ALL RIGHT IT'S 2 PM.
I'LL GO AHEAD AND LAUNCH INTO
THE INTRODUCTION HERE, I'M ROSS
TALOHA OF NATIONAL INSTITUTE OF
DIABETES DIGESTIVE AND KIDNEY
DISEASES. AND I'D LIKE TO
WELCOME YOU ALL TO THE 2024
JOHN DALY LECTURE, WE'VE HELD
THIS ANNUAL LECTURE SINCE 2009,
A YEAR AFTER THE PASS OF JOHN DALY.
THE NIDDK, RESEARCH PROGRAM
IN THE LABORATORY OF BIOORGANIC
CHEMISTRY ARE PLEASED TO NOW
PRESENT THE 12TH EDITION OF THIS
AWARD TO DR' CAROL BOULEY. THIS
EVENT SERVES TO HONOR JOHN'S
MEMORY BY RECOGNIZING LEADING
RECEPTOR NATURAL PROUKT RESEARCH
SUCH A CAROLE ANN BEWLEY, WHO
TYPIFIES JOHN DALY'S SCIENTIFIC
IDEALS IN MORE WAY THAT ONE.
J OHN DALY WAS A PIONEER IN
NATURAL PRODUCTS AND
PHARMACOLOGICAL AND FRIEND,
DOMINI AND IDDK CHEMISTS. HE
CAME TO NIH AS AN ORGANIC
CHEMISTRY POSTDOC WORKING UNDER
BERNARD WITCRRP DRUING THIS
TIME. HE LEARNED PHARMACOLOGY
FROM THE N., I. M. H. NOBEL
LAUREATE JULIUS AXELROD. HE WAS
WIDELY KNOWN FOR HIS WORK ON
NATURAL PRODUCTS APPLIED TO
MODULATE CELLULAR PROCESSES. HE
INTRODUCED MANY ESSENTIAL TOOLS
TO PERFORM ECOLOGICAL RESEARCH.
SUCH AS SPORTS GLEN, WHICH WAS
USED IN MY VERY OWN LABORATORY
THIS WEEK, HE EXPLORED
INTRACELLULAR SIGNALING INDUCED
BY NATURAL SUBSTANCES FROM
PLANTS AND ANIMALS, WHICH HE
COLLECTED FROM JUNGLES IN S AND
CENTRAL AMERICA, MADAGASCAR AND
AUSTRALIA. THIS. YEERSZ
RECIPIENT, DR. CAROLE ANN
BEWLEY, IS WORLD LEADER IN
NATIONAL PRODUCTS AND CHEMICAL
BIOLOGY RESEARCH. SHE IS
CURRENTLY THE CHIEF OF THE
LABORATORY AT BIOORGANIC
CHEMISTRY WITHIN THE NIDDK
INTRAMURAL RESEARCH PROGRAM,
WHERE SHE HAS SERVED SINCE 2019.
NOTABLY, CAROL SERVES IN THIS
ROLE THAT WAS PREVIOUSLY
OCCUPIED BY JOHN DALY HIMSELF.
CAROL. ... COMPLETED HER
DOCTORAL STUDIES AT SCRIPPS
INSTITUTION OF OCEANOGRAPHY AT
UCSAN DIEGO1995 IN SUBSEQUENTLY
TRAINED IN NMR ANALYSIS OF
BIOMOLECULES USING NUCLEAR
MAGNETIC RESONANCE METHODS IN
THE LABORATORY OF METHODS IN
THE LABORATORY OF CHEMICAL
PHYSICS IN MR SECTION WITHIN
NIDDK SHE BEGAN AS A TENURE
TRACK INVESTIGATOR IN THE
LABORATORY OF BIOORGANIC
CHEMISTRY IN 1999 AND IN 2006
INITIATED HER ROLE AS SECTION
CHIEF OF THE NATIONAL PRODUCTS
CHEMISTRY LABORATORY, HER
INTERESTS INCLUDE THE
IDENTIFICATION CHARACTERIZATION
AND MECHANISTIC STUDIES OF
NATURAL PRODUCTS, AS WELL AS
STUDIES OF CARD HYDRATE BINDING
PROTEINS, CAROLE HAS MADE
IMMENSE, CONTRIBUTIONS TO THE
SCIENTIFIC COMMUNITY THROUGH HER
SERVICE ON JOURNAL EDITORIAL
BOARDS, REVIEW PANELS, BOTH
WITHIN THE NIH AND BEYOND WITHIN
SCIENTIFIC SOCIETIES DR. BEWLEY
HAS PUBLISHED ROUGHLY 75
RESEARCH ARTICLES COVERING
DIVERSE TOPICS AND NATURAL
PRODUCT CHEMISTRY ASSESSMENTS OF
PROTEIN STRUCTURE USING
BIOPHYSICAL METHODS AND STUDIES
OF HIV PROTEIN. CAROLE, HAS HAD
A PROFOUND IMPACT ON MAKING THE
SCIENTIFIC COMMUNITY A MORE
COLLEGIAL AND INTERESTING PLACE
ON A PERSONAL NOTE. I AM VERY
PROUD TO SHARE WORKPLACE WITH A
GOVERNMENT SCIENTIST, AS
DEDICATED AND THOUGHTFUL AND
DISTINGUISHED AS DR. BEWLEY,
CAROLE'S MADE AN IMMENSELY
POSITIVE IMPACT ON MY RESEARCH
PROGRAM, AND I'VE HEARD SIMILAR
STORIES FROM OTHER YOUNG,
INDEPENDENT SCIENTISTS. CAROL'S
LECTURE TODAY IS TITLED CHEMICAL
DIVERSITY FROM NATURE AND
ECOLOGICAL AND BIOLOGICAL
SYSTEMS. QUESTIONS CAN BE
SUBMITTED THROUGH THE SUBMIT
LIVE FEEDBACK OPTION ON THE
VIDEO CAST AFTER THE LECTURE IN
A QUESTION AND ANSWER PERIOD. WE
WILL HOLD A RECEPTION WITH
CAROLE OUTSIDE THIS HALL, IT IS
NOW ANY DISTINCT HONOR AND
PRESSURE TO INVITE DR. CAROLE
ANN BEWLEY TO RECEIVE THE PLAUE,
AND DELIVER THE 2024 JOHN DALY
LECTURE.
[APPLAUSE]
>> THANK YOU.
[APPLAUSE]
>> OKAY, THANKS EVERYONE FOR
COMING AND THANK YOU ROSS FOR
THE VERY NICE INTRODUCTION.
AND IT'S REALLY AN HONOR TO BE
ABLE TO GIVE THIS LECTURE IN
HONOR OF JOHN DALY.
I WAS LUCKY ENOUGH TO STILL
OVERLAP WITH HIM FOR SEVERAL
YEAR BEFORE HE PASSED AFTER I
JOINED THE LAB OF ORGANIC
CHEMISTRY.
TODAY I'M GOING TO TALK ABOUT
NATURAL PRODUCTS.
FOR THOSE WHO DON'T KNOW THIS
LECTURE SERIES ALTERNATES BACK
AND FORTH FROM YEAR TO YEAR ON
PHARMACOLOGY, AND THEN NATURAL
PRODUCTS.
SO I WAS SELECTED FOR NATURAL
PRODUCTS. SO IT'S A LITTLE BIT
OF AN UNCONVENTIONAL LECTURE FOR
ME. I DON'T GET TO TALK ABOUT
THESE AREAS MUCH, SO LOOKING
FORWARD TO THIS TALK.
OKAY, SO WHY NATURAL PRODUCTS?
THIS IS CHART TAKEN FROM PIE
CHART, THAT NEWMAN AND CRAIG
FROM THE NCI PUBLISHED SOME
YEARS AGO NOW, THEY CARRIED OUT
THESE DECADE REVIEWS ON THE
ROLE OF NATURAL PRODUCTS AND
DRUG DEVELOPMENT AND DRUG DIS
COVERRY, AND WE ALL KNOW NOW
THAT PHARMACEUTICALS ARE BIASED,
TOWARDS CONTAINING EITHER
NATURAL THEY COME EITHER FROM
NATURAL PRO DUCTS. THEY HAVE
NATURAL PRODUCT SCAFFOLDS. THEY
WERE THE SYNTHETIC COMPOUNDS
THAT WERE INSPIRED BY STRUCTURES
OF NATURAL PRODUCT, OR THEY
MIGHT CONTAIN PHARMACOPHORES
FROM NATURAL PRODUCTS. SO
DEPENDING ON HOW YOU TALLY THIS
OUR DRUGS TEND TO BE REPRESENTED
AROUND 65% BY NATURAL PRODUCTS.
AND HERE IS AN OVERVIEW OF... IS
THIS SHOWING UP?
OF ACTUAL DRUGS THAT WE USE IN
THE CLINIC, AND THERE IT GOES.
OKAY.
SO AND COMING FROM DIVERSE
SOURCES AND PLANTS AND FUNGI,
AND MARINE, INVERTEBRATES, CONE
SNAILS, ETCETERA.
SO JOHN ACTUALLY STARTED
WORKING ON NATURAL PRODUCTS
AFTER HE CAME TO THE NIH HE WAS
SYNTHETIC ORGANIC CHEMIST, AND
AS ROSS MENTIONED IN THE
INTRODUCTION, HE JOINED THE LAB
OF BERNHARD WITKOP, WHO WAS
WORKING ON TOXINS FROM FROGS.
AND JOHN ACTUALLY CAME TO THE
NIH TO WORK ON THIS COMPLEX
ALKALOID, KNOWN AS BITRAKATOXIN
IT'S ALKALOID THAT SOUTH
AMERICANS USED IN POISON BLOW
DARTS FOR HUNTING.
WHEN THEY RAN OUT OF MATERIAL,
JOHN WAS INVITED TO GO TO SOUTH
AMERICA TO TRY AND FIND COLLECT
MORE FROGS TO GET MORE MATERIAL.
SO THIS IS A LITTLE BIT OF AN
UNUSUAL FORAY INTO THE NATURAL
PRODUCTS.
AND I SORT OF TOOK A DIFFERENT
TURN, I ACTUALLY WORKED AT THE
SALK INSTITUTE AS POST BACK
AFTER UNDER GRADUATES AND I
LEARNED TO SCUBA DIVE, AND I'M
GOING TO TAKE YOU ON A LITTLE
BIT OF A... LET'S SEE.
UNDER WATER EXCURSION.
SO WHEN SCUBA DIVING ON CORAL
REEFS YOU CAN QUICKLY SEE HOW
MUCH DIVERSITY THERE IS IN THE
SPECIES AND THE DIFFERENT
ORGANISMS. SO HERE IS BARREL
SPONGE, AND SOFT CORALS WAVING
IN THE CURRENT, AND ALGAE ALL
OVER THE SUBSTRATE.
AND AS YOU ARE GOING TO SEE
RIGHT HERE.
LET ME SEE IF I CAN -- SOMETIMES
SEE SPONGES, THIS IS ACTUALLY
TWO DIFFERENT SPECIES OF
SPONGESES THAT ARE LIVING IN
CLOSE ASSOCIATION WITH ONE
ANOTHER, AND SEE THESE TYPES OF
MUTUALIST RELATIONSHIPS ALL OVER
THE CORAL REEFS, AND IF YOU
LOOK, SOMETIMES WE SEE SPONGES
WHOSE SURFACES ARE COMPLETELY
CLEAR, AND OTHERS THAT DARK RED
ONES COVERED WITH DIFFERENT TYPE
OF BACTERIA AND ALGAE, AND
BIOFILMS ETC.
AND ALSO WHEN SCUBA DIVING YOU
CAN QUICKLY SEE IF YOU GO ON
WALL OR REEF LIKE THIS,
LITERALLY EVERY CM OF THE REEF
IS COVERED WITH A LIVING
ORGANISM, SOMEHOW THESE ALL LIVE
TOGETHER, AND SUPPORT THIS WHOLE
CORAL REEF COMMUNITY.
SO IT'S THIS WHOLE COMMUNITY
THAT I BECAME CONSIDERED IN
BEFORE GOING TO GRAD SCHOOL.
LET'S SEE -- OOPS, I DON'T WANT
THAT.
OKAY.
AND SO, IF YOU... AS I MENTION
IF YOU LOOK AT A LOT OF
DIFFERENT MARINE SPONGES THAT
ARE SHOWN OVER ON THE RIGHT
SIDE, YOU CAN SEE THAT THESE
SURFACES AGAIN ARE COMPLETELY
UNFOULED, WHEN YOU TOUCH THEM
THEY ARE ACTUALLY SMOOTH TO THE
TOUCH, AND DON'T HAVE ANY KIND
OF BIOFILM SO IF YOU ARE A
CHEMIST, YOU THINK POSSIBLY
COULD BE CHEMISTRY INSIDE OF
THOSE ORGANISMS THAT ARE
CONTROLLING EITHER
ANTIBACTERIAL, ANTIFUNGAL, ETC
THIS BECAME MOTIVATION FOR ME TO
GRAD SCHOOL TORE DO MARINE
NATURAL PRODUCT CHEMISTRY.
FURTHER, IFFIC LOOK INSIDE
SPONGES, NOT ALL SPONGESES BUT
MANY THEY HAVE EXTREMELY COMPLEX
MICROBIOMES. AND SO THIS IS A
SPONGE CALLED ANALISOLI I WORKED
ON YEARS AGO, AND IF FI‘EEZE IT
AND CRACK IT OPEN, YOU SEE THAT
IT IS JUST PACKED WITH THESE
FILAMENTOUS BACTERIA. AND YEARS
AND YEARS AGO I DID SOME FIELD
WORK AND PULVERIZED THIS SPONGE
IN AN OLD SCHOOL OMEGA MIXER
THAT HAD A JUICER FILTER PAPER
LIKE A CENTRIFUGE AND TAKE THE
PULVERIZED TISSUE OUT TO DO SELL
SEPARATIONS, IN THE FIELD IN A
VERY PRIMITIVE CENTRIFUGE. AND I
WAS ACTUALLY ABLE TO MAKE
REALLY CLEAN PELLETS OF THESE
FILAMENTOUS BACTERIA. AND AT THE
TIME WE ALREADY SUSPECTED THAT
THE CHEMISTRY WAS COMING FROM
BACTERIAL SANDBION, NOT FROM THE
SPONGE, BECAUSE WE WERE
ISOLATING THE CYCLIC PEPTIDES
THAT HAD ALL OF THESE NON
PROTONACIOUS AMINO ACIDS. THEY
HAD D AMINO ACIDS , BETA, AMINO
GROUPS, AND HYDROXYLATIONS ,
AND AND UNUSUAL PLACES,
ETCETERA, THAT SORT OF HINTED TO
IT BEING BACTERIAL ORIGIN.
SO WE'RE VERY LUCKY HERE AT NIH
TO HAVE ACCESS TO THE NCI OPEN
REPOSITORY UP IN FEDERICK THAT
IS OPEN TO 10'S OF THOUSAND OF
DIFFERENT MARINE EXTRACTS AS
WELL AS PLANTS AND THAT
REPOSITORY OF COURSE HAS BEEN
MINED FOR YEARS, FOR ANTICANCER
COMPOUNDS AND JUST A FEW OF THEM
ARE THAT HAVE BEEN DISCOVERED
OVER THE YEARS ARE SHOWN HERE
AND YOU CAN SEE THE SPECIES AND
KINGDOM DIVERSITY IN THE PIE
CHART.
SO WE WANTED TO SIMPLY DO A
SCREEN FOR ANTIMICROBIAL
COMPOUNDS USING NCI OPEN
REPOSITORY, BECAUSE THAT HAD NOT
BEEN DONE, SO WE JUST PICKED
10,000 MARINE EXTRACTS AND A
POST-DOC AND A FORMER SUMMER
STUDENT CARRIED OUT THE SCR
SCREENAND WE SIMPLY TESTED
AGAINST GRAM-NEGATIVE AND
GRAM-POSITIVE BACTERIA.
AND GRAM POSITIVE HITS WERE VERY
HIGH, WE GOT ABOUT 500 HITS, AND
THAT'S NOT UNCOMMON, BUT
SURPRISINGLY, AGAINST ECOLI,
GRAMM NEGATIVE PATHOGEN WE ONLY
GOT ABOUT 30 WHICH IS A 0.3%
HIT RATE. SO THAT'S EXTREMELY
LOW SO WE WENT ON TO DO
ISOLATION STRUCTURE ELUCIDATION
ETCETER‘A ON ALL OF THE
COMPOUNDS THAT ALL THEIR
EXTRACTS THAT HAD COMPOUND THAT
IS WERE KILLING GRAM-NEGATIVE
BACTERIA.
AND FOUND, WITH THE EXCEPTION OF
ONE SPONGE, AND ALL THESE WERE
SPONGESES AS WELL, EVERY
COMPOUND WAS RICH IN AMINE
CONTAINING MOLECULES.
IN A COUPLE OF SLIDES, DON'T
WANT TO GO TO THIS PART OF OUR
RESEARCH, BUT JUST WANT TO GIVE
YOU ALL AN IMPRESSION OF WHAT'S
OUT THERE.
AND WHAT WE CAN LEARN FROM THE
MARINE NATURAL PRODUCTS.
SO I'M GOING TO TELL YOU THIS
QUICKLY ABOUT THE WORK THAT WE
DID ON THIS P-10.
SIMPLY AN AMID VERSION OF
BLASTOYTE AND S, AND ACCEPT
TROTR EMITOZO
ALKALOID THAT IS COMMON IN ONE
GROUP OF MARINE SPONGES. SO TO
GET AT THIS THIS AMID VERSION OF
PLASTIC CYDEN WAS ACTUALLY
ABOUT 20 TIMES MORE POTENT AND
ACTUAL
ACTUALLY.... GAVE US ACTIVITY
AGAINST GRAM NEGATIVE BACTERIA
WHEREAS THE OTHER ONE, THE THE
NATURAL PRODUCT DID NOT. AND SO
TO GET AT THIS KATHY H, IN THE
LAB RAISED RESISTANT MUTANTS AND
JUST BY YOU KNOW COMPETITION
WITH INCREASING AMOUNT OF
COMPOUND.
AND THEN JACK DAVEISON DID
COMPARATIVE GENOMICS ON THE
MUTANT AND IS WHAT WE
SURPRISINGLY FOUND IS THAT THE
MUTANTS WERE ACTUALLY DELETING
THE NOR-AGEING, NOR-A IS THE
TRANSPORTER THAT GIVES
RESISTANCE TO FLUOROQUINOLONE
ANTIBIOTICS. THIS IS IN STA
STAFFAUREUS BY THE WAY.
AND STRANGELY WHEN THE
STEPHORIOUS WERE SUBJECTED TO
THIS COMPOUND THEY WERE DELETING
THE EFFLEX PUMP, WHICH IS
ACTUALLY WHAT MAKES THEM
RESISTANT TO FLUOROQUINOLONES.
SO IN THE STUDY WE ACTUALLY
LEARNED THIS NOR-A TRANSPORTER
IS ACTUALLY AN IMPORTER FOR THIS
CLASS OF COMPOUNDS WHILE IT'S
AN EXPORTER FOR FLUOROQUINOLONES
AND THIS IS JUST ONE EXAMPLE OF
MECHANICNISTIC STUDIES WE DONE
IN OUR LAB.
WE'VE ALSO BEEN VERY INTERESTED
IN THIS GROUP OF POUNDS, SEPTRON
IS A WELL KNOWN EXAMPLE.
THIS IS WORK DONE IN LAB BY
TORI KLEIN A CURRENT POST-DOC
AND PREVIOUS WORK WE DONE IN
COLLABORATION WITH CHRISTIAN
MELANDERS LAB, WE SHOWED THAT
WHEN DRUG RESISTANT BACTERIA ARE
SUBJECTED TO SEPTRIN AND EXAMPLE
METHICILLIN THE PRESENCE OF
SEPTRIN WILL ACTUALLY RESTORE
ANTIBODY BIOTIC SUSCEPTIBILITY
TO THE DRUG THAT THE BACTERIA
ARE RESISTANT TO. SO WE'VE BEEN
VERY INTERESTED IN TRYING TO
FIGURE OUT THE MECHANISM FOR HOW
THIS COULD TAKE PLACE.
SO TORI'S DONE AN AMAZING JOB
WITH THIS PROJECT.
PRIOR TO HER ARRIVAL WE DID A
CHEMICAL GENETIC SCREEN WITH
JEFF PIETROWSKI AND UNIVERSITY
OF WISCONSIN AND FOUND THAT THE
RESISTANT MUTANTS TO SCEPTRON
WERE USUALLY DEFICIENT IN
SOMETHINGTO DO WITH CELL WALL
BIOSYNTHESIS, OR MEMBRANE,
PERMEABILITY, AND SO TORY'S
DONE A LOT OF MICROSCOPY AND
T.TMETC, IF YOU WANT TO LEARN
MORE ABOUT THIS STORY, AND HOW
SCETPRON IS WORKING SHE'S
SPEAKING AT L2L IN JANINE 2025.
MORE ECOLOGICAL AREA OF FOCUS,
WHEN WE ARE OUT DIVING WE LOOK
FOR OUTLIERS, OR ORGANISMS OR
THINGS THAT LOOK UNUSUAL, OR
SHOULDN'T BE THERE.
THIS IS WHAT HAPPENED WHEN WE
FOUND THIS ALGAE.
THIS IS IN US VIRGIN ISLANDS IN
ROUND BAY. SO IT'S AT THE EAST
END OF THE ISLAND.
AND JUST SNORKELING, AND FOUND
MOUNDS AND MOUNDS OF THIS ALGAE.
THIS IS UNUSUAL, JUST A CORAL
REBEL SUBSTRATE, AND HAS VERY
HIGH CURRENT, VERY UNUSUAL FOR
SOMETHING TO COME AND GROW TO
THIS DENSITY ON THAT KIND OF
SUBSTRATE.
HMM... I HAVE A TIMER.
OKAY.
SO WE WERE INTERESTED IN THIS
ORGANISM.
IT'S NOT FORWARDING...
SORRY ABOUT THIS.
THANK YOU.
OKAY, SO INTERESTED IN THE
CHEMISTRY IN THIS ORGANISM.
AND SO WE COLLECTED SOME, AND WE
ENDED UP ISOLATING THESE
MOLECULES, SO THESE ARE HIGHLY
UNUSUAL, A NEW SCAFFOLD THAT HAS
NOT BEEN SEEN BEFORE, OR SINCE.
SO I DON'T SEEM TO HAVE A
POINTER HERE.
BUT, LET ME JUST SEE.
THAT KIND OF WORKS.
WANT TO POINT OUT A COUPLE
THINGS HERE, THIS MOLECULE IS
HIGHLY STRAINED AND THERE IS
THIS ETHER RIGHT HERE, THAT'S
ACTUALLY IN AN ORTHO POSITION
THAT MAKES THE MOLECULE STRAINED
AND ACTUALLY FOLDS INTO A BASKET
SHAPE, AND ALSO HAS THIS
2-CHLORU-BUTIN GROUP THAT
ABSOLUTELY ESSENTIAL FOR
ANTIBIOTIC ACTIVITY. SO WE ALSO
WERE LUCKY IN THAT WE ISOLATED,
I THINK EIGHT OR NINE DIFFERENT
ANALOGS OF THIS CHRYSOPHANTINE
FAMILY. SO THIS WAS THE MOST
ABUNDANT AND THE MOST ACTIVE. WE
ALSO ISOLATED LINEAR SO NON
MACROCYCLIC COMPOUNDS AND ALSO
ONES WHERE THIS STRAINED ETHER
WAS NOT PRESENT, IT WAS ACTUALLY
IN THE METAPOSITION.
SO ANYONE ONE OF THOSE CHANGES,
THE ACTIVITY, THE ANTI MICROBIAL
ACTIVITY GOES WAY DOWN.
SO... IN WORK WE WERE DOING
MECHANICNISTICLY TO FIGURE OUT
HOW THE ANTIBIOTICS MIGHT WORK,
WE NOTICED MICROSCOPICALLY,
THAT THESE COMPOUNDS WHERE
BACTERIA AND THEY WERE ALSO
CAUSING BALLOONING OF CELLS, SO
THAT'S A HINT THEY COULD BE
DISRUPTING MECHANISMS OF CELL
DIVISION, SO WITH WE LOOKED AT
VARIOUS COMPOUNDS THAT INHIBITED
THE CYTOSKELETAL PROTEIN FIT-C,
AND SO COMPOUNDS HERE AND
HERE'S JUST A X-RAY CRYSTAL
STRUCTURE OF FITZY. FIT-C, IS
ACTUALLY PRECEDED. IT'S A
TUBULIN HOMOLOG BUT SINCE IT'S
BACTERIAL IT ACTUALLY PRECEDED
TUBULIN. SO IT'S ALSO A G T P
ACE AND WHEN G T P BINDS IT
ACTUALLY INDUCES HEAD TO TAIL
POLYMERIZATION AND
PROTOFILAMENT FORMATION. AND SO
WE WERE CURIOUS IF THEY WERE
ACTUALLY FIT-C INHIBITORS.
AND SO IN A SERIES OF
DIFFERENT EXPERIMENTS, WE
SHOWED THAT INDEED IF WE MAKE
PROTOFILAMENTS AND ADD OUR
COMPOUND IT DISCORRUPTS PROTOPY
FILAMENT FORMATION. WE CAN
INHIBIT GTPAS ACTIVITY AND
REALLY. ... INTERESTINGLY, WE
CARRIED OUT MOLECULAR DOCKING OF
THIS. IS THE THREE DIMENSIONAL
STRUCTURE SHOWN IN WHITE AND
BLUE HERE. ACTUALLY JUST WHITE,
THE BLUE IS GDP AND BINDING SITE
IN THE BACK, AND CARRY OUT
SOMETHING CALLED SATURATION
TRANSFER DIFFERENCE NMR.
SO WHAT WE'RE DOING IN THIS
EXPERIMENT WE TAKE THE FIT-C
PROTEIN IN NMR TUBE AND GDP
BOUND TO IT IN GDP BINDING SITE
AND TIE TRAIT IN THE
CRESTOPHANTN AND FIND IT BINDS
AND DISPLACE GDP IN THE BINDING
SIGH, AND CAN ACTUALLY QUANTIFY
THE DISTANCES, AND QUANTITY THE
EFFECT, AND THAT'S DIRECTLY
PROPORTIONAL TO THE DISTANCES OF
ANY GIVEN PROTON THAT WE'RE
OBSERVING FROM THE PROTEIN, AND
THAT'S JUST SHOWN HERE IN THE
MAGNITUDE OF THE LITTLE GRAY
BALLS. AND IF WE WERE TO LOOK
ON THE MAP THAT OUT ON TO THE
MODEL IT FITS PERFECTLY WITH
AREAS WHERE THERE IS NO SGD,
SOLVENT EXPOSED AND THOSE WITH
LARGEST ONE ARE BOUND DEEP IN
THE BINDING SITE.
SO JESSICA KEFFER POST A FORMER
GRADUATE STUDENT IN THE LAB.
WANTED TO SEE WHETHER THIS COULD
ACTUALLY WORK INSIDE OF LIVE
BACTERIA, THERE ARE MANY TIMES
WHERE NATURAL PRODUCT MIGHT
EXERT EFFECTS IN AN ENZYME
ASSAY OR IN VITRO. BUT THEN THAT
DOESN'T TRANSFER TO BACTERIA OR
MAMMALIAN CELLS WHAT HAVE YOU.
SO AN PERMEABLE E COLI CARRYING
FITSY Y. F. P. AND YOU CAN JUST
SEE HERE WHEN SHE ADDS NATURAL
KRISOPLRANTINE A, OR EVEN THE
SYNTHETIC HEMICRISOPHANTINE THAT
WE GOT WITH A COLLABORATIONWITH
PETER WIFF AT UNIVERSITY OF
PITTSBURGH, BOTH OF THOSE
COMPOUNDS DISRUPT THE Z-RING
FORMATION IN BACTERIA IN LIVE
CELLS.
THIS WAS ESTABLISHED THAT IT IS
INDEED AN ACTUAL FIT-C INHIBIT
TORE.
WE WANTED TO GET MORE OF THIS
COMPOUND TO CARRY OUT ADDITIONAL
STUDIES.
IDEALLY ANIMAL STUDIES BUT WE
WERE OUT OF COMPOUND, WHEN WE
WENT TO BACK TO COLLECT THE
ALGAE IT WASN'T THERE, SAID
THIS HAPPENS A LOT WITH NATURAL
PRODUCTS. AND SO WE STARTED TO
LOOK IN THE LITERATURE, THIS
CTAYLORI THIS STRANGE ALGAE HAD
BEEN OBSERVED WORLDWIDE, BUT
VERY SPOTTY, AND VERY
SPORADICALLY.
IN THE MORE POPULAR LITERATURE
I CAME ACROSSHARMFUL ALGAE NEWSH
THIS TITLE. WHAT IN THE WORLD IS
C I CAME ACROSS I CAME ACROSS T
DOING
DOING SARDINIA AND THE TURIAN S
DOING IN MEDITERRANEAN AND YOU
CAN SEE THAT THE THE HUGE
BLOOMS THAT IT'S MAKING BOTH YOU
KNOW UNDERWATER THIS IS -- LET
ME SEE, UNDERWATER EXACTLY LIKE
I HAD SEEN.
AND ALSO SHOULD SAY, WHEN THIS
ALGAE STARTS TO GROW AND GAIN
MASS, IT STARTS TO RESPIRATE AND
MAKES BUBBLES OF AIR, TRAPPED IN
THE M-SHEATHE THAT IT EXUDES AND
WHAT HAPPENS THE AIR PRESSURE IS
ENOUGH TO MAKE IT ACTUALLY RISE
FROM THE BOTTOM OF THE SEA TO
THE TOP JUST LIKE A BALLOON, AND
WHAT WAS HAPPENING IN THE
MEDITERRANEAN WAS GETTING THESE
HUGE MASSES OF IT FLOATING UP,
AND ALSO CAME ACROSS THIS...
THESE SLIDES, THAT WHERE THIS
WAS HAPPENING OFF THE COAST OF
SARDINIA AND BECOMING A HUGE
PROBLEM FOR TOURISM AS YOU COULD
IMAGINE IF YOU GET IN THE WATER
AND SEE THIS.
SO THEY'VE BEEN TRYING TO CLEAN
THIS UP, AND WE HAVE
COLLABORATION TO MONITOR THESE
ALGAE OUTBREAKS WITH RESEARCHER
IN ITALY.
BECAUSE WE WERE UNABLE TO GET
MORE MATERIAL, JACK DAVISON
FORMER POST-DOC IN THE LAB
WANTED TO TAKE ON THE RISKY
CHALLENGE OF ACTUALLY TRYING TO
CULTURE THIS ALGAE IN THE LAB,
AND A SUCCESSFUL IN DOING THIS
AND ACTUALLY DID WHOLE GENOME
SEQUENCING A TRANSCRIPTOMICS
WITH THE GOAL OF TRYING TO
FIGURE OUT IF WE COULD
UNDERSTAND HOW THESE MOLECULES
WERE BEING BIOSYNTHESIZED AND
TO PRODUCE MORE IN VITRO IN THE
LAB AND SO JACK WAS ABLE TO SHOW
IN THE LAB THE ALGAE ACTUALLY
DID MAKE DIFFERENT
HEMICRESTOPHANTINE ANALOGS, AND
NOW BROMINATED INSTEAD OF
CHLORINATED AND HE COULD GET THE
LINEAR MOLECULE BUT WE WERE
NEVER ABLE TO GET THE
CRISOPHANTINE A. THAT WE HAD
WANTED TO SO WORK THAT'S
CONTINUES TO THIS DAY BUT WHAT
WAS REALLY INTERESTING IS THAT
WHEN JACK DID THE WHOLE GENOME
SEQUENCING HE'S ESSENTIALLY
DOING META GENOMICS BECAUSE
THERE ARE A LOT OF BACTERIA THAT
COME WITH THE ALGAE COLLECTED IN
THE FIELD, SO HE HAD THE CTALORI
HE GOT FROM A JAPANESE CULTURE
COLLECTION THAT IS AXENIC BE
IT'S NOT, AND SOME THINGS WON'T
GROW XENOGLY, AND THEN WE ALSO
HE ALSO DID THE SAME SEQUENCING
ON THE FIELD COLLECTED AND WHAT
HE COULD SEE INTERESTINGLY IS
THAT THERE IS ASTABILITY
STABLE MICROBIOME ASSOCIATED
WITH CTALORI AND WE HAVEN'T
REALLY FOLLOWED UP WHETHER THOSE
ASSOCIATED BACTERIA MIGHT HAVE
FIT-C HOMOLOGS THAT ARE
RESISTANT TO THESE KINDS OF
COMPOUNDS ARE NOT SO ANYWAY THAT
IS WORK FOR THE FUTURE. SO IF WE
COME TO THE MORE MODERN AREA
NATURAL PRODUCT RESEARCH, AND
LIKE EVERY FIELD GENOMIC IS
PLAYING A HUGE ROLE, NATURAL
PRODUCTS ARE AMAZING, IN MY
OPINION, SO IN EVERY ASPECT, BUT
ALSO IF YOU LOOK AT THE BACTERIA
THAT PRODUCE MOST OF THE NATURAL
PRODUCTS WE'RE FAMILIAR WITH.
SO WOULD BE ANTIBIOTICS AND DO
WHOLE GENOME SEQUENCES, THIS IS
A STRAIN FROM OUR LAB.
EVERY ONE OF THESE COLORED
RECTANGULAR BARS IS BIOSYNTHETIC
GENE CLUSTER THAT ENCODES FOR
A NATURAL PRODUCT. SO SOMETIMES
THESE CAN ENCODE UP UP TO 30 OR
EVEN 40, DIFFERENT COMPOUNDS IN
A SINGLE GENOME. AND WHAT'S
AMAZING IS THAT BACTERIA HAVE
ACTUALLY ACQUIRED OR EVOLVED
THESE BIOSYNTHETIC GENE
CLUSTERS IN A CLUSTERED WAY. SO
THE ENZYMES APPEAR CONSECUTIVELY
IN A CHAIN ON THE DNA OF THE
ORGANISM
SO THE WHOLE PROMISE OF
GENOMICS IN NATURAL PRODUCTS HAS
FEATURED THIS SCHEME, AND SO
INSTEAD OF GOING OUT AND
COLLECTING SOMETHING AND
ISOLATING AND WORKING BACKWARDS
WE WOULD LIKE TO WORK FORWARD TO
TAKE AN ORGANISM AND HAVE IT'S
GENOME AND LOOK AT BIOSYNTHETIC
GENE CLUSTER OF INTEREST, THEN
GO EXAMINE THE ENZYMATIC
ASSEMBLY LINES WHICH I'VE SHOWN
HERE. SO THIS IS THE
BIOSYNTHETIC GENE CLRRSTER JUST
SHOWED IN GENES. SOME OF THESE
GENES ARE HUGE, AND MIGHT
CONTAIN UPWARDS 10 OR 12 OR EVEN
20 DIFFERENT ENZYMES, AND THOSE
ENZYMES WORK AS A CHEMICAL
ASSEMBLY LINE DEPICTED HERE,
AND THEN WE WOULD GO TO A
STRUCTURE BECAUSE SOMETIMES
BIOINFORMATICALLY WE KNOW ENOUGH
ABOUT THESE ENZYMES FUNCTION
THAT WE CAN EVEN PREDICT STEREO
CHEMISTRY OF ANY GIVEN CARBON,
CHIRAL CARBON.
SO THIS WHOLE PATHWAY HERE, IS
JUST FROM A PROJECT IN OUR LAB
THAT WHERE WE DISCOVERED THESE
POTENT ANTIFUNGAL COMPOUNDS,
CALLED THE ARIDOMISENSE AND I'M
NOT GOING TO TALK ABOUT THOSE
TODAY, BUT THAT WAS SORT OF THE
PROMISE OF NATURAL PRODUCT
DISCOVERY IN THE GENOMICERA.
UNFORTUNATELY IT DOESN'T REALLY
WORK LIKE THAT.
BUT IT'S A NICE DIAGRAM.
SOME YEARS AGO.
WE WANTED TO TAKE ADVANTAGE OF
POWER OF GENOMICS, AND MYSELF
TOGETHER WITH CLIFF BARRY AND
NIAID AND EUGENEK AND WERE
AWARDED ONE OF TRANS-NIH
INNOVATOR AWARDS, AND SO WHAT
WE HAD PROPOSED TO DO WAS TO TO
LOOK FOR ANTI MYCOBACTERALS
ANTI TB DRUGS AND CLIFF HAD THE
ECOLOGICAL IDEA THAT SINCE
MYCOBACTERIA ORIGINATED ITS
THOUGHT AT SORT OF AEROBIC
ANAEROBIC INTERFACES.
AND IF YOU LOOK DEEP INTO CORES
IN BOGS FOR EXAMPLE, AND SO THEY
WENT AND ISOLATED BACTERIA FROM
THOSE BOG CORES FROM THAT
INTERFACE.
AND DID THE STANDARD CULTURE,
AND BACTERIAL ISOLATES, AND
EXTRACTING AND TESTING AGAINST
MYCOBACTERIUM TUBERCULOSIS AND
THEY FOUND THAT THESE THIS
UNUSUAL STRAIN AS A GRAM
NEGATIVE TRAIN, AND THEY
ISOLATED CHRYSOBACTERIA, AND
WERE SHOWING ANTI TB ACTIVITY.
SO WE WERE OUR OUR ROLE WAS
GOING TO BE TO LOOK AT THE
CHEMISTRY AND TRY TO FIGURE OUT
WHAT COMPOUNDS MIGHT BE
PRESENTSO WITH A WE FOUND WITH
THE BACTERIA IS THEY ALL
CONTAINED BIOSYNTHETIC GENE
CLUSTERS THAT ENCODED FOR THESE
KNOWN MOLECULES, CALLED
MICROVIRIDENS, SO MICROVIRIDENS
ARE HISTORICALLY KNOWN FROM TO
BE PRODUCED BY CYANOBACTERIA
SHOWN HERE, AND THIS IS GENOME
OF ANABINA SPECIES, AND AND SO
THESE GRASPITIDES ARE KNOWN AS
RIBOSOMALY ENCODED
POST-RANSLATIONALLY MODIFIED
PEPTIDES OR RIPS.
AND RIPS ARE QUITE AMAZING,
BECAUSE THEY ARE ACTUALLY, THEY
START WITH A PEPTIDE PRECURSOR
SYNTHESIZED BY THE RIBOSOME, SO
THAT'S USUALLY THE A-GENE IN
BIOSYNTHETIC GENE CLUSTER, AND
THAT VERY SMALL BGC WILL HAVE
TAILORING ENZYMES, SO OTHER
ENZYMES THAT ARE GOING TO CARRY
OUT CHEMISTRY ON THAT RIBOSOMALY
ENCODED PEPTIDE.
FOR THE GRASPITIDE FAMILY THEY
ARE KNOWN FOR CONTAINING TWO
ATPGRASPLIGASES, A HUGE SUPER
FAMILY, AND BIND ATP AND CARRY
OUT CHEMICAL REACTIONS OFTEN
LIGATIONS.
AND THESE BGC'S, USUALLY HAVE
SOME KIND OF PROTEASE IN THEM,
TO THE BGC TO CLEAVE THE PRODUCT
AND THEN A TRANSPORTER TO
SHUTTLE IT OUT OF THE CELL
SO WHAT THIS LOOKS LIKE FOR THE
CYANO BACTERIA IS SHOWN ON THE
TYPE LINE HERE AND HERE.
SO I JUST SPLIT UP THE WHOLE
PRECURSOR PEPTIDE SEQUENCE.
SO YOU GET LEADER, PEPTIDE, AND
THEN A CORE.
FOR THE CYANOBACTERIA THERE IS
SIMPLE CORE RIGHT HERE, ACTUALLY
ENCODING FOR THE NATURAL PRODUCT
RIGHT HERE, TRICYCLIC SO
CONTAINS TWO OMEBGA LACTONES
AND ONE OMEGA LACTAM AND THEN.
... WHAT WE SAW IN THE
CHRYSOBACTERIA THAT HAD ACTIVITY
AGAINST MTB THIS VERY, VERY LONG
CORE, THAT PEOPLE HAVE NEVER
SEEN BEFORE IN A GRASPITIDE TYPE
GENE CLUSTER, SO WE LOOK AT BGC
WE AGAIN SEE THE GRASLIBASES AND
STRANGELY WE DON'T SEE ANY
TRANSPORTERS OR PROTEASES.
SO WE SET OUT TO FIGURE OUT WHAT
THE PRODUCTS MIGHT BE, AND JUST
SO YOU KNOW THESE A
ATP-GRASPLIGASES ARE MAKING AN
LACTONE BOND BETWEEN SIR AND
THRIANINIE NRRCLEOPHILE THE
HYDROXYL IS A NUCLEOPHILE OR
ASPARTATE OR GLUTAMATE AS THE
ELECTROPHILE THAT HAS BEEN
ACTIVATED BY A PHOSPHATE WITH
THE ATP GRASP-LIGASE.
SO JENNY IN OUR LAB, SOLVED ALL
THESE QUESTIONS, BY MAKING
SEVERAL DIFFERENT PEPTIDE
CONSTRUCTS THESE WOULD ALL
CONTAIN THE LEADER AND DIFFERENT
VERSION OF THE CORE.
AND HAVING INTRODUCING TEDPOTIER
CLEAVAGE SITES, IN DIFFERENT
AREAS ALONG THE PEPTIDE, AND WE
GOT VERY LUCKY WITH THIS.
HAPPEN TO BE IN GOOD PLACES.
SO WHEN WE DID MSMS
ECONSEQUENCES WE ARE WE WERE
ABLE TO ELUCIDATE WHERE THOSE
MACROCYCLES WERE FORMING. SO
WHEN WE WOULD INCUBATE THAT
PRECURSOR PEPTIDE WITH AN ATP
GRASP GLYCASE. IN THIS CASE THE
CDNC GENE, WHAT WE WOULD SEE IS
LOSS OF 6 WATER MOLECULES, SO
KNOW 6 DEHYDRATIONS HAD TO HAVE
BEEN TAKING PLACE AND THAT SIX
MACROLACTONES WERE PROBABLY
BEING FORMED, AND THROUGH MSMS
SEQUENCING OF EACH ONE OF THESE
PRODUCTS. WE COULD ACTUALLY
FIGURE OUT THIS WAS THE PRODUCT
BGC WAS PRODUCING.
SO THIS WAS UNUSUAL, THIS HAS
NOT BEEN SEEN BEFORE IN ANY
NATURAL PRODUCTS.
IT'S A LITTLE BIT STRANGE.
AND I CAN JUST SAY, AND WORK
I... WON'T TALK ABOUT, IF WE GO
ON AND FORM THE THIRD RING,
PROTEAE, INHIBITION, GOES DOWN,
THEY CONTINUE TO GET
INCREASINGLY MORE POTENT.
SO, TO UNDERSTAND THIS, SO HOW
HOW MIGHT THIS ATPGRALIGASE, A
SINGING ATPGRASLIGE IN THE
BIOSYNTHETIC GENE CLUSTER YET
WE CAN GET THIS REALLY COMPLEX
CHEMISTRY HAPPENING ON SINGLE
RIBOSOMAL PEPTIDE. SO TO FIGURE
THIS OUT JENNY SOLVED THE
CRYSTAL STRUCTURE OF THE CDNC
THAT ATP GRASP LIKE ACE IN THE
PRESENCE OF ADP AND WHAT I DID
NOT MENTION EARLIER IS ENCODE IN
PRECURSOR PEPTIDE, SEQUENCE
LEADER PART THE N TERMINAL PART
OF THE PRECURSOR PEPTIDE.
THERE'S A CONSERVED LEADER
SEQUENCE THAT'S COMMON TO THIS
WHOLE GRASPIDE FAMILY AND THIS
WAS THE FIRST STRUCTURE WHERE WE
HAD LEADER-BOUND, AS WELL AS ADP
AND THE PEPTIDE SUBSTRATE THAT'S
GOING TO BECOME THAT CORE.
AND SO WE WERE LUCKY IN THAT THE
STRUCTURE THAT SHE SOLVED WAS
ACTUALLY ASYMMETRIC HOMODIMER SO
MOLECULE WAS INACTIVE AND SO IT
WAS JUST OPEN WITH THE LEADER
BOUND AND A DP. BUT NO SUBSTRATE
BOUND, AND OTHER MOLECULE,
ACTUALLY CORE BOUND, WHERE THE
CHEMISTRY, THE MACROLUCTUM
CYCLIZATIONS ARE TAKING PLACE
AND THOSE ARE JUST SHOWN HERE
AND HERE RESPECTIVELY.
SO THIS ENZYME UNDER GOES A HUGE
CONFIRMATIONAL CHANGE, WHEN THAT
CORE PEPTIDE, THE SUBSTRATE IS
BOUND, THAT SORT OF LOOP AND
HELICAL REGION ACTUALLY TRAVELS
A FULL 90 DEGREES, ACROSS TO
GRAB THE SUBSTRATE.
AND... THE CHANGE IS SO DRAMATIC
THE SECONDARY STRUCTURAL
ELEMENTS IN THAT REGION,
ACTUALLY CHANGE AS WELL.
AS SHOWN DOWN HERE.
SO THIS IS JUST A CLOSE UP OF
THE ACTIVE SITE, AND WHAT'S VERY
NICE, AS WELL, IS THAT THE
RESOLUTION WAS GREAT IN THIS
CRYSTAL STRUCTURE, WE COULD
ACTUALLY SEE THE N-TERMINAL
REGION OF THAT CORE, THE 3-ANE,
TOWARDS US, AND C-TERMINUS WHICH
IS DIRECTED BACK INTO THE
SLIDES.
AND THE LEADER, WHICH WOULD BE
UP HERE WAS BOUND AS WELL, SO WE
COULD SEE, ORIENTATION OF HOW
THAT ENTIRE PRECURSOR PEPTIDE
WOULD BE BOUND TO THE
ATBGRASPLYGASE.
AND SO WHAT WE ALSO LEARNED
FROM THIS AND ALSO MUCH MANY
MRRTAGENESIS EXPERIMENTS THAT I
WON'T TALK ABOUT IS THAT THIS
ONE RESIDUE ARGININE 123 MAKES
THESE TWO HYDROGEN BONDS TO THIS
CONSERVED ASPARTATE AND
GLUTAMATE. AND SO THIS ASPARTATE
IS GOINGTO BECOME ONE OF THE
MACROLACTONE BONDS IF YOU LOOK
AT ARGININE OVER... SEE IF I CAN
FIND IT.
HERE, JUST A SEQUENCE ALIGNMENT
OF THE CRYSLER BACTERIA VERSUS
CYANOBACTERIA, ARGININE ONLY
OCCURS IN THE CARCIO BACTERIA
AND IT'S REPLACED BY AN ACIDIC
RESIDUE IN THE CYANOBACTERIA. SO
JENNY'S DONE ALL OF THE SWAPPING
YOU MIGHT IMAGINE, AND THE
SINGLE ARGININE IS RESPONSIBLE
FOR THE SPECIES SPECIFICITY AND
THE MACRO LACTONE FORMATION IN
THE CHRYSOBACTERIA. SO IT'S SORT
OF ACTUALLY. ... ACTING AS AN
ANCHOR FOR THE SUBSTRATES TO
BIND.
SO WE HAVE THIS MODEL NOW, IN
THE KINETIC EXPERIMENTS I WON'T
SHOW YOU, WE HAVE THIS MODEL
WHERE THE ARGININE AND SUBSTRATE
COMES IN, THE ARGININE SORT OF
ANCHORS THROUGH THOSE TWO ACIDIC
RESID
RESIDUESAND ATP BINDS, ATP AND
ENZYME CLOSE AND IS MAKES IT
MACROCYCLE OPENS MOVES ETCETERA
AND JUST GOES DOWN THE LINE
INTHIS ITERATIVE PROCESS.
AND BELIEVE THIS OR SHOULD SAY
KNOW THIS TO BE TRUE.
IN KINETIC STUDIES WE'VE SHOWN
YOU NEVER GET AN MACROCYCLE ON
THE C TERMINAL REGION OF THE
PEPTIDE BEING FORMED BEFORE THE
N TERMINAL. SO IT'S ABSOLUTELY
ITERATIVE AND JUST GOES DOWN THE
CHAIN.
SO, A LONG TERM GOAL IN OUR LAB
IS TO SOLVE STRUCTURES AND TO
UNDERSTAND AND DEFINE THIS
ENTIRE PATH WAY.
IT'S STILL EARLY DAYS, ACTUALLY
IN THIS AREA, IN NATURAL
PRODUCTS, SO WE DON'T KNOW... WE
DON'T HAVE A STRUCTURAL PICTURE
OF ANY BIOSYNTHETIC GENE CLUSTER
WORKING THROUGH THE ENTIRE PATH
WAY, AND THAT'S WHAT WE'RE
WORKING ON NOW.
SO JENNY WAS ABLE TO SOLVE THE
CRYSTAL STRUCTURE OF THE SECOND
ATBGRASLIGSE THAT INSTALLS THAT
MACRO LACTAM AND MAKES WHAT
WE'LL CALL THE MATURE , OF IN
THE CORE, AND JUST RECENTLY,
WE'RE LOOKING FOR THE PROTEASE
TO ACTUALLY MAKE THE PREASSUMED
PROTEAES THAT SHOW CLEAVE,
CONSERVED GG MOTIF. AND THAT'S
VERY COMMON IN A CROSS BIOLOGY.
IT'S NOT JUST IN THESE
BIOSYNTHETIC GENE CLUSTERS, BUT
THERE IS NO PROTEASE IN THIS
GENE CLUSTER. SO WE'VE BEEN VERY
INTERESTED IN TRYING TO FIGURE
OUT HOW THIS THIS FINAL PRODUCT
MIGHT BE CLEAVED, EXPORTED FROM
THE CELL TO GIVE THE MATURE
PRODUCT AND THAT'S JUST WORK ON
GOING, IN THE MEANTIME, JENNY
SOLVED THE STRUCTURE OF THIS
ALPHA BETA HYDROLASE WHICH IS
HIGHLY UNUSUAL. THOSE NEVER
OCCUR IN THE CYANOBACTER‘IAL
BIOSYNTHETIC GENE CLUSTERS ONLY
IN THESE CARCIO BACTERIA. AND
WHAT SHE FOUND IS RATHER THAN
CLEAVING THIS GGMOTIF IT'S
ACTUALLY CLEAVING THIS CONSERVED
LEADER, AND VERY INTERESTING
BECAUSE THAT THAT LEADER IS
CENTRAL TO ACTIVATE THE
ATPGRASPLIGASE SO IT THERE'S
SOME REGULATION GOING ON, WITH
THIS ACTIVITY IN THIS
BIOSYNTHETIC GENE CLUSTER. AND
THAT'S, THAT WORK THAT'S GOING
ON IN THE LAB.
SO COMING BACK TO JOHN DALY'S
CAREER AND INTEREST, I THINK
PEOPLE PROBABLY KNOW, AT LEAST
THE OLDER PEOPLE KNOW, THAT JOHN
WAS A GREAT EXPLORER.
I ALREADY TOLD YOU HE STARTED
HIS FORAY IN NATURAL PRODUCTS,
GOING TO THE JUNGLES AND
COLLECTING THESE DENDROBATED
FROGS. AND HE KEPT THE FI‘OGS.
HE BROUGHT THEM BACK TO THE LAB,
NIH AND BUILDING EIGHT. HIS
OFFICE HAD A CHEMICAL FUME HOOD
IN IT.
WASN'T REALLY EVEN AN OFFICE,
AND HAD THIS TERRARIA IN HIS
OFFICE WHERE HE KEPT THE FROGS,
AND FIND OVER THE YEARS, THE
FROGS WOULD LOSE THEIR POISONS
IN THE SKIN, SO ALWAYS SUSPECTED
THAT AT LEAST FOR SOME FROGS,
CLEARLY ARE ABLE TO DE NOVO
SYNTHESIZE THESE ALKALOIDS. BUT
OTHERS ARE PROBABLY GETTING
THEM FROM THEIR DIET. AND
THERE'S EXAMPLES OF THAT THROUGH
OUT BIOLOGY AND ECOLOGY.
IN THE LATER YEARS OF HIS
CAREER, HE WAS VERY FIXATED ON
WHAT COULD BE THE SOURCES OF
THESE TOXINS, AND HE ACTUALLY
WAS GOING BACK TO THE RAINFOREST
WHERE HE WOULD FIND THE HIGHEST
NUMBER OF FROGS, AND WOULD CRAWL
ON THE GROUND, AND BRINGS TARPS
AND VIALS AND WOULD LITERALLY
COLLECT EVERY SINGLE ANT OR
ARTHROPOD, AND WHATEVER INSECT
HE COULD FIND, AND BRING BACK TO
THE LAB AT THAT TIME, THE
MASSSPECTROMETERS WERE NOT SO
SENSITIVE, YOU COULD RUN A
SAMPLE ON THAT SMALL OF A RUN OF
MASSSPEC ON THAT SMALL OF
SAMPLE, SO HE WAS DOING WHAT
CALL, COMBINATORIAL
BIOPROSPECTING. SO HE WAS
ACTUALLY COMBINING DOZENS OR
HUNDREDS OF LITTLE INSECTS AND
RUNNING MASS SPEC ON THEM, AND
LOOKING FOR ALKALOIDS, HE SUE
EXPECTED MIGHT BE PRESENT BASED
ON WHAT FROGS LIVE THERE.
AND WHAT ALKALOIDS HE ALREADY
DISCOVERED FROM THOSE FROGS.
KIND OF REMARKABLE TO THINK
BACK, THAT HE WAS THINKING ALONG
THOSE LINES, BECAUSE TODAY, THE
MASS SPECTROMETRS WOULD BE
SENSITIVE ENOUGH, PROBABLY TO
TELL THE ALKALOID IN A SINGLE
INSECT.
AND WHAT MANY NATURAL PRODUCTS
PEOPLE DO TODAY, IS NONE OF THIS
ECOLOGY OR COLLECTING ETC,
CULTURALING TAKES PLACE, IT'S
ALL DONE ON THE COMPUTER THROUGH
BIOINFORMATICS AND GENOME
MINING, SO KIND OF LOSING THAT
CONNECTION WITH NATURE.
SO TODAY IF SOMEONE WERE
TACKLING THIS PROBLEM, I SUSPECT
THEY TAKE ALL THE ORGANISM AND
IS DO GENOME SEQUENCING, AND
SHOTGUN SEQUENCES ON THEM SXO
HOPE THEY COULD MAYBE FIND SOME
KIND OF CHEMICAL CUES IN THE
ENZYMES THAT ARE ENCODED IN THE
GENOMES, I WILL SAY PLANTS AND
INSECTS ARE THE NO EASY, THEY
DON'T GIVE THE WHAT BACTERIA
DO. BUT IT'S REALLY FUN TO THINK
BACK ON HOW WHAT JOHN WOULD BE
DOING TODAY, IF YOU WERE STILL
DOING RESEARCH. SO WITH THAT, I
JUST WANT TO THANK THIS IS NOT
ALL THE COMPREHENSIVE SLIDE, OF
CURRENT, BUT MENTIONED PEOPLE
OR HOW THEIR NAMES ON SLIDES
ALONG THE WAY AND I'VE HAD A
GREAT GROUP OF STUDENTS AND
POST-DOCS ALL THROUGH MY CAREER
HERE, AND UNDERGRADS AND THEN
THANKS SOME OF MY COLLABORATORS
THAT AYE MENTIONED AND DURING
THE TALK AND LONG TIME SUPPORT,
AND LBC, AND BENEFICIARIES
ACROSS NIH, FUNDING PERMITS
THAT WHERE PARK SERVICES LET US
GO ON DO THIS WORK IN THEIR
WATERS, AND THEIR LANDS AND
FUNDING OF COURSE, AND I OF
COURSE HAVE TO THANK NIDDK FOR
SUPPORTING NATURAL PRODUCT AND
IS CHEMISTRY IN A VERY BIG WAY
FOR ALL THESE DECADES AND ALL OF
YOU FOR YOUR ATTENTION.
[APPLAUSE]
>> ALL RIGHT, ANY QUESTIONS
FROM THE AUDIENCE FOR CAROLE?
I'LL START OUT WITH ONE.
THE SOURCES YOU ISOLATE THESE
NATURAL PRODUCTS FROM ARE SO
COOL.
AND WHEN I LOOK AT VIDEO YOU
SHOW IN THE PICTURES IT JUST
SEEMS SO OVERWHELMING TO ME, DO
YOU HAVE A SENSE OF WHETHER,
LIKE, IS THERE A WAY TO
PRIORITIZE WHERE YOU THINK
UNEXPECTED NATURAL PRODUCTS
MIGHT COME FROM.
YOU MENTIONED THINGS THAT
SHOULDN'T BE THERE.
DO YOU HAVE THOUGHTS ON THAT?
>> M-HM, I HAVE A LOT OF
THOUGHTS.
[LAUGHTER] >> THAT'S SORT OF
HOW WE STARTED WHEN I STARTED MY
LAB.
SO WE PRETTY MUCH FOCUSED ON 2
THINGS, ONE COLLECT ORGANISMS --
IN THE CASE OF THE ALGAE, I
NEVER SEEN ANY ALGAE THAT LOOKED
LIKE THAT YEARS AND YEARS OF
DIVING, AND SPONGES WE CAN LOOK
FOR THINGS WITH SMOOTH SURFACES
THAT ARE LOOKED AT LIKE THEY'RE
CHEMICALLY DEFENDED. SO WHY IS
THIS SPONGE RIGHT NEXT TO IT
COMPLETELY BIOFELT AND IT'S NOT
I WILL SAY THAT BECAUSE I HAD
DONE SO MUCH WORK WITH SYMBIOSIS
AND I KNEW WHAT SPECIES ARE EVEN
AT THE LEVEL OF ORDER OF SPONGES
CONTAINED HUGE POPULATIONS OF
BACTERIAL SYMBIOTES. THAT'S WHAT
WE STAR GETTED EARLY ON IN ANY
LAB.
>> VERY COOL.
>> CONGRATULATIONS, CAROLE, SO
WELL DESERVED, AND I KNOW THAT
JOHN WOULD HAVE BEEN GLOWING TO
WATCH YOUR LECTURE.
I'M WONDERING ABOUT THE
TRANSLATIONAL ROLE OF NATURAL
PRODUCTS.
SO FOR A WHILE, THE
PHARMACEUTICAL INDUSTRY WAS VERY
EXCITED ABOUT NATURAL PRODUCT
AND THEN WANED.
IS IT COMING BACK, AND DO YOU
HAVE ANY THOUGHT ABOUT HOW TO
ENHANCE THE ROLE OF NATURAL
PRODUCTS IN DRUG DISCOVERY?
>> YEAH, WHAT KEN IS REFERRING
TO, ALL OF THE DRUGS THAT ARE
ACTUALLY, WELL, MOST, THAT CAME
FROM NATURAL PRODUCTS, MOST OF
THEM CAME FROM THE
PHARMACEUTICAL INDUSTRY, SO
PHARMA -- EVERY SINGLE LARGE
PHARMA COMPANY USED TO HAVE VERY
LARGE NATURAL PRODUCTS DIVISION,
AND HAVE COLLECTIONS AND
MICROBIAL COLLECTIONS, SOME OF
THE WORK WE DID EARLIER WOULD BE
THROUGH THESE COOPERATIV GRANTS
BETWEEN NIH AND EXTRAMURAL
RESEARCHERS AND PHARMA COMPANY
HAD TO BE INVOLVED TO DO THE
SCREENING.
THAT WAS THE OLD DAYS, AND THEN
IT BECOMES VERY EXPENSIVE.
AND THE OTHER PROBLEM IS YOU
START TO REISOLATE COMPOUND THAT
ARE ARE ALREADY KNOWN, AND OF
COURSE PHARMA DOESN'T WANT TO
DO.
WHAT'S HAPPENED IN THE LAST
DECADE MAYBE 7 OR 8 YEARS,
PHARMA HAS BEEN DONATING THEIR
COLLECTIONS TO ACADEMIC LABS OR
CENTERS, AND TRYING TO MAKE
THOSE AVAILABLE, SIMILAR TO HOW
THE NCI OPEN REPOSITORY DOES.
AND THERE'S REALLY BEEN A MOVE
TO I ALLUDED TO, TO DO SHOTGUN
SEQUENCING ON SAY
50,000MICROBIAL STRAINS AND TRY
TO ASSEMBLE THOSE AND LOOK FOR
NEW MOLECULES, SO THEY ARE OFTEN
REFERRED TO AS ORPHAN GENE
STRUCTURES OR CRYPITIC
COMPOUNDS, AND IDENTIFY THOSE,
AND USE SYNTHETIC BIOLOGY TO
MAKE THEM IN THE LAB.
ONE THING I DID NOT MENTION IN
MY TALK IS THAT THE REASON THAT
WE CAN DO THAT... SIMILAR TO THE
GRASPATIDES IF WE WANTED TO LOOK
FOR MORE MORE GRASPATIDES IN
FACT THAT'S WHAT WE DID WITH THE
KRRNIN LAB IS YOU CAN DO A
BIOINFORMATIC SEARCH ON THAT
PRECURSOR PEPTIDE AND AN ATP,
GLASPITIDE, THE COULD OCCURRENCE
OF THOSE TWO THINGS, IF GENOME
AND IF YOU GET A HIT WITH A
BIOSYNTHETIC GENE CLUSTER YOU
CAN GO AFTER THAT EXACTLY WHAT
WE'VE BEEN DOING FOR THAT
FAMILY, SO LIKEWISE YOU CAN DO
IT FOR OTHER CLASSES AS WELL AND
THAT'S THE FORMS THAT TECHNOLOGY
THAT MANY STARTUPS ARE USING, SO
WOULD SAY IT'S GOING FROM PHARMA
TO STARTUPS NOW. YEAH. AND
THERE ARE SOME THAT ARE ARE
HAVING GREAT SUCCESS WITH THAT.
YEAH. SO JACK DAVISON THE
FORMER POSTDOC IN MY LAB GOT
SWALLOWED UP IN A HEARTBEAT ONCE
LIFEMINE PHARMACEUTICALS STARTED
UP, AND THEY WERE GOING TO WORK
ON FUNGIA AND ALGAE AND JACK HAD
THAT EXPERIENCE AND HE WAS GONE.
>> HI, CAROLE, THAT WAS
BEAUTIFUL, I HAD A QUESTION
ABOUT THE STRUCTURE ELUCIDATION
OF NATURAL PRODUCTS AND PLEASE
EDUCATE ME THERE.
UNLESS PUREFIED TO CERTAIN
EXTENT STRUCTURAL ELUCIDATION BY
NMR OR SOME SORT OF THINGS LIKE
THAT NOT SO STRAIGHTFORWARD, DO
YOU THINK ANALYTIC CHEMICAL
WOULD GET TO A STAGE WHERE WE
WOULD BE ABLE TO LECDATE
STRUCTURES OF NATURAL PRODUCTS
IN MORE COMPLEX MIXTURE WITHOUT
NECESSARILY HAVING TO THEM TO
ALMOST HOMOGENEITY. I'M JUST
CURIOUS TO KNOW YOUR THOUGHT ON
THAT
>> SO ONE THING I DID MENTION,
I WANTED TO MENTION TOO, THE
POSTDOCS SPENT HUNDREDS OF HOURS
SLOWLY SITTING IN THE MORE
COMPLEX POUND, AND NEVER GOES ON
TO A SLIDE.
THERE IS START TO GO BE ENOUGH
NMR LIBRARIES OUT THERE, SMALL
MOLECULE ONES, THAT YOU CAN
START TO DO SOME MATCHING,
>> SO ARE THERE AI-BASED
METHODS BEING DEVELOPED.
>> YEAH, PEOPLE ARE TRYING TO
DO THAT.
>> I SEE, FASCINATING.
THANK YOU.
>> I WAS GOING TO ASK, THIS
QUESTION, RELATED, BUT, THEN
ASKED.
THE COMPLEX FORMATION OF WHAT
YOU SAW A AND B UNITS COMBINING,
USUALLY, IT IS YOU MUST CHANGE
THE CONFIRMATION OF AN A UNIT OR
B UNIT SO THEY CAN INTERACT WITH
SMALL MOLECULES.
AND I THINK YOU NOTICE THERE
WAS ATP BINDING, AND THAT IS
IMPORTANT TO KNOW, IF IT WAS THE
CONFIRMATION CHANGE.
AND THE TWO WERE BINDING
TOGETHER, IS THERE INFORMATION
ABOUT THAT?
>> I THINK WHAT YOU ARE ASKING
IS WHETHER WE SEE PART OF... IF
THE ADP BINDING OR ATP BINDING
IS WHAT IS INDUCING THE LARGE
CONFIRMATION CHANGE.
>> YES.
>> SO THAT'S TRUE, SMELL, BUT
DEPENDS ON THE ATPLIGASE.
SOME YOU GET QUITE A DRAMATIC
CHANGE, SIMPLY BY COMBINING ATP,
AND IN FACT IN THE
CYANOBACTERIAL ENZYME THERE'S
ONE CRYSTAL STRUCTURE OF THE
ASSIGN A BACTERIAL ENZYME BUT IT
DOESN'T HAVE A T P I THINK THEY
SIMPLY... THEY DON'T UNDERGO
HUGE CONFIRMATIONAL CHANGE JUST
BINDING ATP, AND EVEN BINDING
THE LEADER THEY STILL HAVE
THAT... WHATEVER PALM OF THE
LIGASE THERE.
IT'S NOT UNTIL THE CORE, WHERE
THE CHEMISTRY TAKE PLACE, DOES
THE ENZYME CLOSE.
SO I THINK QUITE UNUSUAL, FOR
THIS FAMILY OF ENZYMES, AT LEAST
WHAT OUR CRYSTAL STRUCTURES TELL
US.
>> LIKE THE SYNTHESIS IS A
VERY BASIC EXAMPLE OF A
CONFIRMATION CHANGE INDUCED BY
SUBSTRATE SO THAT THEY'RE
BINDING OF AN OTHER .
>> CAROLE, GREAT TALK, THE
SECOND STORY WHEN YOU WENT BACK
AND REISOLATED AND COULDN'T FIND
THE PRODUCT.
DO YOU HAVE A SENSE IF THAT'S...
WERE MISSING THE BACTERIA THAT
HAD THE CYCLIZATION OR MULTIPLE
BACTERIA ARE REQUIRED TO MAKE
THE STRUCTURE, OR IS THAT
ANSWERABLE QUESTION,
>> IN THE ALGAE, SO I THINK
JACK AND I WOULD AGREE, WE THINK
THE ALGAE IS MAKING IT.
WE DON'T THINK THE BACTERIA
MAKING IT.
>> WELL IT COULD BE BACTERIAL
SYRNBIOTE THAT'S PROVIDING
SOMETHING YEAH WE JUST DON'T
KNOW ENOUGH ABOUT IT.
>> ALL RIGHT.
IF THERE ARE NO MORE QUESTIONS,
JOIN ME IN THANKING CAROLE ONCE
AGAIN FOR A FANTASTIC DAILY
LECTURE.
[APPLAUSE]
FANTASTIC DALY LECTURE.
WE'LL HAVE RECEPTION OUTSIDE IN
THE SEMINAR HALL, SO PLEASE JOIN
US, THANK YOU!