARTICULAR INTERFACE DOES NOT OCCUR IN MAMMALIAN CYPS. SPECIFIC AIM 1 CENTERS ON THE USE OF A CLEAVABLE FUSION OF THE CYP121 HOMODIMER IN COMBINATION WITH ENZYME KINETICS, 19F-NMR, AND LOSS OF FUNCTION MUTATIONS TO TEST THE HYPOTHESIS OF MONOMER-TO-MONOMER CROSS-TALK ACROSS THE DIMER INTERFACE. IN AIM 2, VIRTUAL DRUG SCREENING WILL BE USED TO DEVELOP POTENTIAL PHARMACOPHORES LOCATED NEAR THE DIMER INTERFACE. THE SHORT-TERM GOAL OF THIS WORK IS TO LAY THE FOUNDATION FOR A ROBUST INVESTIGATION OF CANDIDATE SMALL-MOLECULE BINDING SITES ON THE PROTEIN SURFACE THAT INHIBIT WITHOUT BINDING THE ACTIVE SITE; THE LONG-TERM GOAL IS TO DEVELOP NOVEL CLASSES OF NON-COMPETITIVE CYP121 INHIBITORS THAT AVOID THE POTENTIAL FOR DRUG-DRUG INTERACTIONS IN PATIENTS BEING TREATED FOR TB.