Project Grant R43AR082722

METAGENOMIC DISCOVERY AND OPTIMIZATION OF NOVEL ENDOLYSINS TARGETING CUTIBACTERIUM ACNES TO TREAT ACNE VULGARIS - PROJECT SUMMARY ACNE VULGARIS (ACNE) AFFECTS UP TO 50 MILLION PEOPLE IN THE U.S. ANNUALLY AND CAN HAVE SIGNIFICANT NEGATIVE CONSEQUENCES ON PSYCHOSOCIAL FUNCTIONING INCLUDING HIGHER RATES OF ANXIETY, LOW SELF-ESTEEM, DEPRESSION, AND SUICIDAL IDEATION. CUTIBACTERIUM ACNES PLAYS A CENTRAL ROLE IN ACNE PATHOGENESIS AND IT IS NOW UNDERSTOOD THAT A DECREASE IN C. ACNES PHYLOTYPE DIVERSITY AND AN INCREASE IN THE HOMOGENEITY OF THE PATHOGENIC PHYLOTYPE IA1 TRIGGERS INNATE IMMUNE STIMULATION AND ACNE PROGRESSION. TOPICAL AND ORAL ANTIBIOTICS TO TARGET C. ACNES REMAIN PART OF FIRST-LINE TREATMENTS FOR ACNE, BUT CONTINUED USE OF ANTIBIOTICS POSES SIGNIFICANT CHALLENGES INCLUDING EXACERBATION OF ANTIBIOTIC RESISTANCE AS WELL AS COLLATERAL DAMAGE TO THE HEALTHY COMMENSALS IN THE GUT AND SKIN MICROBIOMES. GIVEN THESE DRAWBACKS, NOVEL ANTIMICROBIAL AGENTS THAT CAN PROVIDE ALTERNATIVES TO ANTIBIOTICS AND SELECTIVELY TARGET C. ACNES WITHOUT DAMAGING BENEFICIAL BACTERIA ARE NEEDED. ENDOLYSINS ARE PHAGE-ENCODED ENZYMES THAT CAN DEGRADE BACTERIAL CELL WALLS. EXOGENOUSLY ADDED ENDOLYSINS CAN QUICKLY LYSE THEIR TARGET BACTERIA AND BECAUSE THEY BIND VERY SPECIFIC EPITOPES IN TARGET CELL WALLS, THEY CAN HAVE LYTIC SPECIFICITY DOWN TO A SINGLE SPECIES OR EVEN SUB-SPECIES. GIVEN THESE PROPERTIES, ENDOLYSINS HOLD ENORMOUS POTENTIAL AS HIGH-SPECIFICITY SKIN MICROBIOME MODULATORS. HOWEVER, THE DIVERSITY OF ENDOLYSINS KNOWN TO TARGET C. ACNES IS LOW AND THESE ENZYMES SUFFER FROM LOW ACTIVITY AND LOW SOLUBILITY. RECENT METAGENOMIC ANALYSES DEMONSTRATE THAT CUTIBACTERIUM SP. ARE FOUND UBIQUITOUSLY IN THE SOIL AND THAT THESE ENVIRONMENTS CAN PROVIDE A RICH, UNTAPPED SOURCE OF CUTIBACTERIUM DIVERSITY. AT TOPAZ BIOSCIENCES, WE HAVE DEVELOPED A PROPRIETARY METAGENOMIC PLATFORM FOR THE DISCOVERY AND OPTIMIZATION OF ENDOLYSINS. WE HAVE PREVIOUSLY LEVERAGED THIS PLATFORM TO DEVELOP ENDOLYSINS AGAINST STAPHYLOCOCCUS AUREUS THAT ARE MORE ACTIVE, MORE THERMOSTABLE, AND HAVE A BROADER PH RANGE THAN BENCHMARK ENZYMES. IN THIS PHASE I PROPOSAL, WE WILL LEVERAGE THIS PLATFORM TO EXPAND THE DIVERSITY OF ENDOLYSINS KNOWN TO HAVE ACTIVITY AGAINST C. ACNES AND THEN EXPLOIT THIS DIVERSITY TO DEVELOP CHIMERIC ENZYMES WITH IMPROVED PROPERTIES. TO ACCOMPLISH THIS, WE WILL TAKE ADVANTAGE OF THE MODULARITY OF ENDOLYSINS TO BUILD A LIBRARY OF ENDOLYSIN "PARTS" - ENZYMATIC DOMAINS (EADS) AND CELL WALL BINDING DOMAINS (CBDS) THAT WE WILL SYSTEMICALLY CHARACTERIZE FOR ANTI-CUTIBACTERIUM ACTIVITY, THERMOSTABILITY, AND PH TOLERANCE. AN INITIAL SET OF "PARTS" WILL COME FROM SEQUENCED CUTIBACTERIUM GENOMES AND PHAGES. THEN, TO SIGNIFICANTLY EXPAND THE DIVERSITY OF EADS AND CBDS, WE WILL COMPUTATIONALLY SELECT ~200 DIVERSE ENDOLYSINS/DOMAINS PREDICTED TO HAVE ANTI-CUTIBACTERIUM ACTIVITY FROM PROPRIETARY AND PUBLIC METAGENOMIC DATABASES FOR ADDITIONAL CHARACTERIZATION. FINALLY, WE WILL LEVERAGE SEQUENCE-FUNCTION INSIGHTS GAINED FROM OUR "PARTS" DEVELOPMENT TO DESIGN CHIMERIC ENDOLYSINS COMPOSED OF EADS AND CBDS WITH THE MOST PROMISING PROPERTIES TO GENERATE ENZYME CANDIDATES THAT DEMONSTRATE IMPROVED ACTIVITY, SOLUBILITY, THERMOSTABILITY, AND PH RANGE.