Onur Atasoylu

Tetrazine Phototriggers: Probes for Peptide Dynamics†

Ultrafast photochemical triggers hold the promise of providing information on the dynamics of peptide and protein folding.[1] Prior to photolysis, bonding to the trigger constrains the peptide to have a narrow structure distribution. Photochemical triggering releases the constraints, permitting the molecule to evolve to a different equilibrium distribution. The structure evolution, even when ultrafast, can be followed by infrared probe or two-dimensional infrared spectroscopy. Fast phototriggering can thus reveal early kinetic events in protein dynamics by providing a means to explore the free energy landscape of folding and misfolding. Several phototriggers have been developed for this purpose,[1,2] but there remain significant challenges. For example, disulfide bonds in peptides can be used as a phototriggers. Deep UV light severs the disulfide bond and releases the structural constraints; such experiments have been carried out in short helical peptides,[1a,b] cyclic peptides,[1d] and β hairpins.[1c] Although disulfide photolysis offers the capability of initiating ultrafast structure equilibration, limitations preclude broad generality of this technique. Homolytic S–S bond scission reveals two reactive radicals that can undergo geminate recombination, as well as reactions with protein sidechains. Moreover, the UV excitation required to dissociate the disulfide bond also excites the peptide backbone. Another example is azobenzene which undergoes fast, reversible photoisomerization. When designed into a peptide a light pulse can be used to cause the system to reversibly shift between significantly different equilibrium configurations.[3]

Interactions of halichondrin B and eribulin with tubulin.

Compounds that modulate microtubule dynamics include highly effective anticancer drugs, leading to continuing efforts to identify new agents and improve the activity of established ones. Here, we demonstrate that [(3)H]-labeled halichondrin B (HB), a complex, sponge-derived natural product, is bound to and dissociated from tubulin rapidly at one binding site per αβ-heterodimer, with an apparent K(d) of 0.31 μM. We found no HB-induced aggregation of tubulin by high-performance liquid chromatography, even following column equilibration with HB. Binding of [(3)H]HB was competitively inhibited by a newly approved clinical agent, the truncated HB analogue eribulin (apparent K(i), 0.80 μM) and noncompetitively by dolastatin 10 and vincristine (apparent K(i)s, 0.35 and 5.4 μM, respectively). Our earlier studies demonstrated that HB inhibits nucleotide exchange on β-tubulin, and this, together with the results presented here, indicated the HB site is located on β-tubulin. Using molecular dynamics simulations, we determined complementary conformations of HB and β-tubulin that delineated in atomic detail binding interactions of HB with only β-tubulin, with no involvement of the α-subunit in the binding interaction. Moreover, the HB model served as a template for an eribulin binding model that furthered our understanding of the properties of eribulin as a drug. Overall, these results established a mechanistic basis for the antimitotic activity of the halichondrin class of compounds.

Total synthesis of (-)-2-epi-peloruside A.

A convergent synthesis of (-)-2-epi-peloruside A has been achieved. Highlights include implementation of multicomponent type I anion relay chemistry (ARC) to unite 2-TBS-1,3-dithiane with two epoxides to construct the eastern hemisphere, a late-stage dithiane union to secure the complete, fully functionalized carbon backbone, and Yamaguchi macrolactonization, which led to (-)-2-epi-peloruside A via an unexpected epimerization at C(2).

Cyclopentane-1,3-dione: A Novel Isostere for the Carboxylic Acid Functional Group. Application to the Design of Potent Thromboxane (A2) Receptor Antagonists

Cyclopentane-1,3-diones are known to exhibit pK(a) values typically in the range of carboxylic acids. To explore the potential of the cyclopentane-1,3-dione unit as a carboxylic acid isostere, the physical-chemical properties of representative congeners were examined and compared with similar derivatives bearing carboxylic acid or tetrazole residues. These studies suggest that cyclopentane-1,3-diones may effectively substitute for the carboxylic acid functional group. To demonstrate the use of the cyclopentane-1,3-dione isostere in drug design, derivatives of a known thromboxane A(2) prostanoid (TP) receptor antagonist, 3-(3-(2-(4-chlorophenylsulfonamido)ethyl)phenyl)propanoic acid (12), were synthesized and evaluated in both functional and radioligand-binding assays. A series of mono- and disubstituted cyclopentane-1,3-dione derivatives (41-45) were identified that exhibit nanomolar IC(50) and K(d) values similar to 12. Collectively, these studies demonstrate that the cyclopentane-1,3-dione moiety comprises a novel isostere of the carboxylic acid functional group. Given the combination of the relatively strong acidity, tunable lipophilicity, and versatility of the structure, the cyclopentane-1,3-dione moiety may constitute a valuable addition to the palette of carboxylic acid isosteres.

Design, synthesis, and biological evaluation of diminutive forms of (+)-spongistatin 1: lessons learned.

The design, synthesis, and biological evaluation of two diminutive forms of (+)-spongistatin 1, in conjunction with the development of a potentially general design strategy to simplify highly flexible macrocyclic molecules while maintaining biological activity, have been achieved. Examination of the solution conformations of (+)-spongistatin 1 revealed a common conformational preference along the western perimeter comprising the ABEF rings. Exploiting the hypothesis that the small-molecule recognition/binding domains are likely to comprise the conformationally less mobile portions of a ligand led to the design of analogues, incorporating tethers (blue) in place of the CD and the ABCD components of the (+)-spongistatin 1 macrolide, such that the conformation of the retained (+)-spongistatin 1 skeleton would mimic the assigned solution conformations of the natural product. The observed nanomolar cytotoxicity and microtubule destabilizing activity of the ABEF analogue provide support for both the assigned solution conformation of (+)-spongistatin 1 and the validity of the design strategy.

Design and synthesis of (+)-discodermolide-paclitaxel hybrids leading to enhanced biological activity.

Potential binding modes of (+)-discodermolide at the paclitaxel binding site of tubulin have been identified by computational studies based on earlier structural and SAR data. Examination of the prospective binding modes reveal that the aromatic pocket occupied by the paclitaxel side chain is unoccupied by (+)-discodermolide. Based on these findings, a small library of (+)-discodermolide-paclitaxel hybrids have been designed and synthesized. Biological evaluation reveals a two- to eight-fold increase in antiproliferative activity compared to the parent molecule using the A549 and MCF-7 cancer cell lines.

The solution structure of (+)-spongistatin 1 in DMSO.

The solution structure of (+)-spongistatin 1 (1) has been determined via 1- and 2-D NMR techniques in conjunction with extensive in silico conformational analysis to comprise a mixture of 4 major rapidly interconverting conformational families.

Design, synthesis, and biological evaluation of EF- and ABEF- analogues of (+)-spongistatin 1.

The design, synthesis, and biological evaluation of two potential (+)-spongistatin 1 analogues have been achieved. The analogues, incorporating tethers (red) in place of the ABCD and the CD components of the (+)-spongistatin 1 macrolide, were designed such that the conformations of the retained skeleton (blue) would mimic the assigned major solution conformation of the natural product The nanomolar cytotoxicity observed for the ABEF analogue provides strong support for the assigned solution conformation.

Abstract 2789: Interaction of (+)-discodermolide-Taxol hybrids with microtubules

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Taxol and discodermolide are both microtubule stabilizing agents that bind to the β-tubulin subunit of microtubules. Based on our results that the hydrophobic binding pocket of β-tubulin is occupied by the Taxol side chain but not by (+)-discodermolide, and that the two drugs act synergistically in cancer cell lines, a small library of (+)-discodermolide-Taxol hybrids was synthesized (J. Med. Chem. 54:6319, 2011). Cytotoxicity assays demonstrated a 2-9-fold increase in antiproliferative activity by the hybrids compared to discodermolide in the human cancer cell lines, A549 and MCF-7. Hybrids containing a tether of 3 carbons that connect discodermolide with the Taxol side chain, exhibited the best activity. Tubulin assembly assays were performed with the two most potent hybrid molecules. Like discodermolide, these two hybrids increased tubulin assembly rapidly without a lag period. Tubulin polymerization studies using purified bovine brain tubulin demonstrated that they also had the greatest effect on the formation of polymerized microtubules. These results also were observed in intact A549 cells and in 100,000 x g supernatants prepared from these cells. We have previously shown that [3H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-231 of β-tubulin. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of purified bovine brain tubulin by 94%, demonstrating the specificity of this photolabeling. Microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, discodermolide and ixabepilone, each at a 5-fold molar excess, inhibited the photolabeling by 24%, 92%, 100% and 41%, respectively, indicating that discodermolide is the most potent inhibitor of the photolabeling. In contrast, two other MSAs, laulimalide and peloruside that are known to bind to a different site in β-tubulin, exhibited stimulatory effects on the photolabeling. Both drugs, at a 5-fold molar excess, increased the labeling by 30-40%. [3H]2-(m-azidobenzoyl)Taxol (0.5 - 20 µM) was used to study the kinetics of the inhibitory effects of the hybrid molecules on photoaffinity labeling of tubulin. Discodermolide-Taxol hybrids inhibited the photolabeling of bovine brain tubulin in a dose dependent manner. The concentrations that inhibited by 50% were lowest for the two most potent hybrid molecules. Therefore, the tubulin polymerization activity and the binding affinity of the hybrids to β-tubulin correlated with their antiproliferative activity. Other biological properties of the discodermolide-Taxol hybrids including senescence and antitumor activity are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2789. doi:1538-7445.AM2012-2789