Lev Lis

Specific inhibition of p97/VCP ATPase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains.

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (kcat/Km) of D1 ATP hydrolysis 280-fold, by increasing kcat 7-fold and decreasing Km about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.

Single-Centered Hydrogen-Bonded Enhanced Acidity (SHEA) Acids: A New Class of Brønsted Acids

Hydrogen bonds are the dominant motif for organizing the three-dimensional structures of biomolecules such as carbohydrates, nucleic acids, and proteins, and serve as templates for proton transfer reactions. Computations, gas-phase acidity measurements, and pK(a) determinations in dimethyl sulfoxide on a series of polyols indicate that multiple hydrogen bonds to a single charged center lead to greatly enhanced acidities. A new class of Brønsted acids, consequently, is proposed.

Phosphonooxymethyl Prodrug of Triptolide: Synthesis, Physicochemical Characterization, and Efficacy in Human Colon Adenocarcinoma and Ovarian Cancer Xenografts.

A disodium phosphonooxymethyl prodrug of the antitumor agent triptolide was prepared from the natural product in three steps (39% yield) and displayed excellent aqueous solubility at pH 7.4 (61 mg/mL) compared to the natural product (17 μg/mL). The estimated shelf life (t90) for hydrolysis of the prodrug at 4 °C and pH 7.4 was found to be two years. In a mouse model of human colon adenocarcinoma (HT-29), the prodrug administered intraperitoneally was effective in reducing or eliminating xenograft tumors at dose levels as low as 0.3 mg/kg when given daily and at 0.9 mg/kg when given less frequently. When given via intraperitoneal and oral routes at daily doses of 0.6 and 0.9 mg/kg, the prodrug was also effective and well tolerated in a mouse model of human ovarian cancer (A2780).

SLC28A3 genotype and gemcitabine rate of infusion affect dFdCTP metabolite disposition in patients with solid tumours

Background:Gemcitabine is used for the treatment of several solid tumours and exhibits high inter-individual pharmacokinetic variability. In this study, we explore possible predictive covariates on drug and metabolite disposition.Methods:Forty patients were enrolled. Gemcitabine and dFdU concentrations in the plasma and dFdCTP concentrations in peripheral blood mononuclear cell were measured to 72 h post infusion, and pharmacokinetic parameters were estimated by nonlinear mixed-effects modelling. Patient-specific covariates were tested in model development.Results:The pharmacokinetics of gemcitabine was best described by a two-compartment model with body surface area, age and NT5C2 genotype as significant covariates. The pharmacokinetics of dFdU and dFdCTP were adequately described by three-compartment models. Creatinine clearance and cytidine deaminase genotype were significant covariates for dFdU pharmacokinetics. Rate of infusion of <25 mg m−2 min−1 and the presence of homozygous major allele for SLC28A3 (CC genotype) were each associated with an almost two-fold increase in the formation clearance of dFdCTP.Conclusion:Prolonged dFdCTP systemic exposures (⩾72 h) were commonly observed. Infusion rate <25 mg m−2 min−1 and carriers for SLC28A3 variant were each associated with about two-fold higher dFdCTP formation clearance. The impacts of these covariates on treatment-related toxicity in more selected patient populations (that is, first-line treatment, single disease state and so on) are not yet clear.

Experimental and Computational Bridgehead C–H Bond Dissociation Enthalpies

Bridgehead C-H bond dissociation enthalpies of 105.7 ± 2.0, 102.9 ± 1.7, and 102.4 ± 1.9 kcal mol(-1) for bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and adamantane, respectively, were determined in the gas phase by making use of a thermodynamic cycle (i.e., BDE(R-H) = ΔH°(acid)(H-X) - IE(H(·)) + EA(X(·))). These results are in good accord with high-level G3 theory calculations, and the experimental values along with G3 predictions for bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[3.1.1]heptane, and bicyclo[4.2.1]nonane were found to correlate with the flexibility of the ring system. Rare examples of alkyl anions in the gas phase are also provided.

Carbon–Hydrogen Bond Dissociation Energies: The Curious Case of Cyclopropene

The ionization energy (IE) of the 3-cyclopropenyl radical (6.00 ± 0.17 eV) was measured in the gas phase by reacting 3-cyclopropenium cation (c-C3H3(+)) with a series of reference reagents of known IEs. This result was combined in a thermodynamic cycle to obtain the heat of formation of c-C3H3(•) (118.9 ± 4.0 kcal mol(-1)) and the allylic C-H bond dissociation energy (BDE) of cyclopropene (104.4 ± 4.0 kcal mol(-1)). These experimental values are well reproduced by high level G3 and W1 computations and reveal that the BDE is similar to that for cyclopropane and the vinyl position of cyclopropene. This is unprecedented and is a reflection of the unusual nature of cyclopropene.

Reformulating Tylocrebrine in Epidermal Growth Factor Receptor Targeted Polymeric Nanoparticles Improves Its Therapeutic Index

Several promising anticancer drug candidates have been sidelined owing to their poor physicochemical properties or unfavorable pharmacokinetics, resulting in high overall cost of drug discovery and development. Use of alternative formulation strategies that alleviate these issues can help advance new molecules to the clinic at a significantly lower cost. Tylocrebrine is a natural product with potent anticancer activity. Its clinical trial was discontinued following the discovery of severe central nervous system toxicities. To improve the safety and potency of tylocrebrine, we formulated the drug in polymeric nanoparticles targeted to the epidermal growth factor receptor (EGFR) overexpressed on several types of tumors. Through in vitro studies in different cancer cell lines, we found that EGFR targeted nanoparticles were significantly more effective in killing tumor cells than the free drug. In vivo pharmacokinetic studies revealed that encapsulation in nanoparticles resulted in lower brain penetration and enhanced tumor accumulation of the drug. Further, targeted nanoparticles were characterized by significantly enhanced tumor growth inhibitory activity in a mouse xenograft model of epidermoid cancer. These results suggest that the therapeutic index of drugs that were previously considered unusable could be significantly improved by reformulation. Application of novel formulation strategies to previously abandoned drugs provides an opportunity to advance new molecules to the clinic at a lower cost. This can significantly increase the repertoire of treatment options available to cancer patients.

Combinatorial Pharmacologic Effects of Gemcitabine and its Metabolite dFdU

Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half‐life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype‐based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose‐dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell‐line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug–metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity.