Glycosaminoglycan Mimetics Research Articles

Impaired instructive and protective barrier functions of the endothelial cell glycocalyx pericellular matrix is impacted in COVID-19 disease.

The aim of this study was to review the roles of endothelial cells in normal tissue function and to show how COVID-19 disease impacts on endothelial cell properties that lead to much of its associated symptomatology. This places the endothelial cell as a prominent cell type to target therapeutically in the treatment of this disorder. Advances in glycosaminoglycan analytical techniques and functional glycomics have improved glycosaminoglycan mimetics development, providing agents that can more appropriately target various aspects of the behaviour of the endothelial cell in-situ and have also provided polymers with potential to prevent viral infection. Thus, promising approaches are being developed to combat COVID-19 disease and the plethora of symptoms this disease produces. Glycosaminoglycan mimetics that improve endothelial glycocalyx boundary functions have promising properties in the prevention of viral infection, improve endothelial cell function and have disease-modifying potential. Endothelial cell integrity, forming tight junctions in cerebral cell populations in the blood-brain barrier, prevents the exposure of the central nervous system to circulating toxins and harmful chemicals, which may contribute to the troublesome brain fogging phenomena reported in cognitive processing in long COVID disease.

Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury. NIH grant P20DK097818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Synthesis and interaction with growth factors of sulfated oligosaccharides containing an anomeric fluorinated tail

Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

Biomaterials and tissue engineering approaches using glycosaminoglycans for tissue repair: Lessons learned from the native extracellular matrix.

Glycosaminoglycans (GAGs) are an important component of the extracellular matrix as they influence cell behavior and have been sought for tissue regeneration, biomaterials, and drug delivery applications. GAGs are known to interact with growth factors and other bioactive molecules and impact tissue mechanics. This review provides an overview of native GAGs, their structure, and properties, specifically their interaction with proteins, their effect on cell behavior, and their mechanical role in the ECM. GAGs' function in the extracellular environment is still being understood however, promising studies have led to the development of medical devices and therapies. Native GAGs, including hyaluronic acid, chondroitin sulfate, and heparin, have been widely explored in tissue engineering and biomaterial approaches for tissue repair or replacement. This review focuses on orthopaedic and wound healing applications. The use of GAGs in these applications have had significant advances leading to clinical use. Promising studies using GAG mimetics and future directions are also discussed. STATEMENT OF SIGNIFICANCE: Glycosaminoglycans (GAGs) are an important component of the native extracellular matrix and have shown promise in medical devices and therapies. This review emphasizes the structure and properties of native GAGs, their role in the ECM providing biochemical and mechanical cues that influence cell behavior, and their use in tissue regeneration and biomaterial approaches for orthopaedic and wound healing applications.

Investigation of the anticoagulant activity of cyclic sulfated glycosaminoglycan mimetics

Thrombotic disorders are among the leading causes of deaths worldwide. Anticoagulants are frequently prescribed for their prevention and/or treatment. Current anticoagulants, which target either thrombin or factor Xa, are plagued with a number of drawbacks, the most important of which is the increased risk of internal bleeding. To develop better antithrombotic agents, the anticoagulant activity of cyclic glycosaminoglycan mimetics was evaluated. Human plasma clotting assays and enzyme inhibition assays were exploited to evaluate the anticoagulant activity of sulfated β-cyclodextrin (SBCD) and its three analogs: sulfated α-cyclodextrin, β-cyclodextrin, and methylated β-cyclodextrin. In normal human plasma, SBCD selectively doubled the activated partial thromboplastin time (APTT) at ∼9 μg/mL, with no effect on prothrombin time (PT) at the same concentration. Likewise, SBCD doubled APTT at ∼9 μg/mL and at ∼8 μg/mL in antithrombin-deficient plasma and heparin cofactor II-deficient plasma, respectively. Interestingly, the three SBCD derivatives were inactive at the highest concentrations tested which highlighted the importance of the sulfate groups and the size of the molecule. Enzyme assays revealed that SBCD inhibits factor XIa (FXIa) with an IC50 value of ∼20 μg/mL and efficacy of near 100%. SBCD did not inhibit other related proteins including thrombin, factor IXa, factor Xa, factor XIIa, factor XIIIa, plasmin, chymotrypsin, or trypsin at the highest concentrations tested demonstrating a significant selectivity. In Michaelis-Menten kinetics, SBCD decreased the VMAX and increased the KM of FXIa hydrolysis of a tripeptide chromogenic substrate indicating a mixed inhibition mechanism. Together, it appears that SBCD is a potent and selective inhibitor of human FXIa with substantial anticoagulant activity in human plasma. Overall, this study introduces SBCD as a promising lead for further development as a safer anticoagulant.

Homogeneous, Synthetic, Non-Saccharide Glycosaminoglycan Mimetics as Potent Inhibitors of Human Cathepsin G.

Cathepsin G (CatG) is a pro-inflammatory neutrophil serine protease that is important for host defense, and has been implicated in several inflammatory disorders. Hence, inhibition of CatG holds much therapeutic potential; however, only a few inhibitors have been identified to date, and none have reached clinical trials. Of these, heparin is a well-known inhibitor of CatG, but its heterogeneity and bleeding risk reduce its clinical potential. We reasoned that synthetic small mimetics of heparin, labeled as non-saccharide glycosaminoglycan mimetics (NSGMs), would exhibit potent CatG inhibition while being devoid of bleeding risks associated with heparin. Hence, we screened a focused library of 30 NSGMs for CatG inhibition using a chromogenic substrate hydrolysis assay and identified nano- to micro-molar inhibitors with varying levels of efficacy. Of these, a structurally-defined, octasulfated di-quercetin NSGM 25 inhibited CatG with a potency of ~50 nM. NSGM 25 binds to CatG in an allosteric site through an approximately equal contribution of ionic and nonionic forces. Octasulfated 25 exhibits no impact on human plasma clotting, suggesting minimal bleeding risk. Considering that octasulfated 25 also potently inhibits two other pro-inflammatory proteases, human neutrophil elastase and human plasmin, the current results imply the possibility of a multi-pronged anti-inflammatory approach in which these proteases are likely to simultaneously likely combat important conditions, e.g., rheumatoid arthritis, emphysema, or cystic fibrosis, with minimal bleeding risk.

SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

Abstract 1808: Role of natural killer (NK) cells in glycoasminoglycan-mediated anti-cancer immunity

Abstract Background: Immune-modulating therapies have limited efficacy in colorectal cancers (CRC). This resistance to treatment is attributed to low neoantigen expression, and the inability of T cells to kill HLA-I negative tumor cells, especially cancer stem cells (CSCs). Advantageously, NK cells can preferentially kill CSCs via stress ligand recognition. We have previously shown that glycosaminoglycans (GAGs) of a specific chain length, and synthetic mimetics thereof, selectively inhibit colon CSCs. In the current study, we evaluated the immunomodulatory role of natural GAGs and synthetic GAG mimetics (SGMs) in eliciting Natural Killer effector functions that regulate the anti-tumor immune response. Methods: 8-12 wk old C57bl/6 mice were treated with SGM or natural GAG (Fucoidan, heparin hexasaccharide (HS06)) and vehicle for 2 wks. Splenocytes were expanded in IL-2-containing media with or without respective GAG/SGMs, and subjected to co-culture with syngeneic MC38 colon cancer cells. The effects of each treatment on immune cell profile and anti-cancer properties were evaluated using a) multiplex flow cytometry Cytek® immune profiling assay and b) MTT assay. In vitro, MACS-enriched human peripheral blood NK cells were stimulated ex vivo with GAG/NSGMs and analyzed for activation using flow cytometry and cytotoxicity towards primary human colon cancer-derived spheroids. PDL-1 expression and CSC marker expression in GAG/SGM-treated MC38 spheroids was analyzed using western blotting and qPCR. Results: Unbiased multiplex flow cytometry revealed a robust activation of NK cells, evidenced by increased CD335 (NKp46) expression in SGM or Fucoidan-treated mice. SGM pulsed splenocytes also showed increased cytotoxicity toward MC38 cells compared to vehicle controls ex vivo. Furthermore, our preliminary findings showed that human peripheral blood NK cells (CD45+CD3-CD56+) showed activation (increased CD69 expression) with both GAGs and SGMs but showed increased proliferation (Ki67+) upon ex vivo stimulation with GAG oligosaccharide HS06 and not polymeric GAG. Furthermore, GAG treatment significantly enhanced activated autologous NK cell-induced apoptosis in primary human tumor-derived spheroids. Mechanistically, a significant reduction in Pdl-1 protein expression was observed in MC38 spheroids concurrent with the downregulation of various CSCs maker genes, e.g., Oct4, Sox2, and c-my when treated with various SGMs in vitro. Conclusions: The work highlights the role of novel SGMs in the activation of NK cells to target CSCs and it has major implications for development of novel immunotherapy strategies in CRC. Citation Format: Pavani Pingali, Jackson Faulx, Aditi Nandi, Umesh R. Desai, Bhaumik B. Patel. Role of natural killer (NK) cells in glycoasminoglycan-mediated anti-cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1808.

Designing Smaller, Synthetic, Functional Mimetics of Sulfated Glycosaminoglycans as Allosteric Modulators of Coagulation Factors.

Natural glycosaminoglycans (GAGs) are arguably the most diverse collection of natural products. Unfortunately, this bounty of structures remains untapped. Decades of research has realized only one GAG-like synthetic, small-molecule drug, fondaparinux. This represents an abysmal output because GAGs present a frontier that few medicinal chemists, and even fewer pharmaceutical companies, dare to undertake. GAGs are heterogeneous, polymeric, polydisperse, highly water soluble, synthetically challenging, too rapidly cleared, and difficult to analyze. Additionally, GAG binding to proteins is not very selective and GAG-binding sites are shallow. This Perspective attempts to transform this negative view into a much more promising one by highlighting recent advances in GAG mimetics. The Perspective focuses on the principles used in the design/discovery of drug-like, synthetic, sulfated small molecules as allosteric modulators of coagulation factors, such as antithrombin, thrombin, and factor XIa. These principles will also aid the design/discovery of sulfated agents against cancer, inflammation, and microbial infection.

Designing Synthetic, Sulfated Glycosaminoglycan Mimetics That Are Orally Bioavailable and Exhibiting In Vivo Anticancer Activity.

Sulfated glycosaminoglycans (GAGs), or synthetic mimetics thereof, are not favorably viewed as orally bioavailable drugs owing to their high number of anionic sulfate groups. Devising an approach for oral delivery of such highly sulfated molecules would be very useful. This work presents the concept that conjugating cholesterol to synthetic sulfated GAG mimetics enables oral delivery. A focused library of sulfated GAG mimetics was synthesized and found to inhibit the growth of a colorectal cancer cell line under spheroid conditions with a wide range of potencies ( 0.8 to 46 μM). Specific analogues containing cholesterol, either alone or in combination with clinical utilized drugs, exhibited pronounced in vivo anticancer potential with intraperitoneal as well as oral administration, as assessed by ex vivo tertiary and quaternary spheroid growth, cancer stem cell (CSC) markers, and/or self-renewal factors. Overall, cholesterol derivatization of highly sulfated GAG mimetics affords an excellent approach for engineering oral activity.

Glycosaminoglycan Mimetics Obtained by Microwave-Assisted Sulfation of Marine Bacterium Sourced Infernan Exopolysaccharide.

Sulfated glycosaminoglycans (GAGs) are fundamental constituents of both the cell surface and extracellular matrix. By playing a key role in cell-cell and cell-matrix interactions, GAGs are involved in many physiological and pathological processes. To design GAG mimetics with similar therapeutic potential as the natural ones, the specific structural features, among them sulfate content, sulfation pattern, and chain length, should be considered. In the present study, we describe a sulfation method based on microwave radiation to obtain highly sulfated derivatives as GAG mimetics. The starting low-molecular-weight (LMW) derivative was prepared from the infernan exopolysaccharide, a highly branched naturally slightly sulfated heteropolysaccharide synthesized by the deep-sea hydrothermal vent bacterium Alteromonas infernus. LMW highly sulfated infernan derivatives obtained by conventional heating sulfation have already been shown to display GAG-mimetic properties. Here, the potential of microwave-assisted sulfation versus that of the conventional method to obtain GAG mimetics was explored. Structural analysis by NMR revealed that highly sulfated derivatives from the two methods shared similar structural features, emphasizing that microwave-assisted sulfation with a 12-fold shorter reaction time is as efficient as the classical one.

Abstract 2653: Synthetic, small molecule glycosaminoglycan mimetics induce novel anti-cancer activity through preferential targeting of a growth factor receptor

Abstract Glycosaminoglycans (GAGs), such as heparan sulfate (HS), have been implicated in tumor initiation, progression and metastasis. Earlier we have shown that a defined GAG sequence of six residues, but not four or eight residues, inhibits colorectal cancer stem cells (CSCs) by inducing activation of p38 MAPK. To transform this sequence into a drug-like molecule, we developed a synthetic mimetic of the GAG sequence, labeled as G2.2, which was also found to selectively target CSCs over bulk adherent tumor cells. Unfortunately, G2.2’s oral bioavailability was low. To improve upon its drug-like properties, we pursued a hypothesis-driven analog design to derive three lipid-modified analogs (LMAs). Across a panel of > 15 patient-derived colorectal cancer (CRC) cell lines, which could be stratified according to their clinically-relevant consensus molecular subtypes, LMAs showed enhanced potency while retaining selectivity against CSCs in spheroid inhibition assays. Both G2.2 and LMAs displayed better inhibition of cell lines with mesenchymal phenotypes and metabolic dysregulation. Microarray-based screening against more than a dozen receptor tyrosine kinases led to identification of IGF1R as a potential receptor of the synthetic GAG mimetics. To ascertain these screening results, biophysical studies were performed to clarify preferred soluble and/or cell surface target receptors. In line with microarray results, G2.2 preferentially bound to IGF1R in comparison to its soluble ligand IGF-1. G2.2 also preferred IGF1R as compared to an alternative receptor FGFR1. Further, LMAs bound to IGF1R with improved affinities as compared to parent mimetic G2.2, which could explain anti-CSC inhibition results. Unexpectedly, detailed fluorescence titrations revealed that IGF1R affinity of LMAs is minimally impacted by salt concentration, suggesting that these GAG mimetics utilize non-ionic forces in binding, which support the selectivity of target engagement. Overall, this work proves a powerful proof of concept that synthetic GAG mimetics, such as G2.2 and LMAs, present a unique class of anti-cancer therapeutics with high potential for selective elimination of the tumor initiating subpopulation of CRC cells in patient tumors. Citation Format: Connor O'Hara, Shravan Morla, Ravikumar Ongolu, Nirmita Patel, Rio Boothello, Bhaumik Patel, Umesh Desai. Synthetic, small molecule glycosaminoglycan mimetics induce novel anti-cancer activity through preferential targeting of a growth factor receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2653.

Pleiotrophin Interaction with Synthetic Glycosaminoglycan Mimetics.

Chondroitin sulfate (CS) E is the natural ligand for pleiotrophin (PTN) in the central nervous system (CNS) of the embryo. Some structures of PTN in solution have been solved, but no precise location of the binding site has been reported yet. Using 15N-labelled PTN and HSQC NMR experiments, we studied the interactions with a synthetic CS-E tetrasaccharide corresponding to the minimum binding sequence. The results agree with the data for larger GAG (glycosaminoglycans) sequences and confirm our hypothesis that a synthetic tetrasaccharide is long enough to fully interact with PTN. We hypothesize that the central region of PTN is an intrinsically disordered region (IDR) and could modify its properties upon binding. The second tetrasaccharide has two benzyl groups and shows similar effects on PTN. Finally, the last measured compound aggregated but beforehand, showed a behavior compatible with a slow exchange in the NMR time scale. We propose the same binding site and mode for the tetrasaccharides with and without benzyl groups.

Abstract 1166: Structurally defined glycosaminoglycan mimetics exhibit preference for proteins of the growth factor family

Abstract Heparan sulfate (HS), a glycosaminoglycans of considerable structural diversity, has been implicated in a wide-variety of complex roles including regulation of cell signaling. Unfortunately, homogeneous HS is a dream that will likely be unfulfilled this century. To combat this, our lab has synthesized an array of non-saccharide glycosaminoglycan mimetics (NSGMs) that act as structural and functional mimetics of HS. A key advantage of our NSGMs is homogeneity, which enables assessing selectivity for recognition of target proteins with a high degree of confidence. A second advantage is the quantitative measurement of the affinity of these interactions, which affords a detailed understanding of the forces governing recognition of different proteins. Previously, we have shown a lead NSGM named G2.2 as a potent and selective inhibitor of colorectal cancer stem cells (CSCs) through phenotypic in-vitro and in-vivo assays. To improve upon the drug-like properties of G2.2, cholesterol-modified analogs (G2C, G5C, G8C) were designed to enhance potency and selectivity for protein target(s). Although the hypothesis is that G2.2 and its lipidic analogs target a ligand and/or a receptor belonging to CSC-relevant growth factors, the preferred target, if any, remains to be identified. More importantly, the relative order of preference in-vitro and its use in predicted in-vivo activity remains to be determined. In this work, we have performed a comprehensive biophysical screening of a panel of potential growth factor targets using biophysical methods to determine binding affinities, followed by quantitative measurement of the relative forces of interactions involved in target recognition. Our studies are complemented by computational experiments that provide a foundation for thermodynamic studies. Analysis of our results indicate that these NSGMs preferentially recognize certain growth factor proteins, suggesting that appropriately designed HS mimetics can be tailored to modulate a particular tyrosine kinase receptor pathway. More importantly, our results convey the principle that despite the presence of multiple sulfate groups, HS mimetics can be designed to exhibit selectivity for certain growth factor proteins. In fact, the sulfate groups on our molecules may actually contribute to the selectivity and potency of recognition, two central principles for the development of drugs. Citation Format: Connor P. O'Hara, Nehru V. Sankaranarayanan, Rio S. Boothello, Bhaumik B. Patel, Umesh R. Desai. Structurally defined glycosaminoglycan mimetics exhibit preference for proteins of the growth factor family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1166.

Antiviral activities of four marine sulfated glycans against adenovirus and human cytomegalovirus

Broad-spectrum antivirals are more needed than ever to provide treatment options for novel emerging viruses and for viruses that lack therapeutic options or have developed resistance. A large number of viruses rely on charge-dependent non-specific interactions with heparan sulfate (HS), a highly sulfated glycosaminoglycan (GAG), for attachment to cell surfaces to initiate cell entry. As such, inhibitors targeting virion-HS interactions have potential to have broad-spectrum antiviral activity. Previous research has explored organic and inorganic small molecules, peptides, and GAG mimetics to disrupt virion-HS interactions. Here we report antiviral activities against both enveloped (the herpesvirus human cytomegalovirus) and non-enveloped (adenovirus) DNA viruses for four defined marine sulfated glycans: a sulfated galactan from the red alga Botryocladia occidentalis; a sulfated fucan from the sea urchin Lytechinus variegatus, and a sulfated fucan and a fucosylated chondroitin sulfate from the sea cucumber Isostichopus badionotus. As evidenced by gene expression, time of addition, and treatment/removal assays, all four novel glycans inhibited viral attachment and entry, most likely through interactions with virions. The sulfated fucans, which both lack anticoagulant activity, had similar antiviral profiles, suggesting that their activities are not only due to sulfation content or negative charge density but also due to other physicochemical factors such as the potential conformational shapes of these carbohydrates in solution and upon interaction with virion proteins. The structural and chemical properties of these marine sulfated glycans provide unique opportunities to explore relationships between glycan structure and their antiviral activities.

Abstract 6353: Cholesterol modification enhances potency and pharmacokinetic properties of a selective cancer stem cell targeting agent

Abstract Increasing evidence supports the concept that tumor-initiating cancer stem cells (CSCs) are responsible for the relapse and reconstitution of formerly treated tumors. Previously, our lab identified a potent and selective inhibitor of colorectal CSCs, named G2.2, from a library of non-saccharide glycosaminoglycan mimetics (NSGMs). To further improve upon the potency, pharmacokinetics, and drug-like properties of G2.2, cholesterol-modified analogs were rationally designed using computational molecular modeling and molecular dynamics studies. A small group of designed NSGMs, labeled as G2C, G5C and G8C, were synthesized for biological evaluation. These agents and the parent NSGM G2.2 were studied for their ability to inhibit the growth of primary spheroids from a panel of 15 human colorectal cancer (CRC) cells representative of the consensus molecular subtypes of human CRC. Spheroid formation frequencies (SFFs) of cell lines were measured using limiting dilution assays (LDAs), and IC50 of inhibition was evaluated from dose-response curves for monolayer growth and primary spheroid growth in the presence of NSGMs. Selectivity of CSC targeting was assessed from the ratio of IC50,monolayer to IC50,spheroid. Pharmacokinetics (PK) and bioavailability in rodents were deduced following IV and PO administrations. In-vivo therapeutic potential of the NSGMs was studied in CSC-induced xenograft models aided by ex-vivo CSC phenotype characterization. Each NSGM preferentially targeted 3D spheroidal growth in comparison to monolayer cellular growth by 12- to 450-fold, suggesting excellent CSC selectivity. Cholesterol modification of G2.2 enhanced in-vitro spheroid inhibition potency across the entire panel of cell lines on average by 3- to 21-fold. G8C showed the highest inhibition potency with an IC50 of 1 to 10 μM across cell lines. Cells with higher SFF were several-fold more sensitive to NSGMs, suggesting that basal expression of CSCs could predict NSGM response. Robust reductions in tumor volumes were observed in mice treated with G5C (100 mg/kg i.p. 3x a week) following an intial-treatment with oxaliplatin and 5-fluorouracil (weekly x3) compared to vehicle controls. This reduction was concomitant with a 3.9-fold reduction in colonic crypt stemness marker LGR5+ cells and 2.6-fold decrease in xenograft-derived tertiary spheroids. No gross or organ specific toxicity was found in animals treated with G5C. Finally, cholesterol modification greatly improved PK and oral bioavailability of parent G2.2. Overall, cholesterol modification of CSC-selective G2.2 was found to enhance in vitro potency against spheroidal growth without compromising selectivity toward tumor initiating CSCs. The G2.2 analogs offered robust tumor reductions in vivo with essentially no off target toxicity, and serve as promising and novel potential cancer therapeutics. Citation Format: Connor P. O'Hara, Rio S. Boothello, Shravan Morla, Alberto Vera, Daniel K. Afosah, Nehru V. Sankaranarayanan, Balaji Nagarajan, Chetna Sharon, Bhaumik B. Patel, Umesh R. Desai. Cholesterol modification enhances potency and pharmacokinetic properties of a selective cancer stem cell targeting agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6353.

Glycosaminoglycans as Multifunctional Anti-Elastase and Anti-Inflammatory Drugs in Cystic Fibrosis Lung Disease.

Neutrophil elastase (NE) is a major protease in the airways of patients with cystic fibrosis (CF) that activates airway inflammation by several mechanisms. NE stimulates epithelial toll like receptors (TLR) resulting in cytokine upregulation and release, upregulates MUC5AC, a major airway mucin, degrades both phagocytic receptors and opsonins resulting in both neutrophil and macrophage phagocytic failure, generates oxidative stress via extracellular generation and uptake of heme free iron, and activates other proteases. Altogether, these mechanisms create a significant inflammatory challenge that impairs innate immune function and results in airway remodeling. Currently, a major gap in our therapeutic approach to CF lung disease is the lack of an effective therapeutic strategy targeting active NE and its downstream pro-inflammatory sequelae. Polysulfated glycosaminoglycans (GAGs) are potent anti-elastase drugs that have additional anti-inflammatory properties. Heparin is a prototype of a glycosaminoglycan with both anti-elastase and anti-inflammatory properties. Heparin inhibits NE in an allosteric manner with high potency. Heparin also inhibits cathepsin G, blocks P-selectin and L-selectin, hinders ligand binding to the receptor for advanced glycation endproducts, and impedes histone acetyltransferase activity which dampens cytokine transcription and High Mobility Group Box 1 release. Furthermore, nebulized heparin treatment improves outcomes for patients with chronic obstructive pulmonary disease (COPD), asthma, acute lung injury and smoke inhalation. However, the anticoagulant activity of heparin is a potential contraindication for this therapy to be developed for CF lung disease. Therefore, modified heparins and other GAGs are being developed that retain the anti-elastase and anti-inflammatory qualities of heparin with minimal to no anticoagulant activity. The modified heparin, 2-O, 3-O desulfated heparin (ODSH), maintains anti-elastase and anti-inflammatory activities in vitro and in vivo, and has little residual anticoagulant activity. Heparan sulfate with O-sulfate residues but not N-sulfate residues blocks allergic asthmatic inflammation in a murine model. Polysulfated hyaluronic acid abrogates allergen- triggered rhinosinusitis in a murine model. Finally, nonsaccharide glycosaminoglycan mimetics with specific sulfate modifications can be designed to inhibit NE activity. Altogether, these novel GAGs or GAG mimetics hold significant promise to address the unmet need for inhaled anti-elastase and anti-inflammatory therapy for patients with CF.

Sulfated Non-Saccharide Glycosaminoglycan Mimetics as Novel Drug Discovery Platform for Various Pathologies.

Glycosaminoglycans (GAGs) are very complex, natural anionic polysaccharides. They are polymers of repeating disaccharide units of uronic acid and hexosamine residues. Owing to their template-free, spatiotemporally-controlled, and enzyme-mediated biosyntheses, GAGs possess enormous polydispersity, heterogeneity, and structural diversity which often translate into multiple biological roles. It is well documented that GAGs contribute to physiological and pathological processes by binding to proteins including serine proteases, serpins, chemokines, growth factors, and microbial proteins. Despite advances in the GAG field, the GAG-protein interface remains largely unexploited by drug discovery programs. Thus, Non-Saccharide Glycosaminoglycan Mimetics (NSGMs) have been rationally developed as a novel class of sulfated molecules that modulate GAG-protein interface to promote various biological outcomes of substantial benefit to human health. In this review, we describe the chemical, biochemical, and pharmacological aspects of recently reported NSGMs and highlight their therapeutic potentials as structurally and mechanistically novel anti-coagulants, anti-cancer agents, anti-emphysema agents, and anti-viral agents. We also describe the challenges that complicate their advancement and describe ongoing efforts to overcome these challenges with the aim of advancing the novel platform of NSGMs to clinical use.

End-functionalised glycopolymers as glycosaminoglycan mimetics inhibit HeLa cell proliferation

A novel glycopeptide mimetic, prepared by end-functionalised conjugation of iRGD peptide on a glycopolymer, could effectively enter HeLa cells and inhibit signalling pathways involved in tumour cell proliferation.

Heparan Sulfate Mimetics Accelerate Postinjury Skeletal Muscle Regeneration.

Although skeletal muscle is capable of complete recovery after an injury, specific situations require support or acceleration of this process, such as in the elderly and athletes, respectively. Skeletal muscle regeneration is due to muscle stem cells (MuSCs) that undergo adult myogenesis, a process sustained by MuSC environment. Although recognized as important, extracellular matrix (ECM) has been overlooked in this process. Matrix-based therapy aims at improving ECM remodeling to support tissue repair. In this context, we investigated the properties of a single injection of the clinical grade glycosaminoglycan mimetics RGTA® (ReGeneraTing Agents) on skeletal muscle regeneration in a context compatible with a clinical application, that is, 3 days after the injury. Our results show that RGTA-treated muscles showed an increase of the number of myonuclei in regenerating myofibers and an increase of the capillarization of the new myofibers. In vitro experiments showed that RGTA directly acts on MuSCs by stimulating their fusion into myotubes and on endothelial cells by stimulating the formation and maturation of vessels in a 3D culture setup. These results indicate that a single administration of RGTA in regenerating muscle stimulated both myogenesis and angiogenesis, thus accelerating skeletal muscle regeneration. Impact Statement Although highly powerful in normal condition, postinjury skeletal muscle regeneration is less efficient in some situations, such as obese, elderly, or resting people. In other context, such as high-performance sport, skeletal muscle regeneration must be shortened but in a way ensuring a full functional recovery. In this context, our results show that a single injection of the clinical grade glycosaminoglycan mimetics RGTA® (ReGeneraTing Agents), in a context compatible with a clinical application, that is, 3 days after the injury, is beneficial for skeletal muscle regeneration, through the stimulation of both myogenesis and angiogenesis.