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Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.

Liquid chro ma tog ra phy–mass spec trom e try was applied to deter mine the action pattern of dif fer ent chon droi tin lyases. Two com mer cial enzymes, chon dro itin ase ABC (Pro teus vul ga ris) and chon dro itinase ACII (Arth ro bac ter au res cens), hav ing action pat terns pre vi ously deter mined by vis cos i me try and gel elec tro pho re sis were first exam ined. Next, the action pat terns of recombinant lyases, chon dro itin ase ABC from Bac te roi des theta i ota omi cron (expressed in Esch e richia coli) and chon dro itin ase AC from Fla vo bac terium hep ar i num (expressed in its original host), were exam ined. Chon droi tin sul fate A (CS-A, also known as chon droi tin-4-sul fate) was used as the sub strate for these four lyases. Ali quots taken at var i ous time points were ana lyzed. The prod ucts of chon dro itin ase ABC (P. vul ga ris) and chon dro itin ase AC (F. hepar i num) con tained unsat u rated oli go sac cha rides of sizes rang ing from disac cha ride to deca sac cha ride, dem on strat ing that both are end o lyt ic enzymes. The prod ucts affor ded by chon dro itin ase ABC (B. thetai ota omi cron) and chon dro itin ase ACII (A. au res cens) con tained pri mar ily unsat u rated disac cha ride. These two ex o lyt ic enzymes showed dif fer ent minor prod ucts, sug gest ing some sub tle spec i fic ity dif fer ences between the actions of these two ex o lyt ic lyases on chon droi tin sul fate A.

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.