Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools.

Targeting of cancer stem cells (CSCs) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here, we show for the first time that G2.2, a sulfated non-saccharide GAG mimetic (NSGM) of heparin hexasaccharide, selectively inhibits colonic CSCs in vivo. G2.2 reduced CSCs (CD133+/CXCR4+, Dual hi) induced HT-29 and HCT 116 colon xenografts’ growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared to vehicle-treated xenograft controls. In fact, G2.2 robustly inhibited CSCs abundance (measured by levels of CSC markers, e.g., CD133, DCMLK1, LGR5, LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly, G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly, G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions, blood coagulation profile, and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro, in vivo, and ex vivo mechanistic studies, we showed that G2.2’s inhibition of CSC self-renewal was mediated through activation of p38 α, uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence, G2.2 represents a first-in-class (NSGM) anti-cancer agent to reduce colorectal CSCs.

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), akey serpin present in human plasma, remain unknown. Using ac omputational strategy on alibrary of 46 656 heparan sulfate hexasaccharides we identified ar are sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, ac losely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly,our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAGsequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.Heparin cofactor II (HCII) is as erine protease inhibitor (serpin) that circulates in human plasma at high levels. Although it has been known to selectively inhibit thrombin for several decades, [1][2][3] its true physiologic function remains to be understood. [4] HCII is known to bind to glycosaminoglycans (GAGs) such as dermatan sulfate (DS) and heparan sulfate (HS), which help mediate its inhibition of thrombin. One of the key reasons for the inability to identify HCIIs biologic role is the lack of knowledge on the specificity of HCII-GAGi nteraction.HCII is an interesting serpin. It bears considerable similarity to antithrombin (AT), another plasma serpin that mediates the anticoagulant action of heparin and fondaparinux, [5,6] two clinically used drugs.A Tand HCII are homologous in primary,secondary and tertiary structure ( Figure S1 in the Supporting Information). Yet, whereas AT binds specifically to fondaparinux sequence in heparin, [7] HCII is considered to bind non-specifically to heparin and HS. Further,a lthough both serpins display at wo-step,i nducedfit, allosteric activation mechanism in inhibiting their target enzymes, [8][9][10] no HS oligosaccharide has been discovered to induce robust activation of HCII.Identifying GAGs equences that selectively target proteins is extremely challenging.Akey reason for this is their structural complexity.H S, ah ighly anionic polymer containing variably sulfated, acetylated and epimerized residues, presents enormous structural diversity that makes comprehensive analysis of all possible sequences difficult. Thus, identifying key "needles" in this haystack, especially with synthesis [11,12] or isolation of oligosaccharides from nature nearly impossible. [13] We had earlier developed ad ual-filter computational algorithm, called combinatorial virtual library screening (CVLS) strategy,torapidly sort HS sequences into "specific" and "non-specific" bins. [14,15] We wondered whether this tool could pinpoint HS sequences that preferentially activate HCII for inhibition of thrombin. Further, we posited that such asequence,ifany,would be different from the pentasaccharide seq...