Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an Ester Bond-Forming Non-ribosomal Peptide Synthetase Module

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on themultifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique nonribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues

Comment on "Theory of tailoring sonic devices: Diffraction dominates over refraction"

Recently N. Garcia et al. (Phys. Rev. E 67, 046606 (2003)) theoretically studied several acoustic devices with dimensions on de order of several wavelenghts. The authors discussed on experimental results previously reported by several of us (F. Cervera et al., Phys. Rev. Lett. 88, 023902 (2002)). They concluded that diffraction and not refraction is the ominating mechanism that explain the focusing effects observed in those experiments. In this Comment we reexamined their calculations and discussed why some of their interpretations of our results are misleading.Comment: 2 pages, 2 figures, a comment on an articl

Vaccines : A rapidly evolving technology - Are the hurdles being addressed?

AbstractVaccination usually works in infectious disease, why not in Cancer? Differences in the potency of microbial and cancer antigens, poor initiation of an immune response due to inadequate expression of tumour associated antigens, weak antigens or tolerance induction and local immune suppression were considered. There is a big difference between a therapeutic and a prophylactic vaccine.The opinion of the expert group was that an improved therapeutic efficacy can hardly be expected by further variation of types of vaccines, schedules, routes of administration and adjuvants alone. A major hurdle for developing therapeutic cancer vaccines is the need to effectively monitor the immune response and to be able to use this in an adaptive trial approach.End-points of assessment should be different from standard treatments as complete response or partial responses are usually low, unless combined with other therapies.In order to focus resources to overcome the hurdles of enhancing the therapeutic efficacy of cancer vaccines the Cancer Vaccine Clinical Trial Working Group, representing academia and the pharmaceutical and biotechnology industries has in a consensus process defined 'A clinical development paradigm for cancer vaccines and related biologics'

Semaphorin 6A knockout mice display abnormalities across ethologically-based topographies of exploration and in motor learning

Semaphorins are secreted or membrane-bound proteins implicated in neurodevelopmental processes of axon guidance and cell migration. Exploratory behaviour and motor learning was examined ethologically in Semaphorin 6A (Sema6A) mutant mice. The ethogram of initial exploration in Sema6A knockout mice was characterised by increased rearing to wall with decreased sifting; over subsequent habituation, locomotion, sniffing and rearing to wall were increased, with reduced habituation of rearing seated. Rotarod analysis indicated delayed motor learning in Sema6A heterozygous mutants. Disruption to the axonal guidance and cell migration processes regulated by Sema6A is associated with topographically specific disruption to fundamental aspects of behaviour, namely the ethogram of initial exploration and subsequent habituation to the environment, and motor learning