Clinical Development of JS-K for the Treatment of Cancer

National Cancer Institute

Grant Number: 1R44CA221570-01
FAIN: R44CA221570
Principal Investigator(s): Joseph Felix Nowoslawski, MDv Project Title: Clinical Development of JS-K for the Treatment of Cancer
Amount: $1,998,606

SK Therapeutics (JSKT) Direct to Phase II Application Nitric oxide (NO) is a potentially powerful therapy against cancer. Spontaneous NO-generating drugs cannot be used to treat malignancies because of the pleiotropic effects of NO. In particular, NO is a potent vasodilator with resultant hypotension. In order to make NO-based cancer therapy possible, it is necessary to develop agents that deliver NO preferentially to malignant cells. In collaboration with Dr. Larry Keefer at the National Cancer Institute, Dr. Paul Shami has developed such a drug using compounds that release NO upon interaction with glutathione (GSH) in a reaction catalyzed by Glutathione S-Transferases (GST). This drug design exploits the over-expression of GST in malignant cells. A lead compound was identified as the most active of this family: O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS- K. JS-K has potent and broad-spectrum anti-cancer activity in vitro and in vivo. Because of challenging solubility and stability properties, the Shami lab has developed a formulation for JS-K using Pluronic P123 micelles. JS-K formulated in P123 micelles (P123/JS-K) was well tolerated in a non-GLP dog toxicology study without the induction of hypotension. JS-K is directly cytotoxic to malignant cells and also an inhibitor of angiogenesis. JS-K is a very efficient consumer of the anti-oxidant tripeptide glutathione (GSH), rendering malignant cells particularly susceptible to oxidative stress. JS-K is not toxic towards normal hematopoietic cells. JS-K is synergistic with cytarabine against acute myeloid leukemia (AML) and with bortezomib against multiple myeloma (MM). Recent data suggest that JS-K may sensitize MM cells to the cytotoxic effect of natural killer (NK) cells, raising the possibility of synergistic combinations with novel immunomodulatory agents. One kilogram of JS-K Active Pharmaceutical Ingredient was produced under cGMP conditions. JSK Therapeutics (JSKT) was founded to lead commercialization of JS-K. The United States Food and Drug Administration has granted orphan drug designation for JS-K for the indications of AML and MM. JSKT plans to develop JS-K initially for the treatment of AML and MM. The objective of this project is to complete pre-clinical animal toxicology studies needed to file an Investigational New Drug (IND) application with the FDA. In 4 aims, an analytical method for the measurement of JS-K in biologic matrices will be developed and validated; GLP toxicology studies in rats and dogs using a schedule of administration that reproduces the prospective clinical schedule will be conducted; GLP lung and central nervous system toxicology studies will be conducted in rats; and a GLP cardiovascular system toxicology study will be conducted in dogs. At the completion of this project, JSKT will submit an IND application to the FDA in order to start Phase I clinical trials for relapsed/refractory AML and MM. This will allow partnering with an established pharmaceutical company or obtaining venture investment in order to complete the clinical development and commercialization of JS-K, thus adding a potent new drug to our armamentarium aimed at diminishing the pain and suffering associated with cancer.

Posted in Grant Opportunities

Speculative Grant Proposal: Cloning the Lichen Prion-Degrading Serine Protease and Testing Its Efficacy in a PrP^Sc–Mediated Chronic Pain Model

Cloning the Lichen Prion-Degrading Serine Protease and Testing Its Efficacy in a PrP^Sc–Mediated Chronic Pain Model
Proposed Funding Mechanism: NIH R01 / DoD PRMRP / CURE / PCBI
Duration: 5 Years
Total Direct Costs (estimated): ~$2,500,000 USD

Abstract

PrP^C is an endogenous inhibitor of NMDA receptor signaling in the spinal dorsal horn, and its loss or dysfunction drives central sensitization in chronic pain states (Gadotti & Zamponi, 2011). We hypothesize that after peripheral nerve injury (e.g., surgery, trauma), misfolded, β-sheet-rich PrP (PrP^C-like) accumulates at axon terminals and/or within synaptic compartments, sequestering functional PrP^C and producing a local functional knockout — disinhibiting NMDA receptors and triggering chronic regional pain.

A serine protease of fungal origin, found in ~50% of tested lichen species (Johnson et al., 2011; 2012; Rodriguez et al., 2012), has been shown to degrade PrP^Sc β-sheets by ≥2 logs in vitro. However, the enzyme has never been cloned, sequenced, or recombinantly expressed. This proposal outlines a complete pipeline to: (1) identify and clone the lichen serine protease gene from Parmelia sulcata; (2) recombinantly express and purify the active enzyme; (3) characterize its kinetics against PrP^Sc from multiple mammalian species; and (4) test its ability to reverse PrP^Sc-mediated chronic pain in a newly developed CRPS rodent model.

If successful, this work will yield the first targeted biological tool for degrading β-sheet PrP aggregates at peripheral nerve endings, opening an entirely new therapeutic axis for chronic regional pain syndromes.

Specific Aims

Aim 1. Identify and Clone the Prion-Degrading Serine Protease Gene from Parmelia sulcata

Rationale: The activity has been confirmed as a serine protease of fungal mycobiont origin (Johnson et al., 2011), but its molecular identity is completely unknown. P. sulcata shows the highest reported activity. No genome sequence is publicly available.

Experimental Plan:

Step	Method	Expected Outcome
1.1	Collect and axenically culture P. sulcata mycobiont (isolate from algal/bacterial partners)	Pure fungal culture confirmed by ITS barcoding
1.2	Extract total RNA from culture at timepoints of peak protease activity (assayed by zymography against recombinant PrP^Sc)	RNA-seq library from active cultures
1.3	Whole-transcriptome sequencing (Illumina NovaSeq 6000, 150 bp paired-end, ~50M reads)	Transcriptome assembly (~15,000–25,000 fungal gene models)
1.4	Bioinformatic filter for candidate serine proteases:	20–50 candidate serine protease sequences
	Pfam domains (Peptidase_S8, Trypsin S1, or Pro-protein convertase S8/S53) signal peptide (SignalP 6.0) → secreted upregulated during peak activity
1.5	Heterologous expression of top 10 candidates in Pichia pastoris (pPICZα vector, methanol-induced)	10 recombinant secreted proteases
1.6	High-throughput activity screen: incubate each recombinant protease with proteinase K–resistant PrP^Sc (purified from RML scrapie mouse brain); measure degradation by:	1 positive hit (or more) — the protease(s) that degrade PrP^Sc
	Western blot (3F4 antibody, proteinase K resistance loss) Thioflavin T fluorescence (β-sheet loss) PMCA (amplification competence loss)

Go/No-Go Criteria:

Go: ≥1 candidate clone shows ≥1 log reduction in PrP^Sc signal within 60 min at 25°C
No-Go: No candidate active → revisit transcriptome for missed candidates, consider activity-guided biochemical purification (Aim 1 alternative path)

Alternative Path (if transcriptome mining fails):

If no recombinant candidate shows activity, fall back to classical biochemical purification:

1. Acetone precipitation of P. sulcata thallus
2. Ion-exchange chromatography (Q Sepharose)
3. Size-exclusion chromatography (Superdex 200)
4. Activity tracking via zymogram gel
5. Mass spectrometry (LC-MS/MS) of active band → peptide fingerprint → degenerate PCR → gene cloning

Aim 2. Recombinant Expression, Purification, and Biochemical Characterization of the Active Enzyme

Rationale: Even a single positive hit requires full characterization to confirm identity and determine suitability for pain model testing.

Experimental Plan:

Step	Method	Expected Outcome
2.1	Scale-up expression in P. pastoris (5 L fermenter), Ni-NTA purification (His-tagged)	10–50 mg purified enzyme
2.2	Biochemical characterization:	Full kinetic profile; confirm serine protease class and subfamily
	pH optimum (3–11, 0.5 pH unit increments) Temperature optimum (10–70°C) Thermostability (Tm by CD or DSF) Ionic strength dependence: 0–1 M NaC Inhibitor profile: Pefabloc SC, PMSF, AEBSF, aprotinin, leupeptin, chymostatin, EDTA, E-64, pepstatin A | Confirm serine protease class; identify subfamily
2.3	Substrate specificity:	PrP^Sc degradation kinetics
	Recombinant PrP (full-length mouse/human, α-helical) vs. PrP^Sc (β-sheet rich, from RML mice, CWD deer, and sCJD patients) Kinetic parameters: V_max, K_m, k_cat, k_cat/K_m Monitor by: Western blot, ThT fluorescence, CD spectroscopy (β-sheet loss → random coil)
2.4	Structural characterization:	High-resolution structure; active site geometry for rational drug design
	Crystallization trials (sitting drop vapor diffusion) Cryo-EM if crystals fail (200–300 kDa range depends on oligomerization) | Active site geometry → rational drug design later
2.5	Infectivity assay (gold standard)	Definitive proof of prion deactivation
	Incubate RML scrapie brain homogenate with recombinant protease (1:100 enzyme:substrate, 37°C, 1 hr) Intracerebral inoculation into tga20 mice (n = 10 per group) Monitor for prion disease symptoms (scrapie incubation time assay) Compare to untreated controls

Go/No-Go Criteria:
Go: ≥2 log reduction in PrP^Sc within 1 hour AND eliminates infectivity (survival ≥ untreated control)
No-Go: Partial degradation but infectivity retained → reconsider mechanism and suitability for therapeutic use

Aim 3. Develop a CRPS Rodent Model with Demonstrable PrP β-Sheet Accumulation at Peripheral Nerve Endings

Rationale: No existing chronic pain model has been characterized for PrP β-sheet aggregates. This model must be validated first.

Experimental Plan:

Step	Method	Expected Outcome
3.1	Model selection: chronic constriction injury (CCI) of the sciatic nerve in adult male/female C57BL/6 mice	Established neuropathic pain model, analogous to surgical nerve damage
3.2	Behavioral phenotyping (days 0–28 post-surgery):	Confirmed chronic pain state
	Mechanical allodynia (von Frey filaments, up-down method)	↓ threshold
	Thermal hyperalgesia (Hargreaves test)	↓ latency
	Cold allodynia (acetone drop)	↑ response
	Weight bearing asymmetry (incapacitance meter)	↑ asymmetry
3.3	PrP β-sheet detection at axon terminals (the central test of the hypothesis):
	A. Immunofluorescence: | PrP co-localized with β-sheet markers
	Harvest ipsilateral DRG (L4-L5), dorsal horn (spinal cord L4-L5), and sciatic nerve distal to injury Stain for: PrP (SAF-84 or 3F4 antibody), β-sheet conformation (OC antibody or antibody 15B3, specific for β-sheet PrP^Sc), axonal marker (NF200), synaptic marker (synaptophysin) Quantify: % PrP-positive puncta that are also OC/15B3-positive
	B. Biochemical detection: | Quantified PrP^Sc-like material
	Dissect sciatic nerve distal to injury (2 mm segment) Proteinase K digestion (50 µg/mL, 37°C, 30 min) Western blot: PrP^Sc detected as PK-resistant band at ~27–30 kDa (PrP^C is fully digested) Quantify band intensity relative to uninjured contralateral nerve
	RT-QuIC (Real-Time Quaking-Induced Conversion): | Seeded amplification of β-sheet PrP
	Use sciatic nerve homogenate as seed Use sciatic nerve homogenate as seed Amplification at 42°C, ThT fluorescence read every 15 min Compare lag time: ipsilateral vs. contralateral vs. sham
3.4	Correlation analysis:	Dose-response relationship
	PK-resistant PrP^Sc amount (from 3.3B) vs. pain behavior (von Frey threshold at day 14) Pearson r correlation, n = 12 per group

Go/No-Go Criteria:
Go: Go: PK-resistant PrP^Sc detected at sciatic nerve endings of CCI mice, with significant correlation (r ≥ 0.6, p < 0.05) between PrP^Sc amount and pain severity
No-Go: No PrP^Sc detected → hypothesis is wrong at the level of simple aggregation → pivot to testing PrP^C loss mechanism (cellular fractionation, surface biotinylation to quantify end-terminal PrP^C)

Aim 4. Test Lichen Protease as a Therapeutic for PrP β-Sheet–Mediated Chronic Pain

Rationale: The ultimate test of the hypothesis — does degrading PrP β-sheets at peripheral nerve endings reverse chronic pain?

Experimental Plan:

Step	Method	Expected Outcome
4.1	Dose-finding study:	Optimal dose and route identified
	Single local injection at the sciatic nerve injury site (perineural), days 1, 7, 14 post-CCI Doses: 0.1, 0.5, 1.0, 5.0 µg in 5 µL sterile PBS n = 6/group Pain behavior measured 24 hr, 72 hr, 7 d post-injection PrP^Sc level measured by biopsy + Western blot at endpoint
4.2	Efficacy study (the main event):	Therapeutic proof-of-concept
	CCI surgery (day 0) Treatment: protease (optimal dose from 4.1) vs. heat-inactivated protease (control) vs. vehicle (PBS) Route: perineural injection at injury site Schedule: days 1, 8, 15, 22 n = 12/group (6 male, 6 female)	Primary: Mechanical allodynia (von Frey) at days 2, 4, 7, 9, 11, 14, 16, 21, 28 Secondary: Thermal hyperalgesia, cold allodynia, weight bearing, spontaneous pain (grimace scale) Biomarker: PrP^Sc level (PK-resistant) in sciatic nerve at day 28
4.3	Mechanism confirmation:	NMDA receptor link confirmed or refuted
	Repeat 4.2 with additional group: protease + MK-801 (NMDA antagonist, 0.1 mg/kg i.p.) If protease works via PrP^Sc degradation → restoring PrP^C function → NMDA inhibition Prediction: protease + MK-801 should NOT be additive (same pathway) If additive → mechanism is independent of NMDA receptors
4.4	Safety assessment:	No off-target nerve damage
	Histology of sciatic nerve (H&E, Luxol fast blue for myelin) at injection site Macrophage/mast cell infiltration (Iba-1, CD68 staining) Locomotor function (rotarod, open field) Serum cytokines (IL-6, TNF-α, IL-1β) — systemic inflammation?

Go/No-Go Criteria for Clinical Translation:
Go: ≥50% reversal of mechanical allodynia (von Frey threshold restored to ≥75% of pre-injury baseline), sustained for ≥7 days after last dose, with no significant nerve damage
No-Go: Partial or no effect → consider: (1) wrong mechanism, (2) poor enzyme stability in vivo, (3) insufficient tissue penetration

Timeline

Budget (Estimated Direct Costs)

Category	Year 1	Year 2	Year 3	Year 4	Year 5	Total
Personnel (PI, postdoc, tech, grad)	$180K	$185K	$190K	$196K	$202K	$953K
Supplies (molecular biology, protein purification, animal housing)	$75K	$100K	$120K	$100K	$80K	$475K
Sequencing (RNA-seq, genome)	$50K	$10K	—	—	—	$60K
Mass spectrometry	—	$15K	$5K	—	—	$20K
Crystallography/cryo-EM (core fees)	—	—	$40K	$30K	—	$70K
Animal purchase and per diem	$10K	$30K	$40K	$40K	$30K	$150K
Behavioral equipment (von Frey, Hargreaves)	$25K	—	—	—	—	$25K
Publication fees / travel	$5K	$5K	$10K	$10K	$15K	$45K
Total Direct	$345K	$345K	$405K	$376K	$327K	$1,798K
Indirect (F&A, 55% on-campus)	$190K	$190K	$223K	$207K	$180K	$990K
Total	$535K	$535K	$628K	$583K	$507K	$2,788K

Potential Pitfalls and Mitigations

Pitfall	Risk	Mitigation
Enzyme not produced by mycobiont alone — requires algal/bacterial partner	Medium	Metagenomic RNA-seq includes all symbionts; heterologous expression in P. pastoris handles eukaryotic or prokaryotic genes
Enzyme is a post-translationally modified complex (glycosylated, requires chaperone)	Medium	Use P. pastoris (glycosylation competent); also try E. coli with refolding; cell-free expression as backup
Recombinant enzyme is inactive	High	Include native purification (Aim 1 alternative path) in parallel; co-expression with Kex2 protease to address post-translational processing
No PrP^Sc detected in CCI mouse sciatic nerve (Aim 3 fails)	High	Refutes the strong hypothesis but allows testing of PrP^C loss-of-function mechanism; pivot Aim 4 to PrP^C replacement therapy
Protease causes nerve damage at injection site	Medium	Tune dose; formulate with controlled-release (PLGA nanoparticles); engineer active-site mutants with reduced off-target proteolysis
PrP^Sc is intracellular — protease cannot reach it	Medium	Test cell-penetrating peptide fusion (TAT, penetratin); use liposomal formulation; focus on extracellular synaptic PrP^Sc pool
Protease is immunogenic (foreign fungal protein)	Medium	Humanize or engineer a minimized catalytic domain; test single vs. repeated dosing; measure anti-drug antibodies

Innovation Statement

This proposal is innovative on multiple levels:

1. First cloning of a lichen prion-degrading protease — a 15-year gap in the literature addressed directly
2. First demonstration of PrP β-sheet aggregates at peripheral nerve endings in chronic pain — a fundamentally new mechanism for CRPS
3. First biological (non-small-molecule) therapy for chronic regional pain — a topical/injectable enzyme that degrades the pathological substrate itself, rather than modulating downstream signaling
4. Cross-disciplinary convergence — lichenology × prion biology × pain neuroscience × protein engineering — a combination never before attempted

Significance and Impact

If successful, this work would:

1. Identify the lichen serine protease — filling a 15-year gap and opening the door to engineering a thermostable, human-tolerated variant
2. Validate a new pain mechanism — PrP β-sheet aggregation at injured peripheral nerve endings as a driver of chronic regional pain
3. Provide a first-in-class topical therapeutic — a β-sheet-degrading enzyme applied at the time of surgery to prevent chronic pain, analogous to antibiotic prophylaxis for surgical site infection
4. Offer a tool for other β-sheetopathies — the same enzyme could be tested in Alzheimer’s, Parkinson’s, and other protein aggregation diseases where peripheral pathology contributes to symptoms

Final Note

This proposal rests on a single, testable proposition:

If PrP β-sheet aggregates at peripheral nerve endings cause chronic pain, then specifically degrading those β-sheets with a targeted protease should resolve the pain.

The lichen serine protease — if it can finally be cloned — is the ideal tool for this experiment. Its existence in nature (evolved to degrade prions in the environment) provides the perfect starting point for an enzyme therapy that has no equivalent in the current pain pharmacopoeia.

Posted in Grant Opportunities