Optical Control of Proteasomal Protein Degradation with a Photoswitchable Lipopeptide.

Photolipids have emerged as attractive tools for the optical control of lipid functions. They often contain an azobenzene photoswitch that imparts a cis double-bond upon irradiation. Herein, we present the application of photoswitching to a lipidated natural product, the potent proteasome inhibitor cepafungin I. Several azobenzene-containing lipids were attached to the cyclopeptide core, yielding photoswitchable derivatives. Most notably, PhotoCep4 exhibited a 10-fold higher cellular potency in its light-induced cis-form, matching the potency of natural cepafungin I. The length of the photolipid tail and distal positioning of the azobenzene photoswitch with respect to the macrocycle is critical for this activity. In a proteome-wide experiment, light-triggered PhotoCep4 modulation showed high overlap with constitutively active cepafungin I. The mode of action was studied using crystallography and revealed an identical binding of the cyclopeptide in comparison to cepafungin I, suggesting that differences in their cellular activity originate from switching the tail structure. The photopharmacological approach described herein could be applicable to many other natural products as lipid conjugation is common and often necessary for potent activity. Such lipids are often introduced late in synthetic routes, enabling facile chemical modifications.

Comprehensive Structure-Activity Relationship Studies of Cepafungin Enabled by Biocatalytic C-H Oxidations.

The cepafungins are a class of highly potent and selective eukaryotic proteasome inhibitor natural products with potential to treat refractory multiple myeloma and other cancers. The structure-activity relationship of the cepafungins is not fully understood. This Article chronicles the development of a chemoenzymatic approach to cepafungin I. A failed initial route involving derivatization of pipecolic acid prompted us to examine the biosynthetic pathway for the production of 4-hydroxylysine, which culminated in the development of a 9-step synthesis of cepafungin I. An alkyne-tagged analogue enabled chemoproteomic studies of cepafungin and comparison of its effects on global protein expression in human multiple myeloma cells to the clinical drug bortezomib. A preliminary series of analogues elucidated critical determinants of potency in proteasome inhibition. Herein we report the chemoenzymatic syntheses of 13 additional analogues of cepafungin I guided by a proteasome-bound crystal structure, 5 of which are more potent than the natural product. The lead analogue was found to have 7-fold greater proteasome ß5 subunit inhibitory activity and has been evaluated against several multiple myeloma and mantle cell lymphoma cell lines in comparison to the clinical drug bortezomib.

Concise Chemoenzymatic Total Synthesis and Identification of Cellular Targets of Cepafungin I.

The natural product cepafungin I was recently reported to be one of the most potent covalent inhibitors of the 20S proteasome core particle through a series of in vitro activity assays. Here, we report a short chemoenzymatic total synthesis of cepafungin I featuring the use of a regioselective enzymatic oxidation to prepare a key hydroxylated amino acid building block in a scalable fashion. The strategy developed herein enabled access to a chemoproteomic probe, which in turn revealed the exceptional selectivity and potency of cepafungin I toward the ß2 and ß5 subunits of the proteasome. Further structure-activity relationship studies suggest the key role of the hydroxyl group in the macrocycle and the identity of the lipid tail in modulating the potency of this natural product family. This study lays the groundwork for further medicinal chemistry exploration to fully realize the anticancer potential of cepafungin I.

Recent advances in the chemoenzymatic synthesis of bioactive natural products.

The field of organic chemistry has recently witnessed a rapid rise in the use of chemoenzymatic strategies for the synthesis of complex molecules. Under this paradigm, biocatalytic methods and contemporary synthetic methods are used synergistically in a multistep approach toward a target molecule. In light of the unparalleled regioselectivity and stereoselectivity of enzymatic transformations and the reaction diversity of contemporary organic chemistry, chemoenzymatic strategies hold enormous potential for streamlining access to important bioactive molecules. This review covers recent demonstrations of chemoenzymatic approaches in chemical synthesis, with special emphasis on the preparation of medicinally relevant natural products.

Identification of a lysine 4-hydroxylase from the glidobactin biosynthesis and evaluation of its biocatalytic potential.

We present the functional characterization of GlbB, a lysine 4-hydroxylase from the glidobactin biosynthetic gene cluster. Despite its narrow substrate specificity, GlbB is able to catalyze the hydroxylation of l-lysine with excellent total turnover number and complete regio- and diastereoselectivity. The synthetic utility of GlbB is illustrated by its use in the efficient preparation of a key dipeptide fragment of glidobactin.