Synthesis and Evaluation of a Stable Isostere of Malonyllysine.

Lysine malonylation is a recently characterized post-translational modification involved in the regulation of energy metabolism and gene expression. One unique feature of this post-translational modification is its potential susceptibility to decarboxylation, which poses possible challenges to its study. As a step towards addressing these challenges, we report the synthesis and evaluation of a stable isostere of malonyllysine. First, we find that synthetic substitution of the malonyl group with a tetrazole isostere results in amino acid's resistant to thermal decarboxylation. Next, we demonstrate that protected variants of this amino acid are readily incorporated into peptides. Finally, we show that tetrazole isosteres of malonyllysine can be recognized by anti-malonyllysine antibodies and histone deacylases, validating their ability to mimic features of the endogenous lysine modification. Overall, this study establishes a new chemical strategy for stably mimicking a metabolite-derived post-translational modification, providing a foothold for tool development and functional analyses.

Preparation of Bioderived and Biodegradable Surfactants Based on an Intrinsically Disordered Protein Sequence.

Surfactants, block copolymers, and other types of micellar systems are used in a wide variety of biomedical and industrial processes. However, most commonly used surfactants are synthetically derived and pose environmental and toxicological concerns throughout their product life cycle. Because of this, bioderived and biodegradable surfactants are promising alternatives. For biosurfactants to be implemented industrially, they need to be produced on a large scale and also have tailorable properties that match those afforded by the polymerization of synthetic surfactants. In this paper, a scalable and versatile production method for biosurfactants based on a hydrophilic intrinsically disordered protein (IDP) sequence with a genetically engineered hydrophobic domain is used to study variables that impact their physicochemical and self-assembling properties. These amphiphilic sequences were found to self-assemble into micelles over a broad range of temperatures, pH values, and ionic strengths. To investigate the role of the IDP hydrophilic domain on self-assembly, variants with increased overall charges and systematically decreased IDP domain lengths were produced and examined for their sizes, morphologies, and critical micelle concentrations (CMCs). The results of these studies indicate that decreasing the length of the IDP domain and consequently the molecular weight and hydrophilic fraction leads to smaller micelles. In addition, significantly increasing the amount of charged residues in the hydrophilic IDP domain results in micelles of similar sizes but with higher CMC values. This represents an initial step in developing a quantitative model for the future engineering of biosurfactants based on this IDP sequence.

Self-Assembling Micelles Based on an Intrinsically Disordered Protein Domain.

The self-assembly of micellar structures from diblock polymers that contain hydrophilic and hydrophobic domains has been of great interest for the encapsulation of drugs and other hydrophobic molecules. While most commercially used surfactants are derived from hydrocarbon sources, there have been recent efforts to replace these with biodegradable, nontoxic, biologically synthesized alternatives. Previous examples have primarily examined naturally occurring self-assembling proteins, such as silk and elastin-like sequences. Herein, we describe a new series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles that are 27 nm in diameter after enzymatic cleavage of a solubilizing protein tag. The sequences of the proteins are based on a human intrinsically disordered protein, which has been appended with a hydrophobic segment. The micelles were found to form across a broad range of pH, ionic strength, and temperature conditions, with critical micelle concentration (CMC) values in the low micromolar range, 3 orders of magnitude lower than the CMC of commonly used surfactant sodium dodecyl sulfate (SDS). The reported micelles were found to solubilize hydrophobic metal complexes and organic molecules, suggesting their potential suitability for catalysis and drug delivery applications. Furthermore, the inherent flexibility in the design of these protein sequences enables the encoding of additional functionalities for many future applications. Overall, this work represents a new biomolecular alternative to traditional surfactants that are based on nonrenewable and poorly biodegradable hydrocarbon sources.

Systemic Administration of Cowpea Mosaic Virus Demonstrates Broad Protection Against Metastatic Cancers.

The key challenge in cancer treatment is prevention of metastatic disease which is therapeutically resistant and carries poor prognoses necessitating efficacious prophylactic approaches that prevent metastasis and recurrence. It is previously demonstrated that cowpea mosaic virus (CPMV) induces durable antitumor responses when used in situ, i.e., intratumoral injection. As a new direction, it is showed that CPMV demonstrates widespread effectiveness as an immunoprophylactic agent - potent efficacy is demonstrated in four metastatic models of colon, ovarian, melanoma, and breast cancer. Systemic administration of CPMV stimulates the innate immune system, enabling attack of cancer cells; processing of the cancer cells and associated antigens leads to systemic, durable, and adaptive antitumor immunity. Overall, CPMV demonstrated broad efficacy as an immunoprophylactic agent in the rejection of metastatic cancer.

Pharmacology of a Plant Virus Immunotherapy Candidate for Peritoneal Metastatic Ovarian Cancer.

Due to the increasing incidence of cancer, there is a need to develop new platforms that can combat this disease. Cancer immunotherapy is a platform that takes advantage of the immune system to recognize and eradicate tumors and metastases. Our lab has identified a plant virus nanoparticle, cowpea mosaic virus (CPMV) as a promising approach for cancer immunotherapy. When administered intratumorally, CPMV relieves the immune system of tumor-induced immunosuppression and reprograms the tumor microenvironment into an activated state to launch systemic antitumor immunity. The efficacy of CPMV has been tested in many tumor models and in canine cancer patients with promising results: tumor shrinkage, systemic efficacy (abscopal effect), and immune memory to prevent recurrence. To translate this drug candidate from the bench to the clinic, studies that investigate the safety, pharmacology, and toxicity are needed. In this work, we describe the efficacy of CPMV against a metastatic ovarian tumor model and investigate the biodistribution of CPMV after single or repeated intraperitoneal administration in tumor-bearing and healthy mice. CPMV shows good retention in the tumor nodules and broad bioavailability with no apparent organ toxicity based on histopathology. Data indicate persistence of the viral RNA, which remains detectable 2 weeks post final administration, a phenomenon also observed with some mammalian viral infections. Lastly, while protein was not detected in stool or urine, RNA was shed through excretion from mice; however, there was no evidence that RNA was infectious to plants. Taken together, the data indicate that systemic administration results in broad bioavailability with no apparent toxicity. While RNA is shed from the subjects, data suggest agronomical safety. This data is consistent with prior reports and provides support for translational efforts.

Cellular fate of a plant virus immunotherapy candidate.

Cowpea mosaic virus (CPMV) is a plant virus that is currently being developed for intratumoral immunotherapy. CPMV relieves the immune system from tumor-induced immunosuppression; reprograms the tumor microenvironment to an activated state whereby the treated and distant tumors are recognized and eradicated. Toward translational studies, we investigated the safety of CPMV, specifically addressing whether pathogenicity would be induced in mammalian cells. We show that murine macrophage immune cells recognize CPMV; however, there is no indication of de novo viral protein synthesis or RNA replication. Furthermore, we show that CPMV does not induce hemolysis, platelet aggregation and plasma coagulation amongst other assays in human blood and immune cells. Taken together, we anticipate that these results will reinforce the development of CPMV as an immunotherapeutic platform.

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

In Vivo Fate of Cowpea Mosaic Virus In Situ Vaccine: Biodistribution and Clearance.

Cowpea mosaic virus (CPMV) is a nucleoprotein nanoparticle that functions as a highly potent immunomodulator when administered intratumorally and is used as an in situ vaccine. CPMV in situ vaccination remodels the tumor microenvironment and primes a highly potent, systemic, and durable antitumor immune response against the treated and untreated, distant metastatic sites (abscopal effect). Potent efficacy was demonstrated in multiple tumor mouse models and, most importantly, in canine cancer patients with spontaneous tumors. Data indicate that presence of anti-CPMV antibodies are not neutralizing and that in fact opsonization leads to enhanced efficacy. Plant viruses are part of the food chain, but to date, there is no information on human exposure to CPMV. Therefore, patient sera were tested for the presence of immunoglobulins against CPMV, and indeed, >50% of deidentified patient samples tested positive for CPMV antibodies. To get a broader sense of plant virus exposure and immunogenicity in humans, we also tested sera for antibodies against tobacco mosaic virus (>90% patients tested positive), potato virus X (<20% patients tested positive), and cowpea chlorotic mottle virus (no antibodies were detected). Further, patient sera were analyzed for the presence of antibodies against the coliphage Qß, a platform technology currently undergoing clinical trials for in situ vaccination; we found that 60% of patients present with anti-Qß antibodies. Thus, data indicate human exposure to CPMV and other plant viruses and phages. Next, we thought to address agronomical safety; i.e., we examined the fate of CPMV after intratumoral treatment and oral gavage (to mimic consumption by food). Because live CPMV is used, an important question is whether there is any evidence of shedding of infectious particles from mice or patients. CPMV is noninfectious toward mammals; however, it is infectious toward plants including black-eyed peas and other legumes. Biodistribution data in tumor-bearing and healthy mice indicate little leaching from tumors and clearance via the reticuloendothelial system followed by biliary excretion. While there was evidence of shedding of RNA in stool, there was no evidence of infectious particles when plants were challenged with stool extracts, thus indicating agronomical safety. Together these data aid the translational development of CPMV as a drug candidate for cancer immunotherapy.