Background Human factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long‐standing hypothesis has been that allosterism originating from heparin‐binding site(s) on coagulation enzymes is a promising approach to yield safer agents. Objectives To develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk. Methods We employed a gamut of methods involving synthetic chemistry, biophysical biochemistry, enzyme assays, blood and plasma coagulation assays, and in vivo thrombosis models in this work. Results Sulfated chiro‐inositol (SCI), a non‐saccharide mimetic of heparin, was synthesized in three steps in overall yields of >50%. SCI inhibited FXIa with potency of 280 nmol/L and preferentially engaged FXIa's heparin‐binding site to conformationally alter its active site. SCI inhibition of FXIa could be rapidly reversed by common antidotes, such as protamine. SCI preferentially prolonged plasma clotting initiated with recalcification, rather than thromboplastin, alluding to its intrinsic pathway‐based mechanism. Human blood thromboelastography indicated good ex vivo anticoagulation properties of SCI. Rat tail bleeding and maximum‐dose‐tolerated studies indicated that no major bleeding or toxicity concerns for SCI suggesting a potentially safer anticoagulation outcome. FeCl3‐induced arterial and thromboplastin‐induced venous thrombosis model studies in the rat showed reduced thrombus formation by SCI at 250 μg/animal, which matched enoxaparin at 2500 μg/animal. Conclusions Overall, SCI is a highly promising, allosteric inhibitor of FXIa that induces potent anticoagulation in vivo. Further studies are necessary to assess SCI in animal models mimicking human clinical indications.

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.

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.

<p>The 16 page composite document outlines supplemental methods as well as Figures. Fig S1: a- In vivo tumor formation assay with subcutaneous injections of (2,000) Dual hi HT-29 cells show high tumor forming efficiency of Dual hi cells compared to unsorted cells in athymic NCr nude mice. b- Line graph showing tumor volume progression in NCr nude mice injected subcutaneously with varying amount of (100,000 - 2,000) of FACS-sorted Dual hi (CD133+/CXCR4+) HCT 116 colon cancer cells. The data shows that FACS-sorted CSCs are capable of inducing tumor even at a much lower number of cells. Fig S2: a- The Kaplan-Meier survival curves indicates significant survival in NCr nude mice bearing Dual hi induced HT-29 xenografts treated with 100mg/Kg (p=0.0025) or 200mg/Kg (p=0.014) of G2.2 compared to vehicle treated control. b- Scatter plot representation of flow cytometric analyses of Dual hi CSCs in HT-29 spheroids (100 µM) and xenograft (200 mg/kg) derived cells following labeling with anti-CD133 and anti-CXCR4 antibodies. G2.2-treated HT-29 spheroids (100 µM) and xenograft (200 mg/kg) derived cells show significant inhibition of Dual hi CSCs compared to respective vehicle controls. c- G2.2-treated HT-29 xenograft-derived cells show significant inhibition of CSC self-renewal (2{degree sign} and 3{degree sign} sphere formation) compared to vehicle controls. d- Scatter plot representation of flow cytometric analyses of Dual hi CSCs-induced xenograft derived cells following labeling with anti-CD133, anti-CXCR4, and anti-Annexin V antibodies as well as propidium iodide counterstaining. The Panel 1 shows significant increase in Annexin V+ (apoptotic) cells in G2.2 treated xenografts compared to vehicle controls. Upon further analyses of the G2.2 treated xenograft cells (Panel 2) to determine Annexin V+ cells in each of Dual hi (CSCs) and Dual lo (non-CSCs) population revealed a robust increase in apoptotic cells in Dual hi population only, suggesting that G2.2 selective targets CSC population. e- G2.2-treated primary HT-29 xenograft-derived cells 3{degree sign} colonospheres were retreated with G2.2 in vitro and spheroid growth was measure in the presence of the drug (4{degree sign} spheroids) as well as following withdrawal of the drug (5{degree sign} & 6{degree sign} spheroids) to determine growth and self-renewal respectively of HT-29 xenograft-derived spheroids following a re-challenge with the drug. The box-plot show a robust inhibition of CSC growth/self-renewal by G2.2 following in vivo exposure to the drug (4{degree sign}, 5{degree sign} and 6{degree sign} sphere formation) compared to vehicle controls. f- G2.2 treated secondary HT-29 xenografts-derived cells show significant inhibition of CSC self-renewal (2{degree sign}, 3{degree sign} and 4{degree sign} sphere formation) compared to vehicle controls. g- Western blot analyses of xenografts show robust inhibition of CSC makers (LGR5, DCLK1) and self-renewal factor (BMI1) in G2.2 treated HCT 116 xenografts compared to vehicle controls at days 42 post treatment initiation. The associated bar-graph represents relative densitometry value normalized to GAPDH (housekeeping protein). h- G2.2 treated HCT 116 xenograft-derived cells show significant inhibition of CSC self-renewal (4{degree sign} sphere formation) compared to vehicle controls.Fig S3: a- Flow cytometry analyses of xenograft-derived cells labelled with anti-CD133-APC and CXCR4-PE antibody demonstrating higher proportion of CSCs in Chemotherapy treated mice compared to vehicle treated controls.. b- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) compared to Veh. The xenografts were harvested on day 21 post-FUOX treatment initiation.. c- Scatter plot representation of flow cytometry analyses of HT-29 xenograft-derived cells to determine proportion of Dual hi (CSCs) cells shows, a robust and sustained inhibition of Dual hi cells by CG2.2 compared to CVeh at days 30 and 42 post-FUOX treatment initiation. Numbers in the yellow box represent % of total events in a given quadrant.. d- Unedited uncut western blots along with molecular weight markers. e- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) which was promptly inhibited by CG2.2 compared to both veh. and cVeh treatments. Fig S4: a- Line graph showing activation kinetics of two key mitogen-activated protein kinases, p38 and ERK1/2 (15min → 720min) following G2.2 (100 µM) treatment of HT-29 spheroids - shows early and sustained activation of p38MAPK. Whereas ERK1/2 showed brief activation followed by modest inhibition with G2.2. The dotted line represents basal activation state. b- Western-blot analyses of MAPK shows selective activation (increased levels of phosphorylated form) of p38 but modest inhibition of related MAPK members - JNK and ERK1/2 by G2.2 in HT-29 spheroids. Moreover, activation of p38 by G2.2 (but not its inactive analogs G1.4 and G4.1) was observed selective in HT-29 spheroids enriched in CSCs but not in their monolayer counterparts, suggesting selective activation of p38 MAPK in CSCs by G2.2. Numbers under the blot represent relative densitometric values normalized to vehicle treated controls. c- Immuno-precipitation (IP) with anti-pp38 followed by western blotting (WB) with isoform specific anti-p38 antibody as well as the corresponding bar graph representation of relative densitometry values shows activation of α and β isoforms, inhibition of Î' isoform, and no significant change in γ isoform upon G2.2 treatment (100 μM for 3hrs) in HT-29 spheroids. d- Western Blot analysis shows depletion of p38α in HT-29 cells transfected with p38α siRNA (KD) without any significant effect on the levels of other isoforms. Moreover, p38α KD significantly inhibited G2.2 induced upregulation of pp38. e- Western-blot analyses of CSC (CD133, LGR5, CD44, EpCAM) and self-renewal (CMYC, BMI1) makers shows significant inhibition of these markers by G2.2 (100µM) in scrambled transfected HT-29 spheroids. However, these effects of G2.2 were near-completely reversed in HT-29 spheroids transfected with p38α siRNA, suggesting a critical role of p38α in mediating anti-CSC effects of G2.2. GAPDH served as housekeeping control. The numbers under the blot represent relative densitometric values normalized to GAPDH and vehicle treated controls. f- Western blot analysis and the corresponding bar graph representation of relative expression of CSC markers (CD133, LGR5) and pp38 shows near-complete reversal G2.2's inhibition of levels of CSC markers (24 hours) and its induction of pp38 (3 hours) in the presence of p38α agf. (dominant negative). GAPDH was used as loading control. Fig S5: a- Scatter-plot of flow-cytometric analyses of Dual hi (CD133+/CXCR4+) cells in xenografts treated with vehicle, G2.2 (200 mg/kg), SB (10mg/kg) or SB followed by G2.2 (SG). The data shows that SB, a p38 inhibitor, reversed the effect of G2.2 caused inhibition of CSCs in vivo. Fig S6: a- Average weight of mice treated with 200mg/Kg of G2.2, 3 times a week for 5 weeks compared to vehicle treated controls.. b- High resolution (20X) photomicrograph of G2.2 treated liver shows minor (~ 5%) dropout of hepatocytes (shown with bold black arrows) compared to vehicle controls.</p>

<p>The 16 page composite document outlines supplemental methods as well as Figures. Fig S1: a- In vivo tumor formation assay with subcutaneous injections of (2,000) Dual hi HT-29 cells show high tumor forming efficiency of Dual hi cells compared to unsorted cells in athymic NCr nude mice. b- Line graph showing tumor volume progression in NCr nude mice injected subcutaneously with varying amount of (100,000 - 2,000) of FACS-sorted Dual hi (CD133+/CXCR4+) HCT 116 colon cancer cells. The data shows that FACS-sorted CSCs are capable of inducing tumor even at a much lower number of cells. Fig S2: a- The Kaplan-Meier survival curves indicates significant survival in NCr nude mice bearing Dual hi induced HT-29 xenografts treated with 100mg/Kg (p=0.0025) or 200mg/Kg (p=0.014) of G2.2 compared to vehicle treated control. b- Scatter plot representation of flow cytometric analyses of Dual hi CSCs in HT-29 spheroids (100 µM) and xenograft (200 mg/kg) derived cells following labeling with anti-CD133 and anti-CXCR4 antibodies. G2.2-treated HT-29 spheroids (100 µM) and xenograft (200 mg/kg) derived cells show significant inhibition of Dual hi CSCs compared to respective vehicle controls. c- G2.2-treated HT-29 xenograft-derived cells show significant inhibition of CSC self-renewal (2{degree sign} and 3{degree sign} sphere formation) compared to vehicle controls. d- Scatter plot representation of flow cytometric analyses of Dual hi CSCs-induced xenograft derived cells following labeling with anti-CD133, anti-CXCR4, and anti-Annexin V antibodies as well as propidium iodide counterstaining. The Panel 1 shows significant increase in Annexin V+ (apoptotic) cells in G2.2 treated xenografts compared to vehicle controls. Upon further analyses of the G2.2 treated xenograft cells (Panel 2) to determine Annexin V+ cells in each of Dual hi (CSCs) and Dual lo (non-CSCs) population revealed a robust increase in apoptotic cells in Dual hi population only, suggesting that G2.2 selective targets CSC population. e- G2.2-treated primary HT-29 xenograft-derived cells 3{degree sign} colonospheres were retreated with G2.2 in vitro and spheroid growth was measure in the presence of the drug (4{degree sign} spheroids) as well as following withdrawal of the drug (5{degree sign} & 6{degree sign} spheroids) to determine growth and self-renewal respectively of HT-29 xenograft-derived spheroids following a re-challenge with the drug. The box-plot show a robust inhibition of CSC growth/self-renewal by G2.2 following in vivo exposure to the drug (4{degree sign}, 5{degree sign} and 6{degree sign} sphere formation) compared to vehicle controls. f- G2.2 treated secondary HT-29 xenografts-derived cells show significant inhibition of CSC self-renewal (2{degree sign}, 3{degree sign} and 4{degree sign} sphere formation) compared to vehicle controls. g- Western blot analyses of xenografts show robust inhibition of CSC makers (LGR5, DCLK1) and self-renewal factor (BMI1) in G2.2 treated HCT 116 xenografts compared to vehicle controls at days 42 post treatment initiation. The associated bar-graph represents relative densitometry value normalized to GAPDH (housekeeping protein). h- G2.2 treated HCT 116 xenograft-derived cells show significant inhibition of CSC self-renewal (4{degree sign} sphere formation) compared to vehicle controls.Fig S3: a- Flow cytometry analyses of xenograft-derived cells labelled with anti-CD133-APC and CXCR4-PE antibody demonstrating higher proportion of CSCs in Chemotherapy treated mice compared to vehicle treated controls.. b- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) compared to Veh. The xenografts were harvested on day 21 post-FUOX treatment initiation.. c- Scatter plot representation of flow cytometry analyses of HT-29 xenograft-derived cells to determine proportion of Dual hi (CSCs) cells shows, a robust and sustained inhibition of Dual hi cells by CG2.2 compared to CVeh at days 30 and 42 post-FUOX treatment initiation. Numbers in the yellow box represent % of total events in a given quadrant.. d- Unedited uncut western blots along with molecular weight markers. e- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) which was promptly inhibited by CG2.2 compared to both veh. and cVeh treatments. Fig S4: a- Line graph showing activation kinetics of two key mitogen-activated protein kinases, p38 and ERK1/2 (15min → 720min) following G2.2 (100 µM) treatment of HT-29 spheroids - shows early and sustained activation of p38MAPK. Whereas ERK1/2 showed brief activation followed by modest inhibition with G2.2. The dotted line represents basal activation state. b- Western-blot analyses of MAPK shows selective activation (increased levels of phosphorylated form) of p38 but modest inhibition of related MAPK members - JNK and ERK1/2 by G2.2 in HT-29 spheroids. Moreover, activation of p38 by G2.2 (but not its inactive analogs G1.4 and G4.1) was observed selective in HT-29 spheroids enriched in CSCs but not in their monolayer counterparts, suggesting selective activation of p38 MAPK in CSCs by G2.2. Numbers under the blot represent relative densitometric values normalized to vehicle treated controls. c- Immuno-precipitation (IP) with anti-pp38 followed by western blotting (WB) with isoform specific anti-p38 antibody as well as the corresponding bar graph representation of relative densitometry values shows activation of α and β isoforms, inhibition of Î' isoform, and no significant change in γ isoform upon G2.2 treatment (100 μM for 3hrs) in HT-29 spheroids. d- Western Blot analysis shows depletion of p38α in HT-29 cells transfected with p38α siRNA (KD) without any significant effect on the levels of other isoforms. Moreover, p38α KD significantly inhibited G2.2 induced upregulation of pp38. e- Western-blot analyses of CSC (CD133, LGR5, CD44, EpCAM) and self-renewal (CMYC, BMI1) makers shows significant inhibition of these markers by G2.2 (100µM) in scrambled transfected HT-29 spheroids. However, these effects of G2.2 were near-completely reversed in HT-29 spheroids transfected with p38α siRNA, suggesting a critical role of p38α in mediating anti-CSC effects of G2.2. GAPDH served as housekeeping control. The numbers under the blot represent relative densitometric values normalized to GAPDH and vehicle treated controls. f- Western blot analysis and the corresponding bar graph representation of relative expression of CSC markers (CD133, LGR5) and pp38 shows near-complete reversal G2.2's inhibition of levels of CSC markers (24 hours) and its induction of pp38 (3 hours) in the presence of p38α agf. (dominant negative). GAPDH was used as loading control. Fig S5: a- Scatter-plot of flow-cytometric analyses of Dual hi (CD133+/CXCR4+) cells in xenografts treated with vehicle, G2.2 (200 mg/kg), SB (10mg/kg) or SB followed by G2.2 (SG). The data shows that SB, a p38 inhibitor, reversed the effect of G2.2 caused inhibition of CSCs in vivo. Fig S6: a- Average weight of mice treated with 200mg/Kg of G2.2, 3 times a week for 5 weeks compared to vehicle treated controls.. b- High resolution (20X) photomicrograph of G2.2 treated liver shows minor (~ 5%) dropout of hepatocytes (shown with bold black arrows) compared to vehicle controls.</p>