Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Recent approaches for the synthesis of modified cryptophycins.

Cryptophycins are a family of antimitotic depsipeptides with very high cytotoxicity even against multi-drug resistant (MDR) cancer cells. The first representative was isolated from cyanobacteria Nostoc sp. in 1990. Their bioactivity is based on their interaction with the protein tubulin. Cryptophycins were found to induce apoptosis due to inhibition of the microtubule dynamics. Consequently, cryptophycin analogues are considered as potential antitumour agents. Retrosynthetically, cryptophycins can be subdivided into four building blocks, namely units A-D, to be assembled in the total synthesis. Since the discovery of this compound class, numerous synthetic analogues have been designed for structure-activity relationship (SAR) studies. This review gives a critical overview on cryptophycins, while the main focus lies on the synthetic challenges of recently published cryptophycins, together with emerging concepts on cryptophycin bioconjugation and prodrug design.

Conjugates of modified cryptophycins and RGD-peptides enter target cells by endocytosis.

Tumor targeting anticancer drug conjugates that contain a tumor recognition motif (homing device) are of high current relevance. Cryptophycins, naturally occurring cytotoxic cyclo-depsipeptides, have been modified by total synthesis to provide analogues suitable for conjugation to peptide-based homing devices. An array of functionalized ß(2)-amino acids was synthesized and incorporated into cryptophycins. All analogues proved to be highly active in the cytotoxicity assay using the human cervix carcinoma cell line KB-3-1 and its multidrug-resistant subclone KB-V1. Conformational analysis of cryptophycin-52 and two synthetic analogues was performed by NMR and MD methods to obtain information on the influence of the unit C configuration on the overall conformation. An azide-functionalized cryptophycin was connected by CuAAC to an alkyne-containing fluorescently labeled cyclic RGD-peptide as the homing device for internalization studies. Confocal fluorescence microscopy proved integrin-mediated internalization by endocytosis and final lysosomal localization of the cryptophycin prodrug.

Total synthesis and biological evaluation of fluorinated cryptophycins.

Cryptophycins are cytotoxic natural products that exhibit considerable activities even against multi-drug-resistant tumor cell lines. As fluorinated pharmaceuticals have become more and more important during the past decades, fluorine-functionalized cryptophycins were synthesized and evaluated in cell-based cytotoxicity assays. The unit A trifluoromethyl-modified cryptophycin proved to be highly active against KB-3-1 cells and exhibited an IC(50) value in the low picomolar range. However, the replacement of the 3-chloro-4-methoxyphenyl-substituent in unit B by a pentafluorophenyl moiety resulted in a significant loss of activity.

Antibody-drug conjugates in tumor therapy.

Antibody-drug conjugates (ADCs) for tumor therapy contain a cytotoxic drug conjugated to a monoclonal antibody (mAb) across a linker. A homing device, the mAb enables the recognition of tumor cells by binding to tumor-specific antigens. The linker is usually cleaved inside the tumor cell or close to the cell surface according to a well-designed chemical mechanism or upon in vivo degradation of the immunoglobulin moiety. The potent drug load is then released exerting its cytotoxic activity within the target cell or in its close proximity (bystander effect). This concept enables a highly selective approach to minimize side effects for the patient. In August 2011, Seattle Genetics' ADC Adcetris® (brentuximab vedotin) was approved by the US FDA. This review gives background information on this interesting new technology and presents the relevant, recent patents in this research area. patents by Seattle Genetics, Immunogen and Biotest are discussed owing to their eminent contribution and relevance to the field.

A two step synthesis of a key unit B precursor of cryptophycins by asymmetric hydrogenation.

A novel highly enantioselective two step access to a unit B precursor of cryptophycins in good yields from commercially available starting materials has been developed. The key step is an asymmetric hydrogenation using the commercially available [(COD)Rh-(R,R)-Et-DuPhos]BF(4) catalyst. The synthetic route provides the advantage of less synthetic steps, proceeds with high yields and enantioselectivity, and avoids hazardous reaction conditions.

Approaches for the synthesis of functionalized cryptophycins.

The first syntheses of bioactive cryptophycins functionalized at unit D were accomplished in a one-pot Staudinger reduction/cyclization step. An azido precursor for the lower part of the backbone was introduced to minimize protective group chemistry and enable a very convenient synthesis of cryptophycin-52 and unit D cryptophycin analogues containing an ester or a free carboxylic acid for bioconjugations. Both new cryptophycin derivatives show high biological activity in cytotoxicity assays.

Achiral oligoamines as versatile tool for the development of aspartic protease inhibitors.

Due to the important role that aspartic proteases play in many patho-physiological processes, they have intensively been targeted by modern drug development. However, up to now, only for two family members, renin and HIV protease, approved drugs are available. Inhibitor development, mostly guided by mimicking the natural peptide substrates, resulted in very potent inhibitors for several targets, but the pharmacokinetic properties of these compounds were often not optimal. Herein we report a novel approach for lead structure discovery of non-peptidic aspartic protease inhibitors using easily accessible achiral linear oligoamines as starting point. An initial library comprising 11 inhibitors was developed and screened against six selected aspartic proteases. Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was confirmed by determination of the crystal structure of two derivatives in complex with the HIV-1 protease, and represents a promising basis for the further inhibitor development.