Eva Majerova

Facile stereoselective synthesis of γ-substituted γ-amino acids from the corresponding α-amino acids

Abstract A facile stereoselective method for the synthesis of γ-substituted, γ-amino acids from α-amino acids was developed. The key step of the procedure is complete reduction of the keto functionality of α-amino acyl Meldrums acid by sodium acetoxyborohydride. The resulting amino alkyl Meldrums acid undergoes thermal decarboxylative ring closure to a 5-substituted pyrrolidinone which yields the corresponding γ-amino acid after basic hydrolysis. The overall yield of the procedure ranges from 40 to 65%.

Effect of caspase cleavage-site phosphorylation on proteolysis

Caspases are important mediators of apoptotic cell death. Several cellular protein substrates of caspases contain potential phosphorylation site(s) at the cleavage-site region, and some of these sites have been verified to be phosphorylated. Since phosphorylation may affect substantially the substrate susceptibility towards proteolysis, phosphorylated, non-phosphorylated and substituted oligopeptides representing such cleavage sites were studied as substrates of apoptotic caspases 3, 7 and 8. Peptides containing phosphorylated serine residues at P4 and P1 positions were found to be substantially less susceptible towards proteolysis as compared with the serine-containing analogues, while phosphoserine at P3 did not have a substantial effect. P1 serine as well as P1-phosphorylated, serine-containing analogues of an oligopeptide representing the poly(ADP-ribose) polymerase cleavage site of caspase-3 were not hydrolysed by any of these enzymes, whereas the P1 aspartate-containing peptides were efficiently hydrolysed. These findings were interpreted with the aid of molecular modelling. Our results suggest that cleavage-site phosphorylation in certain positions could be disadvantageous or detrimental with respect to cleavability by caspases. Cleavage-site phosphorylation may therefore provide a regulatory mechanism to protect substrates from caspase-mediated degradation.

Kinetics of the dimerization of retroviral proteases: the "fireman's grip" and dimerization.

All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the “firemans grip,” which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp‐Thr(Ser)‐Gly triplets. It was shown previously that the firemans grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp‐Thr‐Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild‐type and mutant proteases from HIV and myeloblastosis‐associated virus harboring either Ser or Thr in their Asp‐Thr(Ser)‐Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be ∼10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the firemans grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.

First-in-Human Phase 0 Trial of Oral 5-Iodo-2-Pyrimidinone-2′-Deoxyribose in Patients with Advanced Malignancies

Purpose: Iododeoxyuridine (IdUrd), a halogenated nucleoside analog, produced clinical responses when administered as a radiosensitizer via continuous intravenous (c.i.v.) infusion over the course of radiotherapy. We conducted a phase 0 trial of 5-iodo-2-pyrimidinone-2′-deoxyribose (IPdR), an oral prodrug of IdUrd, in patients with advanced malignancies to assess whether the oral route was a feasible alternative to c.i.v. infusion before embarking on large-scale clinical trials. Plasma concentrations of IPdR, IdUrd, and other metabolites were measured after a single oral dose of IPdR. Experimental Design: Eligible patients had advanced refractory malignancies. A single oral dose of IPdR was administered per patient and patients were followed for 14 days for safety assessments; dose escalations were planned (150, 300, 600, 1,200, and 2,400 mg) with one patient per dose level and 6 patients at the highest dose level. Blood sampling was conducted over a 24-hour period for pharmacokinetic analysis. Results: There were no drug-related adverse events. Plasma concentrations of IdUrd generally increased as the dose of IPdR escalated from 150 to 2,400 mg. All patients at the 2,400 mg dose achieved peak IdUrd levels of (mean ± SD) 4.0 μmol/L ± 1.02 μmol/L at 1.67 ± 1.21 hours after IPdR administration. Conclusions: Adequate plasma levels of IdUrd were obtained to justify proceeding with a phase I trial of IPdR in combination with radiation. This trial shows the ability of a small, phase 0 study to provide critical information for decision-making regarding future development of a drug. Clin Cancer Res; 19(7); 1852–7. ©2013 AACR.

Liquid Chromatographic Determination of NSC 737664 (ABT-888: An Inhibitor of Poly(ADP-ribose) Polymerase (PARP)) in Plasma and Urine in a Phase 0 Clinical Trial

Abstract A gradient reversed-phase high performance liquid chromatographic method was developed for determining NSC 737664 (2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide; ABT-888) in human plasma and urine. The chromatographic separation used a mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, and a C18 column (150 mm × 4.6 mm, 5 µ). Quantitation was performed using UV detection at 300 nm. Chromatographic peak identity was confirmed using positive-ion electrospray ionization mass spectrometry. The method was shown to be specific, accurate, and reproducible, and thereby appropriate for monitoring plasma and urine levels of the agent in support of a phase 0 clinical study.

First-in-human study of the epichaperome inhibitor PU-H71: clinical results and metabolic profile

SummaryBackground Molecular chaperone targeting has shown promise as a therapeutic approach in human cancers of various histologies and genetic backgrounds. The purine-scaffold inhibitor PU-H71 (NSC 750424), selective for Hsp90 in epichaperome networks, has demonstrated antitumor activity in multiple preclinical cancer models. The present study was a first in-human trial of PU-H71 aimed at establishing its safety and tolerability and characterizing its pharmacokinetic (PK) profile on a weekly administration schedule in human subjects with solid tumors refractory to standard treatments. Methods PU-H71 was administered intravenously over 1xa0h on days 1 and 8 of 21-day cycles in patients with refractory solid tumors. Dose escalation followed a modified accelerated design. Blood and urine were collected during cycles 1 and 2 for pharmacokinetics analysis. Results Seventeen patients were enrolled in this trial. Grade 2 and 3 adverse events were observed but no dose limiting toxicities occurred, thus the human maximum tolerated dose was not determined. The mean terminal half-life (T1/2) was 8.4xa0±xa03.6xa0h, with no dependency to dose level. A pathway for the metabolic disposal of PU-H71 in humans was derived from microsome studies. Fourteen patients were also evaluable for clinical response; 6 (35%) achieved a best response of stable disease for >2xa0cycles, with 2 patients remaining on study for 6xa0cycles. The study closed prematurely due to discontinuation of drug supply. Conclusions PU-H71 was well tolerated at the doses administered during this study (10 to 470xa0mg/m2/day), with no dose limiting toxicities.