Rapid Discovery of Potent Inhibitors of the Main Protease of SARS-CoV-2

Transcript

• 00:00On behalf of my Co leader,
• 00:02Barbara Burtness and I,
• 00:03I'm pleased to introduce Bill Jorgensen,
• 00:06one of our developmental therapeutic program
• 00:08members and also my long term collaborator.
• 00:11Bill is a graduate of Princeton and Harvard,
• 00:14spent 15 years on the faculty at Purdue,
• 00:17and in 1990 he moved to Yale,
• 00:19where he's currently Sterling
• 00:21Professor in the Chemistry department.
• 00:24Bill is internationally recognized
• 00:25as one of the world leaders in
• 00:28computational chemistry and drug design.
• 00:30His research has been recognized
• 00:32by many honors,
• 00:34and among among those include the American
• 00:36Chemical Society Cope Scholar Award,
• 00:39the ACS Award for computers.
• 00:42Chemical and pharmaceutical research,
• 00:44the ACS Hildebrand Award,
• 00:47the ISTQB P award in computational biology,
• 00:51the Sato International Award from
• 00:54the Pharmaceutical Society of Japan.
• 00:56He's been elected to membership
• 00:58in the International Academy
• 01:00of Quantum Molecular Science,
• 01:02American Academy of Arts and Sciences,
• 01:04and the US National Academy of Sciences.
• 01:07Another recent honor in
• 01:092020 includes one of the 16.
• 01:11Researchers selected for a Nobel Laureate
• 01:15Citation for individuals considered
• 01:18doing Nobel Nobel Class Research
• 01:20that has been cited over 2000 times.
• 01:23Today he's going to tell you a little bit
• 01:26about some of his work on SARS COVID 2.
• 01:29So without further ado,
• 01:31Bill,
• 01:31take it away.
• 01:33Yeah. Well, thank you very much
• 01:35Karen and the pleasure to be here
• 01:37the other side of the campus.
• 01:39So the our work I'll tell you about today
• 01:42is totally a collaboration between my
• 01:45research group and chemistry and Karen's
• 01:47group over here in the the Med school.
• 01:50So I'll talk a little bit in general about
• 01:53computer aided drug discovery and then
• 01:56specifically about our work and finding
• 01:59very potent protease inhibitors for SARS.
• 02:03Move two. So a key element of drug design
• 02:08is the fact of trying to make inhibitors
• 02:12that bind to an enzyme typically.
• 02:15So and we'll be talking about again a small
• 02:19molecule binding to SARS Cove 2 protease.
• 02:22And so this is governed by an equilibrium
• 02:24where you have the protein and water,
• 02:26the inhibitor and water,
• 02:28there's a free energy of
• 02:31binding and then the complex.
• 02:33So the free energy of binding the G because
• 02:36we're working in the constant pressure,
• 02:39constant temperature world is for Gibbs.
• 02:42So it's a Gibbs free energy and I put the
• 02:46stamp of our former colleague Jay Willard.
• 02:50Gibbs here is the father of.
• 02:53Thermodynamics.
• 02:53So the free energy binding just to introduce
• 02:58the concept of a nanomolar inhibitor.
• 03:01So the free energy of binding is given
• 03:04by minus RTL and the dissociation
• 03:06constant if you have a dissociation
• 03:09constant of 10 to the minus nine molar.
• 03:12That would correspond to A1 nanomolar.
• 03:16Inhibitor or an inhibitor that has that KD,
• 03:22one that has a KD of 10 to the
• 03:24minus six would be a micromolar
• 03:26inhibitor and our binder.
• 03:28And the reason I bring this up
• 03:32is that most drugs turn out to be
• 03:36typically one to let's say 20 or
• 03:39so nanomolar in a binding assay.
• 03:42And this all ultimately has to do with
• 03:45the farm human pharmacology and just
• 03:47how big a pill one is willing to take.
• 03:50So this obsession with nanomolar
• 03:53inhibitors just to, you know,
• 03:56reflects this fact,
• 03:58so.
• 03:58Ultimately here we're going to have to
• 04:01do simulations on computer simulations
• 04:04of proteins binding to ligands in water.
• 04:07And so how did this arise?
• 04:09When did it with such things happen?
• 04:12And the answer is,
• 04:13there really wasn't any significant
• 04:16work on doing computer simulations of
• 04:18molecular fluids before the late 1970s.
• 04:21And then of course it grew slowly after that.
• 04:25The problem is you have a lot of particles,
• 04:27you're using classical force.
• 04:29Those describe the interactions,
• 04:31but there's still a lot of particles
• 04:34and you have to.
• 04:35Observe the system over a significant
• 04:38time period.
• 04:39So if you're doing molecular dynamics,
• 04:41we might want to run the molecular dynamics
• 04:44for picosecond hundreds of picoseconds,
• 04:47nanoseconds and this just we didn't
• 04:50have the computer resources to do that.
• 04:53And then making it more complicated
• 04:56by putting a protein into it and
• 04:59describing the energetics of the
• 05:01protein and the water.
• 05:03That really didn't happen until mid.
• 05:061980s and my colleague here,
• 05:08Julian Torrado Rivas and I
• 05:10published one of the first
• 05:13calculations for a protein in water
• 05:16where we did molecular dynamics for
• 05:19100 picoseconds and that was in 1988.
• 05:22So doing the type of calculations
• 05:25we're talking about today is
• 05:28relatively recent phenomenon.
• 05:30This is a picture we'll talk
• 05:32about HIV reverse transcriptase
• 05:33and just to get the sense,
• 05:35I usually give this to less.
• 05:36Sophisticated audiences
• 05:37to point out the yellow,
• 05:39little yellow pieces inhibitor
• 05:41and that's enough to shut down
• 05:44this enzyme and this is an example
• 05:47of one of the compounds that
• 05:49developed through Karens and in
• 05:52our work that is a inhibitor of
• 05:55HIV RT that little molecule.
• 05:57So here's the way we do it.
• 06:00We normally start with an X-ray
• 06:02structure and the first phase of
• 06:05this we're looking for micromolar
• 06:07hit compounds that then we have to
• 06:09do a lot of hard work on to bring
• 06:12them to the low nanomolar level.
• 06:15So we normally start with an X-ray
• 06:17structure and this can be from
• 06:19you know somebody else's work and
• 06:20we remove the ligand that might
• 06:23be in that X-ray structure and
• 06:25then we try to design our new
• 06:27our own inhibitors and that this.
• 06:30Started out we do a virtual
• 06:33screening which is docking.
• 06:36And I'll tell you a little
• 06:37bit more about that,
• 06:38where we literally fly molecules
• 06:40into the protein structure and
• 06:42see which ones look the best.
• 06:45Or are we do denovo design where we
• 06:47use a growing program that I wrote
• 06:50a while back that starts with the
• 06:53little seed core of a molecule of,
• 06:56say benzene you place in the binding site.
• 06:59And then the program will build libraries
• 07:02of compounds starting from that core,
• 07:04growing them out in the binding site.
• 07:06And then you have to.
• 07:08A score of them,
• 07:09evaluate them in the same manner
• 07:12that this invariably gives us
• 07:14these micromolar hit compounds.
• 07:16But we've never been fortunate enough
• 07:18to do this initial part of the work
• 07:21and end up with nanomolar inhibitors.
• 07:23We're close, you know,
• 07:26single digit micromolar.
• 07:27So then the hard part is the lead
• 07:30optimization because we're going to
• 07:32have to refine the micromolar hits by
• 07:35making small changes that we decide.
• 07:38What to do?
• 07:39By a lot of structure building
• 07:41and energy minimizations.
• 07:43So this bond program of mining can
• 07:45rapidly build protein ligand complexes.
• 07:48We can energy minimize them.
• 07:50That's just a fast calculation
• 07:52compared to adding the water
• 07:54doing the molecular anamax.
• 07:56And so we do a lot of the structure building,
• 07:58energy minimization and then for
• 08:02select cases we will do, excuse me,
• 08:07the free energy calculations
• 08:09that are sort of our hallmark.
• 08:12We call them FEP,
• 08:14free energy perturbation calculations.
• 08:16Virtually all pharmaceutical
• 08:17companies today are,
• 08:19you know,
• 08:20jumped on this.
• 08:21Everybody's doing FP calculations
• 08:24for a drug design.
• 08:26So then you have to make a decision on
• 08:29what molecules to synthesize their assay.
• 08:32So you need somebody like Karen to help out
• 08:35in the assaying and the crystallography,
• 08:39the crystallography isn't.
• 08:41Critical,
• 08:42but it sure is helpful if you know very
• 08:45much helps reinforce what
• 08:47the modeling is doing.
• 08:49And also sometimes you'll see that
• 08:51the there the there's a change in
• 08:53the protein structure from what you
• 08:55originally started with that you
• 08:57will see in the crystallography.
• 08:59You don't necessarily see
• 09:00it in the computation.
• 09:01So the crystallography is really helpful.
• 09:06When the HIV area,
• 09:08Karen and I got along for quite a few
• 09:11years without a crystal structures,
• 09:13but then once we certainly
• 09:15current lab start getting them,
• 09:16it certainly made life a lot more confident.
• 09:20So you repeat the cycle until
• 09:23you get the potency you want.
• 09:26All the while we are mindful of properties,
• 09:29so we want the compounds to be drug like.
• 09:33And that requires a having things
• 09:36like reasonable solubility,
• 09:37reasonable cell permeability,
• 09:39no reactive functional groups.
• 09:41So we have software that checks that
• 09:44and then we also do some measurements
• 09:47of solubility and cell permeability,
• 09:50OK.
• 09:50So the FP calculations are done for
• 09:53where you do molecular dynamics or Monte
• 09:57Carlo simulations for protein ligand
• 10:00and a typically a ball of several 1000.
• 10:04Water molecules.
• 10:05And you do a calculation where
• 10:08you're comparing the green ligand,
• 10:11green inhibitor with the blue.
• 10:13So you do calculation.
• 10:15We have protein green legging to give
• 10:17complex protein blue ligand to give complex.
• 10:20And what we do on the computer,
• 10:21it turns out to be easier is to mutate
• 10:25the green leg into the blue unbound
• 10:27in water and then bound protein.
• 10:30And the difference in the two
• 10:33vertical numbers there then gives us
• 10:35the difference in predicted free.
• 10:37Energy binding.
• 10:39And so this type of calculation wasn't
• 10:42done at all before 1985 or just the
• 10:46simple green to blue in water FP calculation.
• 10:50That was something that I.
• 10:53It will take credit for doing the
• 10:56first calculation of that type again.
• 10:58Then there's no software.
• 10:59You had to write all the software,
• 11:01you know the force fields we
• 11:03had to develop etcetera.
• 11:04So it was very different world in 1985, OK.
• 11:08So here are just a little bit on HIV.
• 11:11HIV is still a big problem.
• 11:14Some of the statistics
• 11:16are shown there for 2021.
• 11:18They're,
• 11:19you know,
• 11:19close to 40 million people in the world
• 11:22that are infected with HIV and about 1
• 11:26to 2,000,000 each year are becoming infected.
• 11:30And they're on the order of 650,000 deaths.
• 11:34So that's down quite a bit from what it was.
• 11:38But still,
• 11:39you know,
• 11:40from a very serious problem and
• 11:43a Long story short.
• 11:45We have worked on with Karen on
• 11:48the reverse transcriptase and the
• 11:50so this is a an RNA virus and it
• 11:54has a reverse transcriptase which
• 11:58converts the RNA to DNA which
• 12:01is incorporated into the host
• 12:03cells a genome by HIV integrase.
• 12:06So HIV reverse transcriptase has
• 12:08been the principal target for anti
• 12:11HIV drugs and there are two classes,
• 12:13the nucleosides.
• 12:14And the non nuclear science,
• 12:17Karen has worked on both.
• 12:19So in our collaboration with Karen
• 12:21we've only worked on non nuclear sites,
• 12:23the NRT I and there are allosteric
• 12:26inhibitors. They bind in this little
• 12:29pocket that is about 10 angstroms or
• 12:32so from the polymerase active site.
• 12:35It's one of the few examples of of allosteric
• 12:39inhibitor that's that have become drugs.
• 12:41It's very, very, very very I'm, I'm, I'm.
• 12:44Have to think of it to find others.
• 12:47This is the principal example.
• 12:49The crystal structure.
• 12:51Again a Yale connection.
• 12:53The original crystal structure of HIV
• 12:55RT was done in the sites lab 1992.
• 12:58This is a very big. You know,
• 13:02discovery at the time because the HIV
• 13:05crisis was so severe and it's a big protein,
• 13:08thousand of residues.
• 13:10So Long story short,
• 13:12we've tried to make better
• 13:15non nucleoside inhibitors.
• 13:16The original ones have limitations.
• 13:19They're susceptible to mutations
• 13:21that arise quickly.
• 13:22They also had some undesirable pharmacology.
• 13:26So the way we proceed on
• 13:29HIV is the same with the.
• 13:31COVID and the trick in lead optimization is
• 13:37making systematic changes small changes in.
• 13:42Substituents on rings,
• 13:44the rings themselves and groups
• 13:46that link rings together,
• 13:49and if you know the right changes to make,
• 13:52they can have profound effects.
• 13:54So this is an early HIV compound
• 13:58of of ours that we came about from
• 14:02a de Novo design and Karen's lab.
• 14:05The assay they're running is an infected T
• 14:09cell assay and this compound had an EC50.
• 14:12For inhibiting the reproduction
• 14:15of of of the HIV in the infected
• 14:19cells of 10 micromolar,
• 14:21so 10,000 nanomolar.
• 14:23So that's a reasonable starting place,
• 14:25a small molecule, but we've got to
• 14:28increase the potency by a thousandfold.
• 14:31So I I point out here that if you
• 14:34happen to know to put a cyano group
• 14:36in the four position of this ring,
• 14:38you get a very big boost,
• 14:4050 fold boost to 200 nanomolar.
• 14:44OK.
• 14:45Then if you happen to change
• 14:48the thiazole into a pyrimidine,
• 14:50you get another tenfold boost and
• 14:53you're at 17 nanul. So quite amazing.
• 14:56And then if you happen to know to
• 14:59put a methoxy group and the the
• 15:01three position of the pyrimidine
• 15:03ring here 2 nanomolar.
• 15:05So you have more potency than
• 15:06you need for a drug.
• 15:08So this is all fine and this is
• 15:10what we use the FEP calculations to
• 15:13help us with because these changes
• 15:15are in a sea of possible changes.
• 15:17So we do however scans where we
• 15:20have we have a compound like this,
• 15:22we'll scan in chlorine atoms at each open.
• 15:26To see if we can add a little
• 15:29beef to it and that might have,
• 15:32if we did that it would show that this
• 15:35four position is good for chlorine,
• 15:37well if it's good for chlorine
• 15:39and may also be good or even
• 15:41better for cyano because they're
• 15:42both somewhat electronic drawing.
• 15:44So then we would try siana.
• 15:46But we do these initial scans,
• 15:48we also do heterocycle scans of
• 15:51five and six membered rings because
• 15:53they are obviously affect hydrogen
• 15:55bonding patterns and hopefully
• 15:57that would have picked up that
• 15:59the pyrimidine was the
• 16:00way to go. And then finally we do
• 16:03another substituent scan on the
• 16:05pyrimidine of methyls and chlorines,
• 16:08we would see that substitution and the
• 16:10three position is a good thing and
• 16:13before long we would come to the methoxy.
• 16:15So that's the way it's done.
• 16:17And that's attuned animal or very potent
• 16:19compound we did in collaboration with
• 16:22Eddie Arnold got a crystal structure
• 16:24of that there's quite a quite a bit
• 16:27later and that was the only crystal
• 16:29structure we had until Karen's group
• 16:31started getting some around 2012.
• 16:34OK, so here is just some of the
• 16:37work with Karen.
• 16:39These are all publications on different.
• 16:43And an RTI's and you might say well
• 16:47Gee and from 2006 you have these two
• 16:50national or compound aren't you done,
• 16:51why are you why are you keeping doing
• 16:54this and the answer is that that
• 16:56number is against the wild type virus.
• 16:59But the virus as you know have
• 17:01mutates just like COVID is mutating
• 17:04and there's a whole panel of mutants
• 17:06with the HIV and you need to have
• 17:09efficacy against all of the common
• 17:12mutants with one compound.
• 17:14So it's tough.
• 17:15So that initial compound like initial.
• 17:20Compounds in this class,
• 17:22such as nevirapine,
• 17:23was the first approved drug in
• 17:25this class, like nevirapine.
• 17:27It was good against wild
• 17:29type but not not much else.
• 17:32So these other compounds that
• 17:34I'll just skip to this one,
• 17:36one of our better compounds,
• 17:38we've increased the potency,
• 17:39but we very much increased the
• 17:42performance again mutant panels,
• 17:44so very difficult mutant
• 17:45is a double mutant K10 3N.
• 17:50Y181C and this compound
• 17:52here is A10 animal or EC50,
• 17:56which is you know good and great
• 17:58against that whereas the original
• 18:00compounds here would have had
• 18:02no efficacy against that mutant.
• 18:05And we've gone on,
• 18:07we even see something that looks like
• 18:08a covalent inhibitor which it is.
• 18:10We with cooperation with Karen,
• 18:13we have covalent inhibitors for HIV RT Wild.
• 18:19Type and also the Y181C mutant.
• 18:23But I will go on now to what
• 18:26we did with COVID.
• 18:29So fortunately, because of our work on HIV.
• 18:33We're pretty well positioned just to
• 18:36try to do something when COVID rolled
• 18:39around at the beginning of 2020.
• 18:42So this is the IT again an RNA genome.
• 18:47And it some of the proteins that
• 18:50it encodes are indicated here,
• 18:52and not as many as with the HIV,
• 18:56but you do have.
• 18:59The The There's a proteases here
• 19:01that are sort of papain like protease
• 19:03and then the main protease and what
• 19:06we've worked on is the main protease.
• 19:09There's also,
• 19:09you've probably heard of the
• 19:11RNA dependent RNA polymerase.
• 19:12This is just to reproduce the RNA genome.
• 19:16That's another possible target,
• 19:18and some of the structural
• 19:20proteins are over here.
• 19:21There's the spike and the famous
• 19:24spike that is mutating and causing
• 19:26a lot of problems for the vaccines.
• 19:31So the cycle, the life cycle involves
• 19:36the COVID virus binding to the ACE 2
• 19:41receptors on the cells endocytosis.
• 19:45The RNA genome is unprocessed by a host of
• 19:49ribosomes to give you these two polyproteins.
• 19:53You similar situation with HIV,
• 19:57generating polyproteins that
• 19:58have to be cleaved by HIV. Areas.
• 20:02So here's where if we can stop this
• 20:05proteolysis step, the rest of the
• 20:08reproduction cycle stops and it's,
• 20:11I could say there aren't as many
• 20:14targets here as with the HIV.
• 20:16There's no integrase,
• 20:18no reverse transcriptase.
• 20:19And So what we picked in the beginning
• 20:22of 2020 that we would work on the
• 20:26protease almost because there's
• 20:27hardly anything else to work on
• 20:29and there was a crystal structure.
• 20:31Reported so the first thing we did.
• 20:36So this came about as as you recall,
• 20:40things got serious in late January
• 20:422020 and then in March 2020 is
• 20:45one thing shut down. So we were.
• 20:50Sent out of the lab.
• 20:51You know, we could work from home.
• 20:53If you had special permission,
• 20:54you could work in the lab.
• 20:56But we didn't pursue that.
• 20:58But we decided for working at
• 21:00home that what we could do is we
• 21:02would do docking because we have
• 21:04the crystal structure,
• 21:06a crystal structure of the protease.
• 21:08So we would do docking.
• 21:10And the typical way docking works
• 21:11is you have the crystal structure
• 21:13and you have a library of compounds
• 21:15and these are typically commercially
• 21:18available compounds.
• 21:19There's a.
• 21:20Famous library called Zinc that
• 21:22has up to 100 million compounds
• 21:25and then the computers software
• 21:28combines them and it makes the
• 21:31complexes and then it has to score
• 21:33the complexes which is the weak spot.
• 21:37Often the scoring isn't very
• 21:39accurate but you can then test
• 21:41the high scoring molecules.
• 21:42Well that's a lot of compounds to deal with
• 21:46so I thought well we would do 1st instead.
• 21:50Is to dock known drugs,
• 21:52approved FDA approved drugs.
• 21:53So I happen to keep a library of
• 21:56these in the computer and there are
• 21:59three-dimensional structures of the drugs,
• 22:00which is this is all three-dimensional.
• 22:03And so I asked Muhammad and Julian.
• 22:07To dock the 2000 known drugs to see if
• 22:12we could see get some reasonable hits
• 22:16from that and So what happened was.
• 22:21The docking was done in a consensus fashion,
• 22:25meaning they used four different
• 22:27docking protocols,
• 22:273 different programs and four ways
• 22:30of doing the docking because any
• 22:32one program we don't fully trust.
• 22:35So we're hoping that there will be a
• 22:38consensus where you score well in all four.
• 22:42Protocols. And so we got the list.
• 22:46Excuse me. I don't have code and I've tested.
• 22:53But.
• 22:54We we got the list of the top
• 22:57compounds and then, very importantly,
• 22:59we visualize the predicted poses,
• 23:02the complexes.
• 23:05And based on that visualization,
• 23:07we picked compounds that we think look
• 23:10good in the way they're positioned.
• 23:13And I also was very concerned about
• 23:16the idea that we would possibly be
• 23:20making analogs of these compounds
• 23:22because I didn't expect to have
• 23:25again come up with a 10 nanomolar
• 23:28compound we never have in the past.
• 23:30So we purchased 17 compounds and.
• 23:34Gave them to Karens lab,
• 23:37and Karen had meanwhile obtained
• 23:40the protein, expressed it,
• 23:42and she also had implemented the A fret
• 23:45assay that was from the literature.
• 23:48So she was ready to go.
• 23:50And the 17 compounds arrived.
• 23:54And to our surprise, in Karen's lab,
• 23:5814 of them showed some inhibition of the
• 24:02protease activity of Massar Scope 2 Proteus.
• 24:05So that was.
• 24:06Shocking.
• 24:07And so we were did very well on the
• 24:10compound selection and the most
• 24:12potent compounds are listed here.
• 24:14They were single digit micromolar.
• 24:19And but we had a bunch that were
• 24:22under about 50 micromolar.
• 24:24So that this we published and this is a
• 24:28picture of one of the dock structures.
• 24:31The binding site is you know is
• 24:34meant to accommodate a peptide
• 24:36that's going to get cleaved and we
• 24:39have site sub sites we call S1S1,
• 24:41Prime S2 and then this channel S3S4S5.
• 24:45So here's just a picture of a
• 24:48compound in that binding site.
• 24:50So we published that but of course
• 24:53we were looking very much now.
• 24:55And one of these compounds we're
• 24:57going to take and try to optimize it.
• 25:00And the compound we picked,
• 25:02we were we didn't say what it was
• 25:04going to be in this paper and it was
• 25:06not one of the most potent ones.
• 25:08In fact, it was this one param panel.
• 25:11Which is only 100 to 250 micromolar,
• 25:15so a relatively weak hit.
• 25:18But the fact was I liked the way it looked.
• 25:24And this was the dock structure.
• 25:28I'm orienting them all in the same
• 25:31way as 1S Primus 2 and I felt that
• 25:34the dock structure looked reasonable.
• 25:36Sometimes they they have features,
• 25:39they just say this doesn't feel right.
• 25:41But this looked reasonable.
• 25:43But I could also see that it had
• 25:45features that were not optimal.
• 25:47So looking at it over here,
• 25:49so the yellows are carbons,
• 25:52Reds are oxygens, Blues or nitrogens.
• 25:55I could see features that were not optimal.
• 25:57There's a histidine here and it could,
• 26:00it would be nice if it could form
• 26:03a hydrogen bond with this ring.
• 26:04So you probably want to put a nitrogen
• 26:06in here, this nitrogen of the purity,
• 26:08and that's not doing any good.
• 26:10So we can get rid of that.
• 26:13It's just spacing out into solvent.
• 26:15There's an NH over here that's.
• 26:18I would like to be in a hydrogen bond,
• 26:20but it isn't.
• 26:21Meanwhile,
• 26:21this carbonyl is just interacting
• 26:24with solvent,
• 26:25so maybe I could flip that from
• 26:27moving over left to there.
• 26:29Plus it looked like there was a
• 26:32little space in the meta position
• 26:35of that right ring.
• 26:37So.
• 26:39What happened next was we did some
• 26:42FEP calculations to test those
• 26:44ideas and this is what those are
• 26:46raw data looks like in an Excel
• 26:48sheet so that the the things I'm
• 26:51trying here are for the left ring.
• 26:55I'm going to try different rings.
• 26:57So ring scan where I did 234
• 27:01pyridinyl 4 pyrimidine 2 triazine,
• 27:04so a bunch of different rings there.
• 27:07They also did a calc and that those
• 27:10calculations said that the three pyridine
• 27:13the the negative number here is good.
• 27:16This is the change in free energy
• 27:19of binding relative to benzene.
• 27:21So this was saying go for the three pyrenee.
• 27:25Also I checked that ring flip of the
• 27:28carbonyl and that was very good,
• 27:30minus 4.7 and then over on the right
• 27:33side checking to see if we could
• 27:36put something in that meta position,
• 27:39indeed the meta position when we did
• 27:42a chlorine scan at each position,
• 27:45the meta here shed very good.
• 27:48Looks like we should put a chlorine there.
• 27:50So combining those three ideas
• 27:53led to then the.
• 27:55Three initial compounds
• 27:57that were synthesized.
• 27:58So here.
• 27:59Now I'm aligning everything so you
• 28:01can see the changes from parent panel,
• 28:04the three pyridyl.
• 28:06The carbonyl's been flipped and we've
• 28:09added the chlorine and we've left the
• 28:12the cyano phenyl from parent panel.
• 28:14I also from modeling with my bond program.
• 28:19Again, the,
• 28:19the slow part in all of this is synthesis.
• 28:22So we have plenty of time to do computer
• 28:25work while people are doing synthesis.
• 28:28So it's a natural thing to, you know,
• 28:32look very hard at these structures.
• 28:33And I had looked hard at this and I
• 28:36recognized maybe I could do something
• 28:38over with this ring because there's an
• 28:40edge that will show more clearly here
• 28:43of a loop that could use some hydrogen bonds.
• 28:47And I thought a uracil might work,
• 28:49so I'd modeled.
• 28:50Got with the program complex
• 28:51has looked very good.
• 28:53So we synthesized a uracil and also
• 28:56just this 35 dot clock compound.
• 28:59So this is a very happy day now.
• 29:02Because the potency of those original 3
• 29:06compounds was 10-6 and four micromolar.
• 29:09So here we've gotten a huge boost as
• 29:12expected from the FEP calculations.
• 29:14And this was the wonderful and I'll
• 29:17tell you the timing more in a bit,
• 29:19but this is now June of 2020.
• 29:23So we didn't get back into our lab until May.
• 29:27And now in June we have these,
• 29:31this 4 micromolar. Compound.
• 29:34We've only,
• 29:35and then it came a little later was
• 29:38actually October and Karen's group
• 29:40got a crystal structure for that
• 29:43dichloro compound and it's basically
• 29:46identical to what we've predicted.
• 29:48There's the carbonyl and hydrogen
• 29:51bond we wanted.
• 29:52There's a hydrogen bond between
• 29:54the pyridine and the histidine.
• 29:56We still have the nitrile hydrogen
• 29:58bonded in what he called the oxyanion
• 30:01sort of hole and the dichloro compound.
• 30:05Is again looking very good.
• 30:07Furthermore,
• 30:08we have this channel running N from
• 30:11the upper chlorine there and so we're
• 30:14ready to think about putting some
• 30:16of the something in that Channel.
• 30:19So the next thing was to try to grow
• 30:23substituents into that Channel and
• 30:25just for grins and I mean really not
• 30:28interested in methyl particularly,
• 30:30but just for grins, we did FP
• 30:32calculations for methyl ethyl propyl,
• 30:34O methyl ethyl propyl albuterol and
• 30:37then some ones with a hydroxyl that
• 30:40I figured probably wouldn't be very
• 30:42good problem with hydroxyl is it's
• 30:44very happy unbound said waters around
• 30:47and if you go bound it may be happy.
• 30:49Again, but you're not going to gain much.
• 30:52The way you gain is by having more
• 30:55hydrophobic pieces that are binding into
• 30:58hydrophobic part of the binding site.
• 31:00So this told us.
• 31:04Tried the O propyl compound
• 31:06so we synthesize on.
• 31:08There are two synthetic chemists
• 31:10are working on this so Lizzie and
• 31:14Chun way and so we they made.
• 31:19The proxy compound in both the
• 31:22cyano phenyl and the urea series,
• 31:25and this turned out great,
• 31:29140 nanomolar and 120 animal later on.
• 31:33This wasn't in sequence.
• 31:35We had made the trifluoromethyl analogs
• 31:38to that. They're more hydrophobic.
• 31:40They're probably going to be better
• 31:42binders as they were showing this
• 31:44one even down at 25 an animal,
• 31:47but generally I don't like CF.
• 31:49Big groups and drug like molecules
• 31:51because they really hurt the
• 31:53solubility of the compounds.
• 31:55So, but we're doing very well here 120.
• 31:59An animal or and I'll show
• 32:01you the timing on this,
• 32:02but this,
• 32:03this I think is in August now and
• 32:07Karen's group again got a crystal
• 32:10structure in October and it was exactly
• 32:14as expected including this bent.
• 32:17Hard at the at the end of the Propoxur group.
• 32:19And so it's a Ghosh. We call it a gauche.
• 32:22You've all taken organic chemistry, I'm sure.
• 32:27So that's the course you hated the most,
• 32:30but maybe, maybe not.
• 32:32But there it is.
• 32:33There's this gosche OCC and we
• 32:36had figured that was the case.
• 32:38The modeling told us that because at
• 32:41that terminal methyl fits right in
• 32:43the S4 site of the of that Channel.
• 32:48And so there's a lucine or problem,
• 32:50so hydrophobic site and so put
• 32:54it right in there also.
• 32:56Again, like I said,
• 32:57there's lots of time to do computing
• 33:00and so we considered benzel oxy groups.
• 33:04So you can imagine a benzene ring
• 33:08sitting here and potentially projecting.
• 33:12A substituent into that pocket.
• 33:15So sure enough we did modeling on
• 33:18these benzyl oxy analogs and did a
• 33:21chlorine scan on the fennel which said
• 33:24in a methyl scan and both methyl and
• 33:27chlorine were predicted to be very good.
• 33:30And so those compounds were made and
• 33:34the parent compounds 120 micromolar,
• 33:37but the ortho chloro compound
• 33:4118 an animal compound and this
• 33:44we had in October of of 2020.
• 33:48And Karen's group again got a
• 33:51crystal structure for the bend the
• 33:55parent Benz loxy components and
• 33:59is positioned as one expected.
• 34:02So this is just now a little video.
• 34:06To. Have those. Break this.
• 34:10This is a dimer, so they're two.
• 34:12This is Karen's crystal structure
• 34:16of the propoxur compound and
• 34:19just zeroing in on it.
• 34:22There.
• 34:29OK, so you can.
• 34:34Run it again. So that little
• 34:39molecule is enough to shut down the
• 34:43enzymatic activity of that protein.
• 34:50OK, so this we published and.
• 34:54I was also saying a a second here,
• 34:57we're going to of course the
• 34:58well I've shown you so far
• 35:00is just protease inhibition.
• 35:01We've got to go into cells,
• 35:02infected cells and so that
• 35:05we published 28 compounds.
• 35:07Of course by the results
• 35:09I've talked about so far,
• 35:10we have lots of compounds here
• 35:13under the 50 nanomolar and
• 35:14you can see there are authors,
• 35:16lots of people involved and from
• 35:18the medical school, you know,
• 35:20fair and Isaacs and Brett Lindenbach,
• 35:22grouper and very important.
• 35:24Along with Karen in doing the
• 35:27cell assays that will describe in
• 35:29a Miller in a minute and Scott
• 35:31Miller in chemistry had donated
• 35:33his graduate student Lizzie Stone
• 35:36to help us with the synthesis,
• 35:38along with my postdoc Chunwei Zang.
• 35:42So that was good.
• 35:43We published that in ACS Central
• 35:46science in February 2022.
• 35:47A little later we also replaced
• 35:50the benzyl Oxy with heterocycles.
• 35:52This is a standard, I'd say,
• 35:54medicinal chemistry.
• 35:55This isn't, you know, genius stuff.
• 35:59Heterocycles often have some desirable
• 36:02properties over a substituted benzene.
• 36:05So we published some more compounds
• 36:07in the summer than of a 2021.
• 36:10We also.
• 36:11Tested cell permeability with
• 36:14a pampa assay in our lab and
• 36:18measured aqueous solubility.
• 36:20So now we have uracil's with
• 36:22the hydrogen or with a methyl.
• 36:24So the ones with the methyl are going
• 36:27to have better cell permeability.
• 36:29And so that is an issue because we want
• 36:33to show that we have efficacy and sell assay.
• 36:37So this is where the,
• 36:39again the folks here in the Med
• 36:40school are so important to us.
• 36:42The BSL three facility was used.
• 36:45There's krassimir getting suited up
• 36:49because COVID, of course, is airborne.
• 36:51He has to have a full breathing
• 36:54apparatus and the assays that were done.
• 36:57Karen certainly can describe these far
• 36:59better than I can, but there's one.
• 37:01It's a a plaque assay using infectious virus.
• 37:05And so you have the live these are Vero
• 37:09cells infected with large live SARS Cove two.
• 37:14And there's also then the replicon.
• 37:16Assay and the Republican
• 37:18isn't using infectious virus,
• 37:20but it's giving us a very
• 37:22virtually identical readout.
• 37:24So we're testing our compounds and we have
• 37:28as a as a reference compound remdesivir,
• 37:32which is A1 micromolar EC50
• 37:37and the assays that were done.
• 37:40And long short, we have many
• 37:42compounds that are one micromolar.
• 37:45We also have some compounds.
• 37:46This one's 38 nanomolar EC 50,
• 37:49that's inhibition of the
• 37:52of the protease activity,
• 37:54but it's not active in the replicant housing.
• 38:00And this simply because it
• 38:01doesn't get into the virus.
• 38:03Cell permeability is too low,
• 38:05so the cell permeability is critical.
• 38:08The quite remarkable compound is number 19.
• 38:12So this.
• 38:14Benzyl oxy compound that has a
• 38:17methylated uracil and in the assay it
• 38:21was 80 nanomolar in the infectious
• 38:24virus assay and 175 and the replicon assay.
• 38:29So this became our our lead
• 38:32compound for preclinical work.
• 38:35Now unfortunately in our world we can't,
• 38:37you know we're not Pfizer,
• 38:39so we can't take 10 compounds and put
• 38:41them all into preclinical studies but.
• 38:44We did work on 19 and a pharmaceutical
• 38:48company was very interested in 19.
• 38:52They took 19 and did their own sell
• 38:55assay and they came back and their
• 38:57cell was 15 animals they can confirmed
• 39:00everything that we we had reported.
• 39:03So that compound 19 is a very potent compound
• 39:08in infected cells and Karen's group has
• 39:12been working on the PK, it has very good.
• 39:16Basic PK bioavailability.
• 39:18And they have done with Pretty Kumar
• 39:21some initial mouse studies and this
• 39:25is with these humanized mouse mice,
• 39:27KTH 2 mice.
• 39:29And again Karen could describe
• 39:31the current status of this.
• 39:34But basically we were delighted a very
• 39:37low dose of the compounds that were using
• 39:41and if you don't untreated mouse after
• 39:45six days as this is now fluorescent.
• 39:48Imaging of where the virus is.
• 39:50So initially the virus goes into the lungs,
• 39:53but it makes its way into the brain.
• 39:57And at day six,
• 39:59the mouse is again horribly
• 40:01infected and dies.
• 40:03So we have tested we meaning Karen
• 40:07and pretty by both Ivy and oral.
• 40:12And the results have been very good.
• 40:14There's only one dose and you see
• 40:17protection for four days, you know,
• 40:19completely clean a mouse and even at 6 days.
• 40:22So with the oral, it's,
• 40:25you know, really very clean.
• 40:26So if this was being dosed every day,
• 40:29the feeling is infection that wouldn't go on.
• 40:33So we have very, you know,
• 40:35concur raging data with this compound.
• 40:38There has been some.
• 40:40You know again external interest
• 40:42in this compound, yeah,
• 40:44we think we if we had the resources we
• 40:46can come up with lots of other compounds,
• 40:48but we need support for this and are
• 40:51you know high level because these
• 40:54preclinical studies are are expensive.
• 40:57So just to compare what we've done.
• 41:00Versus others.
• 41:01So first of all our compound is a non
• 41:04covalent inhibitor by most of the other
• 41:07work in this area been covalent inhibitors.
• 41:10Up until recently covalent
• 41:12inhibitors were considered to be.
• 41:15Not desirable because you're always
• 41:18worried about off target activity.
• 41:21But here is how other people progress.
• 41:23So a lot of these things are peptidic.
• 41:25Generally we don't like peptidic inhibitors
• 41:29because they can be proteolysis by many.
• 41:33Proteolytic enzymes that exist
• 41:36in humans so but this is some of
• 41:40the compounds and EC 50 of 720.
• 41:44Remember we're 50 or 80 nanomolar.
• 41:48This is the COVID moon shot that
• 41:49got quite a bit of publicity.
• 41:51This is just the icy 50.
• 41:53They obtained an assay 2400
• 41:57compounds and the best IC50 they
• 42:01obtained is basically 100 nanomolar.
• 42:04At 30,
• 42:05we had made no more than 30 compounds
• 42:08and we were at 18 nanomolar.
• 42:10Another peptide peptide peptide,
• 42:13but this is a PAX lovin.
• 42:17So Pax Lovid is this neurometrix alvir,
• 42:21but you have to include a SIP
• 42:24inhibitor ritonavir.
• 42:25So ritonavir is an HIV protease inhibitor.
• 42:29Not something you probably want
• 42:30to take for a long time and have
• 42:32their side effects of that.
• 42:33Of course you're not going to take
• 42:35packs a little bit for a long time.
• 42:36So I guess it's OK,
• 42:38but on the other hand having to
• 42:41have the SIP inhibitor to keep the.
• 42:45Protease inhibitor from being chewed up.
• 42:48Metabolically.
• 42:49Is clearly not desirable because
• 42:51you don't want to be, you know,
• 42:54can have drug, drug interactions.
• 42:56This is our compound.
• 42:58Again, by comparison,
• 42:59other things that you know.
• 43:02I'm obviously a little bit prejudiced here,
• 43:04but this to me is a tough molecule.
• 43:08All the stereo chemistry going
• 43:10to be tough to synthesize.
• 43:11You have high cost of goods.
• 43:13It's peptic. You worry about that.
• 43:15It is a covalent inhibitor,
• 43:17covalently modifies the cyano,
• 43:19but it's probably reversible.
• 43:21Are covalent.
• 43:22There have been a synthesis
• 43:24issues with the compound.
• 43:27It's also intrinsically not
• 43:28as potent as our compound.
• 43:30It's a EC 50 or 740 whereas we're at you
• 43:35know 10 times more potent with there's no,
• 43:38we don't we know from our preclinical
• 43:41work on off target and SIP
• 43:43activity that we don't have any sip
• 43:46problems with the compound either.
• 43:47So the rest of the story.
• 43:51So why isn't our compound in clinical
• 43:53trials and that's a probably takes me
• 43:57more than the last time I I have here.
• 44:00But the packs of lovin and thermal,
• 44:03Trevor got into clinical trials very
• 44:05quickly because it was sitting on the
• 44:08shelf from the SARS Cove One project.
• 44:10They made a minor modification to
• 44:12make it have better solubility.
• 44:14So it was ready to go and
• 44:17so it's off and running,
• 44:19I doubt seriously it's the best.
• 44:21Drug possible,
• 44:23and there's no way, and well,
• 44:26time will tell the problem for the
• 44:28pharmaceutical companies that they're
• 44:29all in the business of making money.
• 44:32And so the before the end of last fall.
• 44:37People are getting kind of cocky about,
• 44:39you know, covid's under control,
• 44:42the vaccines are working.
• 44:44And then Omicron came along around December
• 44:48of last year and that's changed things a bit.
• 44:53But we'll see who has the, you know,
• 44:57stamina to advance additional protease
• 45:00inhibitors into the clinic because
• 45:02of the cost of the clinical trials.
• 45:05This is a timeline just showing
• 45:06the power I think of our approach.
• 45:09So June 15th all we had was parent panel.
• 45:12By August 3rd we had these six
• 45:15and four micromolar compounds.
• 45:17By.
• 45:18September 2nd we had the proxy
• 45:20140 nanomolar compound.
• 45:22September 10th we had the corresponding of.
• 45:26Benzyl oxy,
• 45:27uracil and then we started getting
• 45:30some crystal structures October 3rd.
• 45:33We had the first crystal structure October
• 45:374th and also the Propoxur compound and
• 45:41but the speed here which we got to the.
• 45:46These sort of loading animal compounds
• 45:49again to get to 18 animal we had
• 45:52synthesized about 30 compounds and a
• 45:54few of them were things we probably 8
• 45:57or 10 of them were real or wild shots.
• 46:02And this synthesis was done by a gun.
• 46:04Postdoc chunwei and graduate student Lizzie.
• 46:07So that's the story and I I hope
• 46:11I've told you a little bit about
• 46:14what Karen and I do and the.
• 46:16Hour of combining the computation with the,
• 46:21you know,
• 46:22reliable assaying and crystallography
• 46:24is such a different world than
• 46:26what we lived in 20 years ago.
• 46:28So just thanking people in my lab notably.
• 46:33And Julian is a long-term associate other
• 46:35so he's a senior research scientist.
• 46:38Anna and Joe were both associate research
• 46:42scientist and other people listed here.
• 46:45Karen of course my.
• 46:47Wonderful collaborator and
• 46:49other Pi collaborators,
• 46:51pretty yosi on our Jack projects and Brett,
• 46:57Brett and Faron on the COVID project.
• 47:01So pleasure to be here with
• 47:02you and thank you very much.
• 47:12What a whirlwind journey.
• 47:13Yeah, amazing.
• 47:15Are there any questions here,
• 47:18Emily? Are you monitoring
• 47:19questions in the chat, Tommy?
• 47:35Yeah, these the COVID compounds
• 47:37are all binding to the active
• 47:41site of the Proteus.
• 47:43So the cysteine, that's a,
• 47:44there's a cysteine,
• 47:45it's a cysteine protease,
• 47:46there's a cysteine it.
• 47:48We're sort of in the middle of all
• 47:50the structures I showed you and
• 47:52that's the active site cysteine.
• 48:02Even fine.
• 48:08Yes, the the Pfizer compound
• 48:12binds in that same site, and it
• 48:15covalently modifies that cysteine.
• 48:18And you? Does not covalent.
• 48:27Yes.
• 48:34Dog.
• 48:40Quite understand, he's asking if
• 48:43in the crystal structure does
• 48:45it bind to the cleavage. Yes,
• 48:48and this is the.
• 48:51The cysteine there cysts 145
• 48:54and this histadine over here.
• 48:58Are the catalytic residues,
• 48:59so our compound sitting right on top of them.
• 49:03And the Pfizer compound covalently modifies
• 49:07that cysteine as do most of the other.
• 49:11There's a very few coat non covalent
• 49:13inhibitors have been reported for this.
• 49:16But we from the getco we wanted
• 49:18to pursue non covalent inhibitors
• 49:20just to avoid the potential
• 49:23issues of covalent inhibitors.
• 49:29So you know, we're we're
• 49:31extremely familiar with the hyper
• 49:33immutability of this virus in the
• 49:36spike protein to evade immunity.
• 49:38I wonder if you've done sort of low dose
• 49:41exposure and if there's a mutational.
• 49:44Response to to a protease
• 49:46inhibitor like this?
• 49:49Yeah, I haven't maybe.
• 49:50I mean, we haven't to my knowledge,
• 49:53unless Karen's been up to
• 49:55something I don't know about,
• 49:57the SARS Cove 1 protease and SARS Cove
• 50:012 protease are extremely identical.
• 50:05They're the only differences are quite
• 50:07far from the the protease active site.
• 50:10So it's it's hoped that there won't be.
• 50:15A lot of mutations possible
• 50:18for the Proteus, however,
• 50:19it hasn't been under pressure.
• 50:21So I think with the Pax lovid treatments
• 50:24we will probably begin to see some
• 50:28mutations closer to the binding site.
• 50:31And there there was a recent
• 50:33paper in science,
• 50:35I believe is it science indicating some
• 50:40mutations that might arise in this Proteus,
• 50:43so it was under some pressure that they.
• 50:46Put it,
• 50:47but we haven't looked into that yet.
• 51:01So this is a this is a related question,
• 51:03but how different is the COVID 2
• 51:06protease active site from that
• 51:08of other common human proteases?
• 51:11Umm. Well, I would say it's it's the
• 51:17COVID active site is quite unique,
• 51:20but it's virtually identical to
• 51:23the SARS Cove one active site,
• 51:25but I don't think there's been a.
• 51:30I don't think that these inhibitors are
• 51:33generally inhibiting other proteases.
• 51:39So I I haven't heard that,
• 51:42so I don't expect it, but if they were,
• 51:45it would certainly be, I'd imagine
• 51:47it'd be assisting a protease would
• 51:49be the ones you'd be looking at.
• 51:53Well, we're we're at the hour.
• 51:55I can't thank you enough for this
• 51:58lucid explanation to a bunch of
• 52:00non chemists was really beautiful.
• 52:02Also on behalf of healthcare
• 52:04workers who, you know,
• 52:05see people with COVID all the time.
• 52:07It's wonderful work.
• 52:07Thank you very much. Thank you.