Plasmin Regulation through Allosteric, Sulfated, Small Molecules

Plasmin, a key serine protease, plays a major role in clot lysis and extracellular matrix remodeling. Heparin, a natural polydisperse sulfated glycosaminoglycan, is known to allosterically modulate plasmin activity. No small allosteric inhibitor of plasmin has been discovered to date. We screened an in-house library of 55 sulfated, small glycosaminoglycan mimetics based on nine distinct scaffolds and varying number and positions of sulfate groups to discover several promising hits. Of these, a pentasulfated flavonoid-quinazolinone dimer 32 was found to be the most potent sulfated small inhibitor of plasmin (IC50 = 45 μM, efficacy = 100%). Michaelis-Menten kinetic studies revealed an allosteric inhibition of plasmin by these inhibitors. Studies also indicated that the most potent inhibitors are selective for plasmin over thrombin and factor Xa, two serine proteases in coagulation cascade. Interestingly, different inhibitors exhibited different levels of efficacy (40%–100%), an observation alluding to the unique advantage offered by an allosteric process. Overall, our work presents the first small, synthetic allosteric plasmin inhibitors for further rational design.


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Direct inhibition of human plasmin by sulfated small molecules. Direct inhibition of plasmin was measured using a chromogenic substrate hydrolysis assay on a microplate reader (FlexStation III, Molecular Devices), as reported earlier [2]. Briefly, to each well of a 96-well microplate containing 85 µL of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.1% PEG8000, and 0.02% Tween80 at 37 °C was added 5 µL potential inhibitor (or vehicle alone) and 5 µL enzyme. The final concentration of the enzyme was 20 nM. After 5 min incubation, 5 µL of 1 mM Spectrozyme PL was rapidly added and the residual enzyme activity was measured from the initial rate of increase in A405. Relative residual enzyme activity (Y, activity in the presence of inhibitor to that in its absence) as a function of the concentration of SPGG derivative was fitted using logistic Equation (1)

Michaelis-Menten kinetics of Spectrozyme PL hydrolysis by plasmin in the presence of molecule (32).
The initial rate of Spectrozyme PL hydrolysis by human plasmin (20 nM) was monitored from the linear increase in absorbance at 405 nm corresponding to less than 10% consumption of the substrate. The initial rate was measured as a function of various concentrations of the substrate (0-400 μM) in the presence of fixed concentration of inhibitor (32) (0-250 μM) or inhibitor (52) (0-150 μM) in 50 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl at 37 °C. The data were fitted by Michaelis-Menten Equation (2) to determine KM,app and VMAX.
(1). 1  General procedure for synthesis of substituted phenyl quinazolin-4(3H)-one (58). To a stirred solution of anthranilamide (1.0 equiv) in anhydrous DMA, substituted benzaldehyde (1.1 equiv) and NaHSO3 (1.5 equiv) was added in a single neck flask attached with a reflux condenser. The reaction mixture was vigorously stirred at 145 °C for 12 h; the reaction mixture was diluted with EtOAC (25 mL) and water (25 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with saturated NaCl solution (25 mL), and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure fallowed by the

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purification of the crude by flash chromatography on silica gel (10%-80% ethyl acetate in hexanes) afforded 2-aryl quinazolin-4(3H)-one. Characterization data were reported earlier in reference 3. General procedure for acetylation of hydroxyls in phenyl quinazolin-4(3H)-one core structure (59). To a solution of phenyl quinazolin-4(3H)-one in dry DCM was added pyridine (2.0 equiv per hydroxyl group) and acetic anhydride (1.0 equiv per hydroxyl group). After stirring for 2 h, the reaction mixture was diluted with EtOAC (25 mL) and water (25mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with saturated 3N HCl (25mL) solution to remove excess pyridine and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure afforded crude product and purified using flash chromatography on silica gel (10%-50% ethyl acetate in hexanes). Characterization data were reported earlier in reference 3.
General procedure for two steps synthesis of N 3 -azide alkyl quinazolinon-4(3H)-one (31a-34a). To a solution of (1.0 equiv) in DMF was added K2CO3 (1.5 equiv) and stirred for two minutes. This was followed by addition of 1-bromo-n-chloroalkane (1.0 equiv) and stirred vigorously for 12 h. After the reaction completed as indicated from TLC the reaction mixture was diluted with EtOAC (25 mL) and water (25 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL) and removal of the solvent under reduced pressure afforded crude chloro-compounds which were directly used for next step without further purification. The chloro compound was then solubilized in DMF in a flask attached to a reflux condenser and sodium azide (1.5 equiv) was added to it. After stirring for overnight at 60 º C, the reaction mixture was diluted with EtOAC (25 mL) and water. The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with saturated NaCl solution (25 mL), and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure afforded the desired crude azides which were further purified using flash chromatography on silica gel (20%-35% ethyl acetate in hexanes). Products used directly in the next reaction.
General procedure for protection of flavonoid by MOM (61). To a solution of flavonoid (1.0 equiv) in DCM, N,N'-diisopropylethylamine (8.0 equiv) and MOM chloride (3.5 equiv) was added under nitrogen. After vigorous stirring at 0 °C for 1 h, the reaction mixture was allowed to warm to room temperature over 2 h and the stirring was maintained for 12 h. The resulting mixture was diluted with water (100 mL), extracted with EtOAC (200 mL), and then the organic layer was washed with water (100 mL) and dried over Na2SO4. The residue obtained after removal of the solvent was purified by flash column chromatography. Characterization data were reported earlier in reference 3.
General procedure for flavonoid propargylation (62). To a solution of MOM-protected flavonoid in DMF was added K2CO3 (1.5 equiv) and allowed this reaction mixture to stir for 2 min fallowed by the addition of propargyl bromide (1.0 equiv). After stirring for 12 h at room temperature, the reaction mixture was diluted with EtOAC (25 mL) and water (25mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with saturated NaCl solution (25 mL), and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure fallowed by purification using flash column chromatography afforded the desired propargylated compounds in quantitative yield. General procedure of MOM deprotection (63). The compound was solubilized in acetone in a flask attached to a reflux condenser and 3N HCl was added to it. After stirring for 12 h at reflux temperature, the reaction mixture was neutralized with NaHCO3 solution and diluted with EtOAC (25 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with saturated NaCl solution (25 mL), and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure afforded the desired crude which was further purified using flash chromatography.
(63). 1  General procedure for copper-catalyzed azide alkyne cycloaddition (1,4-cycloaddition) (31b-34b). To a solution of terminal alkyne (1 equiv.) and azide (1.0 equiv) were suspended in 1:1 mixture of H2O and DMF. Freshly prepared sodium ascorbate solution in water (5 mol %) was added fallowed by CuSO4.5H2O solution in water (1 mol %) was added. The heterogeneous reaction mixture was stirred vigorously for 12 h, at which point it cleared and TLC analysis indicated complete consumption of the reactants. To this reaction mixture, 2 mL of 3% ammonia solution was added for quenching of excess CuSO4.5H2O and stirred for further 10 min. The reaction mixture was diluted with EtOAC (25 mL), stirred for another 10-15 min and then filtered through a Celite bed. The combined reaction mixture was extracted with EtOAc (2 × 25 mL) and removal of the solvent under reduced pressure afforded crude compound which was further purified using flash chromatography.    09 (m, 8 H). 13 79 (m, 4 H), 1.22-1.10 (m, 10 H). 13
General procedure for synthesis of the propargylated quinazolinone monomer (64). To a solution of quinazolinone monomer in DMF was added K2CO3 (1.5 equiv) and allowed this reaction mixture to stir for 2 min fallowed by the addition of propargybromide (1.5 equiv). After stirring for 3 h, the reaction mixture was diluted with EtOAC (25 mL) and water (25 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL). The organic extracts were combined, washed with saturated NaCl solution (25 mL), and dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure afforded the desired propargylated compounds in quantitative yield and sufficient purity (as indicated by TLC) to be directly used in the next reaction without any further purification. The crude recation mixture was then subjected to deacetylation by solubilizing in THF followed by addition of lithium hydroxide monohydrate Li(OH).H2O (2 equiv). After stirring for overnight, the reaction mixture was diluted with EtOAC (25 mL) and water (25mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL) and removal of the solvent under reduced pressure afforded crude deacetylated compounds which were further purified using flash
General procedure for synthesis of bis-azide derivative. Involved in synthesis of sulfated molecule (41). To a solution of alkyne (2.0 equiv) and bis-azide (1.0 equiv) were suspended in 1:1 mixture of H2O and DMF. Freshly prepared sodium ascorbate solution in water (10 mol %) was added fallowed by CuSO4.5H2O solution in water (2 mol %) was added. The heterogeneous reaction mixture was stirred vigorously for 12 h, at which point it cleared and TLC analysis indicated complete consumption of the reactants. To this reaction mixture, 2 mL of 3% ammonia solution was added for quenching of excess CuSO4.5H2O and stirred for further10 min. The reaction mixture was diluted with EtOAC (25 mL), stirred for another 10-15 min and then filtered through a Celite bed. The combined reaction mixture was extracted with EtOAc (2 × 25 mL) and removal of the solvent under reduced pressure afforded crude compound which was further purified using flash chromatography. Characterization data were reported earlier in reference 3.
General procedure for protection of flavonoid by MOM (61, 67, and 68). See above. General procedure for flavonoid dimerization (48a-55a). To a solution of MOM-protected flavonoid (1.0 equiv) in DMF was added K2CO3 (2.5 equiv) and stirred for two minutes. This was followed by addition of di-bromoalkane (0.5 equiv) and stirred vigorously for 12 h. After the reaction completed as indicated from TLC the reaction mixture was diluted with EtOAC (25 mL) and water (25 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 25 mL), organic layer was washed with saturated NaCl solution (25 mL). The combined organic layers were dried over anhydrous Na2SO4, evaporated under reduced pressure to afford crude flavonoid dimers which were further purified using flash chromatography on silica gel (70%-85% ethyl acetate in hexanes). 55a : X = CH 2 CH 2 CH 2 55b : X = CH 2 CH 2 CH 2 55 : X = CH 2 CH 2 CH 2