A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is distinct from host homologues.

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.

Cannabinoformins: Designing Biguanide-Embedded, Orally Available, Peripherally Selective Cannabinoid-1 Receptor Antagonists for Metabolic Syndrome Disorders.

We have designed orally bioavailable, non-brain-penetrant antagonists of the cannabinoid-1 receptor (CB1R) with a built-in biguanide sensor to mimic 5'-adenosine monophosphate kinase (AMPK) activation for treating obesity-associated co-morbidities. A series of 3,4-diarylpyrazolines bearing rational pharmacophoric pendants designed to limit brain penetration were synthesized and evaluated in CB1R ligand binding assays and recombinant AMPK assays. The compounds displayed high CB1R binding affinity and potent CB1R antagonist activities and acted as AMPK activators. Select compounds showed good oral exposure, with compounds 36, 38-S, and 39-S showing <5% brain penetrance, attesting to peripheral restriction. In vivo studies of 38-S revealed decreased food intake and body weight reduction in diet-induced obese mice as well as oral in vivo efficacy of 38-S in ameliorating glucose tolerance and insulin resistance. The designed "cannabinoformin" four-arm CB1R antagonists could serve as potential leads for treatment of metabolic syndrome disorders with negligible neuropsychiatric side effects.

Analysis of the isomer ratios of polymethylated-DOTA complexes and the implications on protein structural studies.

A rigidified and symmetrical polymethylated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand bearing four SSSS methyl groups in both the tetraaza ring and the acetate arms (SSSS-SSSS-M4DOTMA) was prepared. The isomer ratio of SSSS-SSSS-M4DOTMA complexed with a series of lanthanide ions was carefully investigated using RP-HPLC and NMR. A square antiprismatic (SAP) configuration was exclusively observed for the early lanthanides, while the twisted square antiprismatic (TSAP) geometry was preferred as the lanthanide ion size decreases. The late lanthanides preferentially adopted the TSAP geometry. One of the pendant arms was modified with a pyridyl disulfide group (SSSS-SSSS-M8SPy) for cysteine attachment and displayed a similar isomer trend as the parent compound, Ln-SSSS-SSSS-M4DOTMA. Covalent attachment to the ubiquitin S57C mutant showed resonances whose intensities are in agreement with the isomeric population observed by RP-HPLC. Furthermore, the NOE experiments combined with quantum chemical calculations have unequivocally demonstrated that the SAP of Pr-SSSS-SSSS-M4DOTMA and Pr-SSSS-SSSS-M8SPy, as well as the TSAP of Yb-SSSS-SSSS-M8SPy are more stable than their corresponding isomers.

Rational design, synthesis, and evaluation of tetrahydroxamic acid chelators for stable complexation of zirconium(IV).

Metals of interest for biomedical applications often need to be complexed and associated in a stable manner with a targeting agent before use. Whereas the fundamentals of most transition-metal complexation processes have been thoroughly studied, the complexation of Zr(IV) has been somewhat neglected. This metal has received growing attention in recent years, especially in nuclear medicine, with the use of (89) Zr, which a ß(+) -emitter with near ideal characteristics for cancer imaging. However, the best chelating agent known for this radionuclide is the trishydroxamate desferrioxamine B (DFB), the Zr(IV) complex of which exhibits suboptimal stability, resulting in the progressive release of (89) Zr in vivo. Based on a recent report demonstrating the higher thermodynamic stability of the tetrahydroxamate complexes of Zr(IV) compared with the trishydroxamate complexes analogues to DFB, we designed a series of tetrahydroxamic acids of varying geometries for improved complexation of this metal. Three macrocycles differing in their cavity size (28 to 36-membered rings) were synthesized by using a ring-closing metathesis strategy, as well as their acyclic analogues. A solution study with (89) Zr showed the complexation to be more effective with increasing cavity size. Evaluation of the kinetic inertness of these new complexes in ethylenediaminetetraacetic acid (EDTA) solution showed significantly improved stabilities of the larger chelates compared with (89) Zr-DFB, whereas the smaller complexes suffered from insufficient stabilities. These results were rationalized by a quantum chemical study. The lower stability of the smaller chelates was attributed to ring strain, whereas the better stability of the larger cyclic complexes was explained by the macrocyclic effect and by the structural rigidity. Overall, these new chelating agents open new perspectives for the safe and efficient use of (89) Zr in nuclear imaging, with the best chelators providing dramatically improved stabilities compared with the reference DFB.

Quantum chemical calculations and mutational analysis suggest heat shock protein 90 catalyzes trans-cis isomerization of geldanamycin.

The affinity of geldanamycin (GA) for binding to heat shock protein 90 (HSP90) is 50- to 100-fold weaker than is the affinity of the structurally distinct natural product radicicol. X-ray crystallography shows that although radicicol maintains its free conformation when bound to HSP90, the conformation of GA is dramatically altered from an extended conformation with a trans amide bond to a kinked shape in which the amide group in the ansa ring has the cis configuration. We have performed ab initio quantum chemical calculations to demonstrate that the trans-cis isomeriztion of GA in solution is both kinetically and thermodynamically unfavorable. Thus, we propose that HSP90 catalyzes the isomerization of GA. We identify Ser113, a conserved residue outside the ATP binding pocket, as essential for the isomerization of GA. In support of this model, we show that radicicol binds equally well to both wild-type HSP90 and the Ser113 mutant, whereas the binding of GA to the Ser113 mutant is decreased significantly from its binding to wild-type HSP90. Based on this finding, a mechanism of keto-enol tautomerization of GA catalyzed by HSP90 is proposed. The added requirement of isomerization prior to tight binding may explain the enhanced binding affinity of GA for HSP90 in a cell extract versus in a purified form.