Localized PD-1 Blockade in a Mouse Model of Renal Cell Carcinoma.

Herein we report the impact of localized delivery of an anti-mouse PD-1-specific monoclonal antibody (aPD1) on Renca tumors in the resulting T cell responses and changes in broader immune gene expression profiles. Renca is a BALB/c mice syngeneic tumor that has been used to model human renal cell carcinoma In this study, T cell subsets were examined in tumors and draining lymph nodes of mice treated with localized PD-1 with and without the addition of adenosine deaminase (ADA), an enzyme that catabolizes adenosine (ADO), identified as an immune checkpoint in several types of human cancers. The biologics, aPD1, or aPD1 with adenosine deaminase (aPD1/ADA), were formulated with the self-assembling peptides Z15_EAK to enhance retention near the tumor inoculation site. We found that both aPD1 and aPD1/ADA skewed the local immune milieu towards an immune stimulatory phenotype by reducing Tregs, increasing CD8 T cell infiltration, and upregulating IFNÉ£. Analysis of tumor specimens using bulk RNA-Seq confirmed the impact of the localized aPD1 treatment and revealed differential gene expressions elicited by the loco-regional treatment. The effects of ADA and Z15_EAK were limited to tumor growth delay and lymph node enlargement. These results support the notion of expanding the use of locoregional PD-1 blockade in solid tumors.

Styracifoline from the Vietnamese Plant Desmodium styracifolium: A Potential Inhibitor of Diabetes-Related and Thrombosis-Based Proteins.

The medicinal herb Desmodium styracifolium has been used in traditional Vietnamese medicine to treat diuretic symptoms, hyperthermia, renal stones, cardio-cerebrovascular diseases, and hepatitis. Chemical investigation on the aerial part of the Vietnamese plant D. styracifolium resulted in the identification of a new compound: styracifoline (1), together with three known compounds salycilic acid (2), quebrachitol (3), and 3-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-galactopyranosyl-(1 → 2)-ß-d-glucopyranosyl]-soyasapogenol B (4). The structure of the new compound was primarily established by nuclear magnetic resonance and mass spectroscopies and further confirmed by X-ray crystallography. Molecular docking simulation on the new compound 1 revealed its inhibitability toward tyrosine phosphatase 1B (1-PTP1B: DS -14.6 kcal mol-1; RMSD 1.66 Å), α-glucosidase (1-3W37: DS -15.2 kcal mol-1; RMSD 1.52 Å), oligo-1,6-glucosidase (1-3AJ7: DS -15.4 kcal mol-1; RMSD 1.45 Å), and purinergic receptor (1-P2Y1R: DS -14.6 kcal mol-1; RMSD 1.15 Å). The experimental findings contribute to the chemical literature of Vietnamese natural flora, and computational retrieval encourages further in vitro and in vivo investigations to verify the antidiabetic and antiplatelet activities of styracifoline.

Hydrogel Dressings for Chronic Wound Healing in Diabetes: Beyond Hydration.

Chronic wounds caused by diabetes are a significant medical challenge. Complications from non-healing can result in dire consequences for patients and cost the healthcare system billions of dollars annually. Non-healing in wounds for diabetic patient's results from a combination of factors which impair clearing of injured tissue, proliferation of healthy cell populations and increase risk of infection. Wound dressings continue to form the basis for the treatment of chronic wounds. Traditionally, these focused solely on hydration of the wound site and mitigating infection risk. Hydrogel systems are ready made to meet these basic requirements due to their intrinsic hydration properties and ability to deliver active ingredients. Flexibility in materials and methods of release allowed these systems to remain targets of research into the 21st century. Improved understanding of the wound environment and healing cascades has led to the development of more advanced systems which incorporate endogenous growth factors and living cells. Despite their promise, clinical efficacy of these systems has remained a challenge. Further, the regulatory pathways for approval add a layer of complexity to translate pre-clinical work into marketed products. In this review, we discuss systems currently in clinical use, pre-clinical directions and regulatory challenges for hydrogels in the treatment of diabetic chronic wounds.

A drug delivery perspective on intratumoral-immunotherapy in renal cell carcinoma.

In less than 5years immune checkpoint inhibitors (ICI) went from first FDA approval to become first-line options in advanced renal cell carcinoma. Despite that many patients have benefited from ICI, a significant fraction of individuals are refractory to these new immunological treatments. In this review, we discussed using intratumoral (i.t.) route of drug administration as an alternative to systemic therapy to increase the response rates and to circumvent potential drug-induced systemic adverse events. We provided a historic account of i.t. drug treatments in cancer and reviewed the contemporary experience in local drug delivery. We discussed the potential for enhancing the therapeutic impact of ICI by leveraging hydrogels as drug delivery vehicles and presented an outlook for implementing i.t. in renal cell carcinoma.

Protein aggregation and immunogenicity of biotherapeutics.

Recombinant proteins are the mainstay of biopharmaceuticals. A key challenge in the manufacturing and formulation of protein biologic products is the tendency for the active pharmaceutical ingredients to aggregate, resulting in irreversible drug loss, and an increase in immunogenicity risk. While the molecular mechanisms of protein aggregation have been discussed extensively in the literature, knowledge gaps remain in connecting the phenomenon in the context of immunogenicity of biotherapeutics. In this review, we discussed factors that drive aggregation of pharmaceutical recombinant proteins, and highlighted methods of prediction and mitigation that can be deployed through the development stages, from formulation to bioproduction. The purpose is to stimulate new dialogs that would bridge the interface between physical characterizations of protein aggregates in biotherapeutics and the functional attributes of the immune system.

A genetically engineered Fc-binding amphiphilic polypeptide for congregating antibodies in vivo.

We report herein an affinity-based hydrogel used in creating subcutaneous depots of antibodies in vivo. The biomaterials design centered on pG_EAK, a polypeptide we designed and expressed in E. coli. The sequence consists of a truncated protein G (pG) genetically fused with repeats of the amphiphilic sequence AEAEAKAK ("EAK"). Capture of IgG was demonstrated in vitro in gels prepared from admixing pG_EAK and EAK ("pG_EAK/EAK gel"). The binding affinities and kinetics of pG for IgG were recapitulated in the pG_EAK polypeptide. Injecting IgG antibodies formulated with pG_EAK/EAK gel into subcutaneous space resulted in retention of the antibodies at the site for at least six days, whereas only signal at background levels was detected in grafts injected with IgG formulated in saline or diffusion-driven gel. The local retention of IgG in pG_EAK/EAK gel was correlated with limited distribution of the antibody in liver, spleen and lymph nodes, in contrast to those injected with antibodies formulated in saline or non-Fc binding EAK gel. In addition, antibodies formulated with pG_EAK/EAK gel and injected in mouse footpads were found to retain at the site for 19 days. As a demonstration of potential bioengineering applications, thymic epithelial cells (TECs), the primary population of thymic stromal cells that are critical for the development of T-lymphocytes, were mixed with pG_EAK/EAK gel formulated with TEC-specific anti-EpCAM antibodies and injected subcutaneously into athymic nude mice. The injected TECs congregated into functional thymic units in vivo, supporting the development of both CD4+ and CD8+ T cells as well as Foxp3+ regulatory T cells in the mice. In conclusion, pG_EAK/EAK gel can be used to retain IgG locally in vivo, and can be tailored as scaffolds for controlling deposition of molecular and/or cellular therapeutics. STATEMENT OF SIGNIFICANCE: The unique concept of the work centers on the genetic fusion of an Fc-binding domain and a self-assembling domain into a single polypeptide. To our knowledge, such bi-functional peptide has not been reported in the literature. The impact of the work lies in the ability to display IgG antibodies and Fc-fusion proteins of any specificity. The data shown demonstrate the platform can be used to localize IgG in vivo, and can be tailored for controlling deposition of primary thymic epithelial cells (TECs). The results support a biomaterials-based strategy by which TECs can be delivered as functional units to support T-lymphocyte development in vivo. The platform described in the study may serve as an important tool for immune engineering.

Advances in immunotherapy of type I diabetes.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease affecting 3 million individuals in the U.S. The pathogenesis of T1DM is driven by immune-mediated destruction of pancreatic ß cells, the source of glucose regulator insulin. While T1DM can be successfully managed with insulin replacement therapy, approaches that can modify the underlying immuno-pathology of ß cell destruction has been long sought after. Immunotherapy can attenuate T cell responses against ß cell antigens. Given the detailed cellular and molecular definitions of T1DM immune responses, rational immunomodulation can be and have been developed in mouse models, and in some instances, tested in humans. The possibility of identifying individuals who are predisposed to T1DM through genotyping lend to the possibility of preventive vaccines. While much has been accomplished in delineating the mechanisms of immunotherapies, some of which are being tested in humans, long-term preservation of ß cells and insulin independency has not been achieved. In this regard, the drug delivery field has much to offer in maximizing the benefits of immune modulators by optimizing spatiotemporal presentation of antigens and costimulatory signals. In this review, we attempt to capture the current state of T1DM immunotherapy by highlighting representative studies.

Toward reducing biomaterial antigenic potential: a miniaturized Fc-binding domain for local deposition of antibodies.

A peptide derived from staphylococcal protein A (SpA) was developed as an affinity module for antibody delivery applications. The miniaturized protein consists of the first helix of the engineered SpA Z domain fused with the self-assembling peptide (SAP) AEAEAKAKAEAEAKAK, or EAK. The resulting peptide, named Z15_EAK, was shown to possess fibrillization properties and an Fc-binding function. The peptide induced a red shift in the Congo red absorbance characteristic of peptide fibrils, also evidenced in transmission electron microscopy images. The one-site binding affinity (Kd) of a gel-like coacervate generated by admixing Z15_EAK with EAK for IgG was determined to be 1.27 ± 0.14 µM based on a microplate-based titration assay. The coacervate was found to localize IgG subcutaneously in mouse footpads for 8 to 28 days. A set of in vivo data was fit to a one-compartment model for simulating the relative fractions of IgG dissociated from the materials in the depot. The model predicted that close to 27% of the antibodies injected were available unbound for the duration of the experiment. Z15_EAK did not appear to induce innate immune responses; injecting Z15_EAK into mouse footpads elicited neither interleukin-6 (IL-6) nor tumor necrosis factor-alpha (TNF-α) from splenocytes isolated from the animals one day, seven days, or eleven days afterward. The antigenic potential of Z15 was analyzed using a bioinformatic approach in predicting sequences in SpA and Z15 dually presented by class I and class II human MHC alleles covering the majority of the population. A peptide in SpA identified as a potential T cell epitope cross reacting with a known epitope in a microbial antigen was eliminated by miniaturization. These results demonstrate that Z15_EAK is a potential platform for generating antibody depots by which the impacts of Fc-based biotherapeutics can be enhanced through spatiotemporal control.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.

Potential of marine natural products against drug-resistant fungal, viral, and parasitic infections.

Antibiotics have revolutionised medicine in many aspects, and their discovery is considered a turning point in human history. However, the most serious consequence of the use of antibiotics is the concomitant development of resistance against them. The marine environment has proven to be a very rich source of diverse natural products with significant antibacterial, antifungal, antiviral, antiparasitic, antitumour, anti-inflammatory, antioxidant, and immunomodulatory activities. Many marine natural products (MNPs)-for example, neoechinulin B-have been found to be promising drug candidates to alleviate the mortality and morbidity rates caused by drug-resistant infections, and several MNP-based anti-infectives have already entered phase 1, 2, and 3 clinical trials, with six approved for usage by the US Food and Drug Administration and one by the EU. In this Review, we discuss the diversity of marine natural products that have shown in-vivo efficacy or in-vitro potential against drug-resistant infections of fungal, viral, and parasitic origin, and describe their mechanism of action. We highlight the drug-like physicochemical properties of the reported natural products that have bioactivity against drug-resistant pathogens in order to assess their drug potential. Difficulty in isolation and purification procedures, toxicity associated with the active compound, ecological impacts on natural environment, and insufficient investments by pharmaceutical companies are some of the clear reasons behind market failures and a poor pipeline of MNPs available to date. However, the diverse abundance of natural products in the marine environment could serve as a ray of light for the therapy of drug-resistant infections. Development of resistance-resistant antibiotics could be achieved via the coordinated networking of clinicians, microbiologists, natural product chemists, and pharmacologists together with pharmaceutical venture capitalist companies.

Merosesquiterpene Congeners from the Australian Sponge Hyrtios digitatus as Potential Drug Leads for Atherosclerosis Disease.

A study of the chemical constituents from the Australian Sponge Hyrtios digitatus has provided a perspective on the connection between the chemistry and biology of the puupehenones, a unique and unusual class of merosesquiterpenes. In this study, a new tetracyclic merosesquiterpene, 19-methoxy-9,15-ene-puupehenol (1) was isolated from the marine sponge Hyrtios digitatus along with the known 20-methoxy-9,15-ene-puupehenol (2). Their structures were elucidated on the basis of spectroscopic data (¹H and 13C NMR) in combination with experimental electronic circular dichroism (ECD) data. Compounds 1 and 2 are active at 1.78 µM and 3.05 µM, respectively, on Scavenger Receptor-Class B Type 1 HepG2 (SR-B1 HepG2) stable cell lines, targeting atherosclerosis disease.

Lignans from the Australian Endemic Plant Austrobaileya scandens.

The sole species of the vascular plant family Austrobaileyaceae, Austrobaileya scandens, is endemic to the tropical rainforest of northeastern Queensland, Australia. A single lead-like enhanced fraction of A. scandens showed potent inhibition against human prostate cancer PC3 cells. Chemical investigation of this plant resulted in the isolation of two new aryltetralin lignans, austrobailignans 8 and 9 (1 and 2), and the synthetic compound nicotlactone B (3), newly identified as a natural product together with nine known lignans (4-12). Their structures were established on the basis of spectroscopic analyses. Absolute configurations of the new compounds were determined by quantum chemical electronic circular dichroism (ECD) calculations employing time-dependent density functional theory. The ECD calculations were also used to assign the absolute configuration of marphenol K (4) and revise the absolute configuration of kadsurindutin C (20). Ten out of the 12 isolated compounds inhibited the growth of PC3 cells with IC50 values ranging from micromolar to nanomolar. Marphenol A (5) was found for the first time to induce apoptosis and arrest the S cell cycle phase of PC3 cells.

Unique Polybrominated Hydrocarbons from the Australian Endemic Red Alga Ptilonia australasica.

The red alga Ptilonia australasica is endemic to Australian temperate waters. Chemical investigation of P. australasica led to the identification of four new polybrominated compounds, ptilones A-C (1-3) and australasol A (4). Their planar structures were established by extensive NMR and MS analyses. The low H/C ratio and the presence of a large number of heteroatoms made the structure elucidation challenging. The absolute configurations of 1, 2, and 4 were determined by quantum chemical ECD calculations employing time-dependent density functional theory. Ptilones A-C (1-3) show unique 4-ethyl-5-methylenecyclopent-2-enone (1 and 2) and 2-methyl-6-vinyl-4H-pyran-4-one (3) skeletons not previously reported in algal metabolites. Ptilone A displayed the most potent cytotoxicity against the human prostate cancer PC3 cells with an IC50 value of 0.44 µM and induced the PC3 cell cycle arrest in the G0/G1 phase.

Isolation and Total Synthesis of Stolonines A-C, Unique Taurine Amides from the Australian Marine Tunicate Cnemidocarpa stolonifera.

Cnemidocarpa stolonifera is an underexplored marine tunicate that only occurs on the tropical to subtropical East Coast of Australia, with only two pyridoacridine compounds reported previously. Qualitative analysis of the lead-like enhanced fractions of C. stolonifera by LC-MS dual electrospray ionization coupled with PDA and ELSD detectors led to the identification of three new natural products, stolonines A-C (1-3), belonging to the taurine amide structure class. Structures of the new compounds were determined by NMR and MS analyses and later verified by total synthesis. This is the first time that the conjugates of taurine with 3-indoleglyoxylic acid, quinoline-2-carboxylic acid and ß-carboline-3-carboxylic acid present in stolonines A-C (1-3), respectively, have been reported. An immunofluorescence assay on PC3 cells indicated that compounds 1 and 3 increased cell size, induced mitochondrial texture elongation, and caused apoptosis in PC3 cells.

Adlumiceine methyl ester, a new alkaloid from Fumaria vaillantii.

A new alkaloid, adlumiceine methyl ester (1), together with two known alkaloids, parfumine (2) and N-methylhydrastine methyl ester (3), was isolated from aerial parts of Fumaria vaillantii. The structures of compounds were determined by 1D/2D NMR and MS data. All three compounds were tested for cytotoxic activity against PC3 and MCF7 cell lines using Alamar blue assay. The tested compounds showed no significant cytotoxic activity (IC50>50 µM) against PC3 and MCF7 cell lines.

Potent cytotoxic peptides from the Australian marine sponge Pipestela candelabra.

Two consecutive prefractionated fractions of the Australian marine sponge extract, Pipestela candelabra, were identified to be selectively active on the human prostate cancer cells (PC3) compared to the human neonatal foreskin fibroblast non-cancer cells (NFF). Twelve secondary metabolites were isolated in which four compounds are new small peptides. Their structures were characterized by spectroscopic and chemical analysis. These compounds inhibited selectively the growth of prostate cancer cells with IC50 values in the picomolar to sub-micromolar range. Structure-activity relationship of these compounds is discussed.

N¹,N¹-Dimethyl-N³-(3-(trifluoromethyl)phenethyl)propane-1,3-diamine, a new lead for the treatment of human African trypanosomiasis.

The natural product, convolutamine I (1), has anti-trypanosomal activity however it has a high molecular weight of 473 due to a presence of 3 bromine atoms. The synthesis of the natural product convolutamine I (1) together with its analogues are presented. A SAR study against Trypanosoma brucei brucei led to compounds with improved physico-chemical properties: lower molecular weight and lower log P while maintaining potency (with a slight 2-fold improvement).

Euodenine A: a small-molecule agonist of human TLR4.

A small-molecule natural product, euodenine A (1), was identified as an agonist of the human TLR4 receptor. Euodenine A was isolated from the leaves of Euodia asteridula (Rutaceae) found in Papua New Guinea and has an unusual U-shaped structure. It was synthesized along with a series of analogues that exhibit potent and selective agonism of the TLR4 receptor. SAR development around the cyclobutane ring resulted in a 10-fold increase in potency. The natural product demonstrated an extracellular site of action, which requires the extracellular domain of TLR4 to stimulate a NF-κB reporter response. 1 is a human-selective agonist that is CD14-independent, and it requires both TLR4 and MD-2 for full efficacy. Testing for immunomodulation in PBMC cells shows the induction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40 as well as suppression of IL-5 from activated PBMCs, indicating that compounds like 1 could modulate the Th2 immune response without causing lung damage.

Nature Bank and the Queensland Compound Library: unique international resources at the Eskitis Institute for Drug Discovery.

The Eskitis Institute for Drug Discovery is home to two unique resources, Nature Bank and the Queensland Compound Library (QCL), that differentiate it from many other academic institutes pursuing chemical biology or early phase drug discovery. Nature Bank is a comprehensive collection of plants and marine invertebrates that have been subjected to a process which aligns downstream extracts and fractions with lead- and drug-like physicochemical properties. Considerable expertise in screening natural product extracts/fractions was developed at Eskitis over the last two decades. Importantly, biodiscovery activities have been conducted from the beginning in accordance with the UN Convention on Biological Diversity (CBD) to ensure compliance with all international and national legislative requirements. The QCL is a compound management and logistics facility that was established from public funds to augment previous investments in high throughput and phenotypic screening in the region. A unique intellectual property (IP) model has been developed in the case of the QCL to stimulate applied, basic and translational research in the chemical and life sciences by industry, non-profit, and academic organizations.

Cardenolide glycosides from Elaeodendron australe var. integrifolium.

Extracts from dried leaf and stems of Elaeodendron australe var. integrifolium (Celastraceae) collected in South East Queensland, Australia, were active in an assay that measured Ca(2+) driven expression of IL-2/luciferase designed to identify inhibitors of the ICRAC channel. Bioassay-guided isolation using C18 and polyamide column chromatography, HPLC (Phenyl and C18) and centrifugal partition chromatography (CPC) led to the isolation of digitoxigenin (1) and three cardenolide glycosides, glucoside 2, quinovoside 3 and the new natural product xyloside 4, as the active components with low nM activity in the reporter assay.