Designing a Covalent Allosteric Peptide Inhibitor of Caspase-6

Jessica Holechek Author

07/26/2020 Added

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My summer research poster describing the project I have designed.

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[00:00:00.260]My name is Jessica Holecheck
[00:00:01.750]and I will be discussing my project,
[00:00:03.490]Designing a Covalent Allosteric
[00:00:05.120]Peptide Inhibitor of Caspase-6.
[00:00:07.810]Neurodegenerative disorders affect millions
[00:00:09.880]of people worldwide and are caused
[00:00:11.470]by the dysregulation of certain proteins,
[00:00:13.840]for example, caspases, which are a family of proteases
[00:00:16.970]whose dysregulation can lead to buildup
[00:00:19.010]of protein aggregates leading to neuronal degeneration.
[00:00:22.400]Inhibition of disease causing proteins
[00:00:24.380]is studied as a potential therapy for these diseases.
[00:00:27.640]Most inhibitor drugs are designed
[00:00:29.450]to interact with their target proteins
[00:00:31.190]under equilibrium conditions,
[00:00:32.980]allowing the inhibitor to dissociate
[00:00:34.850]from the defective protein and return
[00:00:36.590]to its active conformation.
[00:00:38.450]To prevent this dissociation,
[00:00:40.150]covalent inhibitors have been studied.
[00:00:42.670]However, covalent inhibitors have been thought
[00:00:44.830]to be potentially harmful due to the possibility
[00:00:47.050]of generating off-target toxicity.
[00:00:49.440]Here, you can see that an orthosteric inhibitor
[00:00:51.650]binds directly to the substrate binding site.
[00:00:54.520]While non-covalent inhibitor will bind
[00:00:56.300]through intermolecular interactions,
[00:00:58.050]a covalent inhibitor will react
[00:00:59.640]with the protein to form a covalent bond
[00:01:01.710]permanently preventing this substrate from binding.
[00:01:04.170]While this is great because it increases
[00:01:05.940]the affinity of the inhibitor for the protein.
[00:01:08.340]It can potentially bind proteins
[00:01:09.810]that wasn't intended to inhibit
[00:01:11.560]generating this off-target toxicity.
[00:01:14.100]This is due to the fact that the amino acid sequence
[00:01:16.460]at the active site is highly conserved
[00:01:18.280]among a family of proteins.
[00:01:20.200]Allosteric binding sites are on the outer surface
[00:01:22.350]of the protein and are less conserved.
[00:01:24.630]Because of this covalent allosteric inhibitors
[00:01:27.080]have been studied in order to mitigate off-target toxicity.
[00:01:30.500]Here, you can see a protein kinase
[00:01:32.060]in its active confirmation with ATP bound
[00:01:34.460]to its active site.
[00:01:36.010]Instead of blocking the substrate directly,
[00:01:38.090]the inhibitor changes the confirmation
[00:01:39.860]of the protein by binding to a site
[00:01:41.600]independent of the active site,
[00:01:43.340]preventing the substrate from accessing the active site.
[00:01:46.280]You can also see that this is a reversible process.
[00:01:48.910]However, as I mentioned before,
[00:01:50.950]a covalent inhibitor will form of covalent linkage
[00:01:53.870]permanently changing the conformation.
[00:01:56.540]Up until now, only small molecules have been used
[00:01:58.860]to study covalent allosteric inhibitors.
[00:02:01.290]Peptides have been discovered to successfully
[00:02:03.280]inhibit target proteins with increased selectivity
[00:02:05.770]due to their larger size.
[00:02:07.480]One such interaction is the reversible
[00:02:09.920]allosteric inhibition of caspase-6 by pep419.
[00:02:13.550]Using this as our test system,
[00:02:15.050]we aim to test whether covalent allosteric peptides
[00:02:17.550]can improve upon non-covalent versions.
[00:02:19.970]If this proof of concept study suggests
[00:02:21.950]improved selectivity and efficiency,
[00:02:23.840]it can be applied to other target systems
[00:02:25.770]for potential drug development.
[00:02:27.890]Because of the increase affinity
[00:02:29.520]of covalent inhibitors and the increased selectivity
[00:02:31.950]of allosteric peptide inhibitors,
[00:02:33.960]our goal is to design a novel peptide
[00:02:35.930]that will covalently bind
[00:02:36.980]to caspase-6 at an allosteric site.
[00:02:39.420]We hypothesize that a covalent allosteric peptide
[00:02:41.930]inhibitor will show enhance affinity and selectivity
[00:02:44.980]for caspase-6 relative to presently characterized
[00:02:47.810]small molecules or non-covalent peptides.
[00:02:50.410]This diagram demonstrates
[00:02:51.720]the method of covalent bond formation.
[00:02:53.990]The ligand, pep419 will bind the caspase-6
[00:02:56.750]bringing the electrophilic modification
[00:02:58.670]into close proximity with the nucleophilic lysine.
[00:03:01.800]As the covalent bond forms,
[00:03:03.300]the electrophilic warhead leaves
[00:03:04.820]and you're left with the covalently bound
[00:03:06.430]allosteric peptide inhibitor.
[00:03:08.350]On the left, you can see the intended site
[00:03:10.140]of covalent bond formation,
[00:03:11.440]which takes place at lysine 147 on caspase-6.
[00:03:14.810]To form this bond, we will first modify
[00:03:16.700]the peptide containing three amino alanine,
[00:03:19.240]a nucleophilic unnatural amino acid
[00:03:21.570]to take the place of glutamic acid 17.
[00:03:24.300]This will then be reacted with a modification
[00:03:26.440]consisting of an electrophilic warhead
[00:03:28.340]that will act as a good leaving group
[00:03:29.730]for the reaction, and a hydrocarbon chain
[00:03:32.020]with a specified number of carbons
[00:03:33.670]to bridge the 9.6 angstrom gap
[00:03:35.730]between three amino alanine
[00:03:37.060]and the amine group on the lysine.
[00:03:39.210]There are two different electrophilic warheads
[00:03:40.850]that we will use, a fast reacting N-acyl-N-alkyl sulfonamide
[00:03:44.700]and a slow reacting dibromophenyl benzoate.
[00:03:47.900]Here are the final constructs
[00:03:49.230]that will be bound to the peptide
[00:03:50.560]after peptide synthesis.
[00:03:52.700]On the right are the approximate measurements
[00:03:54.500]of the distance between the peptide backbone
[00:03:56.490]and the reactive site.
[00:03:58.130]Using a linker length of five carbons
[00:04:00.080]will create a 10.6 angstrom modification
[00:04:02.880]long enough to bridge the gap
[00:04:04.190]between the peptide and the protein.
[00:04:06.720]We will first run a series of experiments
[00:04:08.500]monitoring the activity of caspase-6
[00:04:10.500]in the presence of the non-covalent
[00:04:11.970]and covalent pep419,
[00:04:13.680]which is indicated by star,
[00:04:15.440]as well as the negative control,
[00:04:16.890]which is a mutated pep419
[00:04:18.640]that does not bind to caspase-6.
[00:04:21.470]The graph on the left is the theoretical
[00:04:23.250]inhibition curve comparing these peptides.
[00:04:25.740]If one of the design covalently bound
[00:04:27.671]pep419 is a better inhibitor,
[00:04:29.800]it will take a lower concentration
[00:04:31.540]in order to reduce caspase-6 activity
[00:04:33.790]compared to the non-covalent version.
[00:04:36.250]As pep419 E12A is our negative control,
[00:04:38.920]there should be no reduction in percent activity.
[00:04:41.700]Next, we'll monitor this selectivity
[00:04:43.380]of the covalent peptides
[00:04:44.510]by incubating them with caspase-6,
[00:04:46.460]as well as two other caspases seven and nine,
[00:04:49.200]which pep419 has previously shown to weakly inhibit.
[00:04:52.720]If the covalent pep419s have increased selectivity,
[00:04:55.870]this curve could theoretically represent
[00:04:57.700]the inhibition of the covalently binding peptides
[00:05:00.330]with no effect on caspase seven or nine.
[00:05:02.930]After testing the potency
[00:05:04.140]of the covalent peptide inhibitors,
[00:05:06.060]we will characterize their binding
[00:05:07.350]by first incubating them with caspase-6
[00:05:09.740]and analyzing the reaction using mass spectrometry.
[00:05:13.180]This will verify whether or not the peptide
[00:05:15.050]actually covalently bonded to the protein.
[00:05:17.560]We will then compare the labeling efficiency
[00:05:19.410]of peptides containing the two different
[00:05:21.310]electrophilic warheads by analyzing
[00:05:23.540]the ratio of unbound and bound caspase-6 peaks.
[00:05:26.700]Finally, the peak containing the bound peptide
[00:05:28.840]will be collected as a fraction
[00:05:30.260]and the modification site will be verified
[00:05:32.220]through trypsin digest.
[00:05:33.720]Lastly, the inhibition potency of the covalent peptides
[00:05:36.510]will be analyzed in human neuroblastoma cells
[00:05:38.850]by measuring concentrations of cleaved lamin A,
[00:05:41.620]a caspase-6 specific substrate.
[00:05:44.030]Electroporation will be used to transfect
[00:05:46.040]the cells with increasing concentrations
[00:05:47.870]of the covalent, non-covalent,
[00:05:49.570]and negative control peptides.
[00:05:51.450]Caspase-6 will then be activated
[00:05:53.130]by treatment with staurosporine,
[00:05:54.870]which induces apoptosis.
[00:05:57.080]Finally, we will analyze lamin A
[00:05:58.920]presence through a Western blot.
[00:06:00.830]The far right figure is modified
[00:06:02.470]from a previous study comparing the non-covalent
[00:06:05.270]pep419 and it's negative control.
[00:06:07.800]Using the data we gather
[00:06:09.070]we will be able to further assess the potencies
[00:06:11.220]of the covalent peptides in comparison
[00:06:13.360]to these previously characterized non-covalent peptides,
[00:06:16.460]which I've indicated by the addition
[00:06:18.180]of a third row on the Western blot.
[00:06:20.540]Taken together, these assays will allow us
[00:06:22.500]to test whether covalent allosteric peptides
[00:06:24.690]can improve upon the non-covalent versions.
[00:06:27.360]This concludes the project I've designed this summer,
[00:06:29.830]which will be carried out by an incoming graduate student
[00:06:32.280]this upcoming semester.
[00:06:34.040]I would like to thank UNL
[00:06:35.257]and the National Science Foundation
[00:06:37.020]for allowing me to take part in this opportunity.
[00:06:39.480]Thank you for listening.

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Designing a Covalent Allosteric Peptide Inhibitor of Caspase-6

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