Pre-Clinical Toolbox

Our pre-clinical toolbox is collected to accelerate drug discovery and research for academia and industry.

Why a Toolbox?

To enable drug discovery, our team has assembled a pre-clinical toolbox to provide a standardized workflow and easy access to test molecules in relevant disease models at the in vitro and in vivo levels. Our pre-clinical toolbox may be used for research purposes and commercial purpose entities.

Access Tools

Assays

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Cellular Models

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Animal / Organoid Models

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Listing of Research Resources

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ASSAYS

The primary assay for VCP is the binding and enzymatic activity of VCP hexamers. The secondary assays are VCP functions at the cellular level.

Primary assay- The primary assay for VCP is the binding and enzymatic activity of VCP hexamers.

Method: Measurement of ATPase Activity of Valosin-containing Protein/p97,

Reference Protocol: Referred in Suvara K, Honda K, Muroi M, Kondoh Y, Osada H, Watanabe N. Measurement of ATPase Activity of Valosin-containing Protein/p97. Bio Protoc. 2020 Feb 5;10(3):e3516. doi: 10.21769/BioProtoc.3516. PMID: 33654741; PMCID: PMC7842740.

Method: Monitor assembly disassembly of SG: transient transfection of GFP-G3BP1, an SG marker, expose the transfected cells to heat shock (43ºC, 1 hour), recovery (37ºC) is followed at 30 minutes 60 minutes, and 90 minutes with confocal microscopy (Gwon et al., 2021).

Note: SGs are dynamic, reversible biomolecular condensates, which assemble in the cytoplasm of eukaryotic cells under various stress conditions. Formation of SGs typically occurs upon stress-induced translational arrest and polysome disassembly. The increase in cytoplasmic mRNAs triggers the formation of a protein-RNA network.

VCP is a key component of SG disassembly.

Reference: Gwon Y, Maxwell BA, Kolaitis RM, Zhang P, Kim HJ, Taylor JP. Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner. Science. 2021 Jun 25;372(6549):eabf6548. doi: 10.1126/science.abf6548. Epub 2021 Aug 5. PMID: 34739333; PMCID: PMC8574224.

*Secondary Assay

Method: Fluorescence Unfolding Assays.

Note: Assays are carried out at 37 °C. Samples contained 25 nM GFP substrate, 75 nM p97 hexamer, 150 nM adaptor (UN, NSFL1C, or UBXN7), and/or 250 nM GroEL trap in unfolding assay buffer (25 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM TCEP, 2 mM ATP). Control experiments indicated that the levels of p97, adaptor, and GroEL used were at or near saturation). Other nucleotides and p97 inhibitors were present at 2 mM and 10 μM, respectively, when indicated. Kinetic experiments were carried out on a Fluoro-Log 3 (Horiba Jobin Yvon) with excitation at 488 nm and emission at 509 nm. Relative fluorescence was calculated by normalizing the fluorescence signal to that at time 0. Unfolding rates were calculated by fitting curves to an exponential decay model in Prism (GraphPad).

Reference: Blythe, Emily E et al. “Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy.” Proceedings of the National Academy of Sciences of the United States of America vol. 114,22 (2017): E4380-E4388. doi:10.1073/pnas.1706205114

*Secondary Assay

Method: Cell viability is monitored every 3 or 4 days by staining with Calcein AM Viability Dye (Thermo, cat# 65-0853-81), acquiring and analyzing images with ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices).

Reference: Wang F, Li S, Wang TY, Lopez GA, Antoshechkin I, Chou TF. P97/VCP ATPase inhibitors can rescue p97 mutation-linked motor neuron degeneration. Brain Commun. 2022 Jul 6;4(4):fcac176. doi: 10.1093/braincomms/fcac176. PMID: 35865348; PMCID: PMC9294923.

*Secondary Assay

Method: The MTT assay is a measure of the metabolic activity of the cells analyzed; the more metabolic activity in the sample, the higher the signal.

Note: The underlying mechanism of the assay has also not been fully understood and there are still controversies and uncertainties on some of the aspects such as what additional organelles, enzymes, and molecules are involved in MTT reduction, the origin of extracellular formazan crystals, the cytotoxic effect of the MTT reagent itself, and how the assay measurements represent cell viability, metabolic activity, and/or treatment toxicity.

To access: Several commercial kits available (i.e. Thermo Fisher, Sigma Aldrich and Abcam).

Reference: Stockert J.C., Horobin R.W., Colombo L.L., Blázquez-Castro A. Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 2018;120:159–167. doi: 10.1016/j.acthis.2018.02.005.

*Secondary Assay

PATIENT-DERIVED CELLULAR MODELS

Our approach to in vitro models is to make standardized models available to all researchers and host biobanks with patient samples.

Cure VCP Disease has contracted with iXcells to develop patient-derived IPSCs.

Cell Lines Available: R155H and R159C, derived from patient blood, accompanied by whole genome sequencing, and karyotyping.

Method: Created from the derivation, activation, and expansion of PBMCs; Custom iPSC generation and characterizations from PBMCs

Custom Differentiation: Generation of Skeletal Myoblasts; 8-10 million scale, per line; including ICC marker staining (MyoD and Desmin) and validation of myotube formation (MHC and MyoG)

Custom Motor Neuron Differentiation: 8-10 million scale; per line; including ICC marker staining (HB9/TUJ-1 on DIV2, CHAT/MAP2 on DIV7).

To access: Complete this request form

CRISPR engineered IPSCs - R155H motor-neurons are available at Jackson Lab project.

To access: Contact Jackson Labs.

VCP patient samples are available by request:

Patient fibroblasts are available at Coriell Institute for Medical Research
Blood and urine samples are available at Cure VCP Disease / CombinedBRAIN Biorepository
Other samples are available at MRC Centre Neuromuscular Biobank.

If the repository does not meet your current research needs, any researcher may contact Cure VCP Disease directly and our organization may be able to facilitate introductions between researchers.

The University of Maryland Brain and Tissue Bank gratefully accepts brain and other tissue donations from individuals with neurologic, development, movement and psychiatric disorders as well as from individuals with none of these underlying diseases to serve as unaffected controls.

Our researchers have worked with the NIH and the University of Maryland to establish a process so of ensuring that donations are properly collected and stored so that each donor's gift aids the advancement in VCP research .

For patients interested in donating UMB SOM - BTB: Tissue Donor Registration Form (umaryland.edu)

For investigators Investigators | University of Maryland School of Medicine (umaryland.edu)

ANIMAL / ORGANOID MODELS

Our approach to in vivo models is to evaluate existing VCP models and explore funding and grant opportunities for new models.

Method: CRISPR/Cas9-engineered Drosophila knock-in models.

Reference: Wall JM, Basu A, Zunica ERM, et al. CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases. Dis Model Mech. 2021;14(7):dmm048603. doi:10.1242/dmm.048603

Characteristics: Degeneration, progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function, nuclear morphology defects, and sex-specific phenotypic differences in several mutants, listed below. N384H was embryonically lethal.

To access: Contact Alyssa E Johnson at Louisiana State University, Department of Biological Sciences, Baton Rouge, LA 70803, USA.

Method: Systemic CRISPR/Cas9-mediated VCP knockout zebrafish

(crRNA:tracrRNA/Cas9 targeting exon 3)

Characteristics: Lethal at the embryological stage of 84 hpf. Shows heart and skeletal muscle pathology.

Reference: Voisard, Philipp et al. “CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo.” International journal of molecular sciences vol. 23,12 6722. 16 Jun. 2022, doi:10.3390/ijms23126722

Constitutive VCP knockout mouse

Note: Constitutive VCP knockout in mice causes early embryonic lethality.

Reference: Müller J.M., Deinhardt K., Rosewell I., Warren G., Shima D.T. Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality. Biochem. Biophys. Res. Commun. 2007;354:459–465. doi: 10.1016/j.bbrc.2006.12.206.

VCP conditional knockout mouse (cKO)

Method: Cross VCP gene (VCPFL/FL) with CaMKIIα-Cre mice that express Cre recombinase under the CaMKIIα (calcium/calmodulin-dependent protein kinase II alpha) promoter (VCPFL/FL:CamkCre, termed VCP conditional knockout [cKO]).

Characteristics: Decrease in both nuclear and cytosolic VCP in neurons from the CA1 region of the hippocampus and cortex in VCP cKO mouse brains. Develop the full spectrum of FTLD-TDP with neuronal degeneration, behavioral changes, TDP-43 inclusions, and lysosomal pathology. Other mouse models of FTLD-TDP pathology have been generated.

To access: Contact Chris Weihl

Reference: Wani, Abubakar, et al. "Neuronal VCP Loss of Function Recapitulates FTLD-TDP Pathology." Cell Reports, vol. 36, no. 3, 2021, p. 109399, https://doi.org/10.1016/j.celrep.2021.109399. Accessed 14 Mar. 2023.

Cardiac-specific dominant-negative VCP transgenic (DN-VCP TG) mouse

Reference: Sun, Xiaonan, et al. "Functional Inhibition of Valosin-Containing Protein Induces Cardiac Dilation and Dysfunction in a New Dominant-Negative Transgenic Mouse Model." Cells, vol. 10, no. 11, 2021, https://doi.org/10.3390/cells10112891. Accessed 14 Mar. 2023.

R155C VCP knockin mouse

Characteristics: Showed decreased plasma lactate, serum albumin, and total protein concentrations, platelet numbers, and liver-to-body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals.

Reference: Clemen, Christoph S et al. “The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?.” Biochemical and biophysical research communications vol. 503,4 (2018): 2770-2777. doi:10.1016/j.bbrc.2018.08.038

R155H and A232E knockin mouse

Characteristics: Developed pathology that is limited to muscle, brain and bone, recapitulating the spectrum of disease in humans with IBMPFD. The mice exhibit progressive muscle weakness and pathological examination of muscle shows classic characteristics of inclusion body myopathy including rimmed vacuoles and TDP-43 pathology. The mice exhibit abnormalities in behavioral testing and pathological examination of the brain shows widespread TDP-43 pathology.

To access: Available for purchase at Jackson Lab

Reference: Custer, Sara K et al. “Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone.” Human molecular genetics vol. 19,9 (2010): 1741-55. doi:10.1093/hmg/ddq050

VCPR155H/+ knockin mouse

Characteristics: Developed significant progressive muscle weakness, progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies, and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons.

Reference: Nalbandian, Angèle, et al. "A Progressive Translational Mouse Model of Human VCP Disease: The VCP R155H/+ Mouse." Muscle & Nerve, vol. 47, no. 2, 2013, p. 260, https://doi.org/10.1002/mus.23522. Accessed 14 Mar. 2023.

VCPR155H/+ knockin mouse under a muscle-specific promoter

Characteristics: Weakness progressively starting at 6 months of age. Abnormal muscle pathology, which included coarse internal architecture, vacuolation, and disorganized membrane morphology with reduced caveolin-3 expression at the sarcolemma developed coincident with the onset of weakness. Show an increase in ubiquitin-containing protein inclusions and high-molecular-weight ubiquitinated proteins, markers of UPS dysfunction.

Reference: Weihl, Conrad C et al. “Transgenic expression of inclusion body myopathy associated mutant p97/VCP causes weakness and ubiquitinated protein inclusions in mice.” Human molecular genetics vol. 16,8 (2007): 919-28. doi:10.1093/hmg/ddm037

General remark on VCP knockin and transgenic mouse models:

Models show a late manifestation of neuro-muscular pathologies mostly after 18 months, with TDP-43 pathology from 13 to 15 months, and a reduction in motor neurons after 20 months.

We are currently exploring new animal models. Please contact armelle.pindon@curevcp.org if you would like to discuss or learn more

LISTING OF RESEARCH RESOURCES

A listing of where to buy research products and reference compounds available for VCP research.

KUS 121:

Phase I/II

At Kyoto university

Conditions: Central retinal artery occlusion

Indications: Treatment of non-arteritic central retinal artery occlusion

Mechanism of action: VCP inhibitor

CB5339:

Phase I

At CLEAVE therapeutics

Conditions: Acute myeloid leukemia, Cancer, solid tumor, Myelodysplasia, Non-Hodgkin lymphoma,

Indications: In adult patients with relapsed or refractory acute myeloid leukemia; with locally advanced or metastatic solid tumors; with aggressive lymphoma.

Mechanism of action: VCP inhibitor