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Chlorofusin, its seven chromophore diastereomers, and key analogues were comparatively examined for inhibition of MDM2–p53 binding revealing that the chromophore, but not simple replacements, contributes significantly to the natural products properties, and that this contribution is independent of its relative and absolute stereochemistry.
The tumor suppressor p53 is an important part of an innate cancer defense mechanism and acts as a transcription factor that initiates cell cycle arrest and apoptosis in response to stress such as DNA damage.1–4 The activity of p53 is modulated by MDM2 (HDM2), which tightly binds p53 preventing it from acting as a regulator of cell division5–7 and targeting it for nuclear export and degradation.8,9 Overexpression of MDM2 has been implicated in many cancers, defining the p53–MDM2 interaction as an attractive target for therapeutic intervention.4 An X-ray crystal structure of the N-terminal domain of MDM2 bound to a 15-residue transactivation domain of p53 revealed the structural details of their complex that is mediated by the interaction of three hydrophobic residues of a p53 α-helix with a hydrophobic cleft of MDM2. Molecules that bind the hydrophobic cleft of MDM2 disrupt this protein–protein interaction with p53, restoring its regulatory function and inhibiting tumor growth.4,11
Chlorofusin (1, Figure 1) was isolated from the fungal strain Microdochium caespitosum12 and reported to disrupt the MDM2–p53 interaction by directly binding to the N-terminal domain of MDM2 (IC50 = 4.6 μM, KD = 4.7 μM).12–14 Thus, chlorofusin has been regarded as an exciting lead for antineoplastic intervention that acts by a rare disruption of a protein–protein interaction although the structural details of this inhibitory interaction derived from MDM2 binding have yet to be established.15 On the basis of extensive spectroscopic and degradation studies, the chlorofusin structure was proposed to consist of a densely functionalized, azaphilone-derived chromophore linked through the terminal amine of ornithine to a 27-membered cyclic peptide composed of nine amino acid residues.12
Although the studies permitted the identification of the cyclic peptide structure and connectivity, the two asparagine residues Asn3 and Asn4 were only established to have opposite stereochemistries (L and D) and their respective assignments were not possible. Similarly, the spectroscopic studies conducted by Williams provided the structure and an assigned relative stereochemistry for the unusual azaphilone-derived chromophore, but did not permit an assignment of its absolute stereochemistry. In early studies, we reported the synthesis of the two cyclic peptide diastereomers bearing either the L-Asn3/D-Asn4 or D-Asn3/L-Asn4 stereochemistry and correlation of the former with the spectroscopic properties (1H and 13C NMR) of the natural product.16 Concurrent with this disclosure, Searcey reported the synthesis of the L-Asn3/D-Asn4 diastereomers incorporating either a D-ADA8 or L-ADA8 residue confirming its stereochemical assignment,17 and recently Nakata18 has reported a synthesis of this cyclic peptide.
Most recently, we reported studies leading to a reassignment of the relative stereochemistry for the chlorofusin chromophore as well as the assignment of the chromophore absolute configuration (4R,8S,9R).19 In this work and in addition to natural chlorofusin, the remaining seven chlorofusin chromophore diastereomers including the (4R,8R,9R)/(4S,8S,9S)-diastereomers proposed by Williams12 as well as a diastereomer misinterpreted as the natural product by Yao20 (4S,8R,9S) were synthesized as key analogues of the natural product and for comparison of their spectral properties with that reported for chlorofusin. In addition to providing unambiguous support for the structural reassignment, this provided the opportunity to explore and define key structure–activity relationships (SAR) for the inhibition of p53–MDM2 (p53–HDM2) binding. Notably, there is no apparent structural relationships between the residues found in the chlorofusin cyclic peptide and the p53 α-helix mediating the binding to the hydrophobic cleft of MDM217 and the nature of chlorofusin interaction with the N-terminal domain of MDM2 established by surface plasma resonance (SPR)13 remains to be defined. Herein we report the assessment of 1–8 and several additional key analogues and partial structures of chlorofusin for inhibition of p53–MDM2 binding.
The assay adopted to examine chlorofusin and its analogues was an ELISA competition assay described by Searcey.21 In this assay, the analogues are examined for their ability to disrupt the binding of MDM2 (available from EMD Biosciences) and a biotinylated SGSG-p53 peptide (17–27) that is immobilized on streptavidin-coated 96-well plates. A control p53 peptide (SGETFSDLWKLLPEN) as well as nutlin-1, a well established standard, produced IC50’s comparable to those reported (4.1 μM and 0.13 μM, respectively). Significant in the selection of this assay is the complete lack of any detectable background signal in the absence of immobilized p53 peptide. This permitted reproducible detection and accurate quantitation of inhibited binding by weak inhibitors without the complication of a significant background signal. Notably and over the course of several years, we examined multiple assays for monitoring p53–MDM2 binding and the Searcey assay proved by far to be the most reliable. Additionally, we were not able to convince potential collaborators to adopt the original DELFIA modified ELISA used to identify 112 and we were unable to secure the necessary proteins needed to reproduce the assay ourselves. Attempts to use more contemporary assays that enlist fluorescent quantitation failed since the intrinsic fluorescence of chlorofusin was found to interfere with the binding measurements. Using the Searcey assay, chlorofusin and each of its seven chromophore diastereomers proved to be effective inhibitors of p53–MDM2 binding (Figure 2). Chlorofusin was the most effective of the compounds evaluated displaying a potency that is comparable to that reported (IC50 = 8μM), but all exhibited activity within 3-fold of the natural product. Three additional chromophore diastereomers (3, 5, and 8) were equipotent with chlorofusin including two that are in the unnatural 4S series. Most notable of these is 5 in which the chromophore is enantiomeric with that of the natural product. These surprising results, in which the chromophore relative and absolute stereochemistry play little or no role, are especially interesting given that the simplified derivatives 9–12 of the cyclic peptide, in which the chromophore was simply removed (9, R = NH2) or replaced with large hydrophobic amine protecting groups, proved to be >10-fold less potent displaying IC50’s >100μM. The inactivity of such derivatives of the chlorofusin cyclic peptide has been disclosed previously and was confirmed herein.16,17,21 The relative inactivity of 10, bearing a Cbz group in place of the chromophore, is noteworthy given that it may be viewed as an achiral unfunctionalized replacement.
Just as significant, the entire series of partial structures that contain each chromophore diastereomer bearing a N-butyl, N-benzyl, and Nε-linked FmocHN-Thr-Orn-OBn or H2N-Thr-Orn-OBn all displayed IC50’s >100μM and each was >10-fold less active than chlorofusin (Figure 2). This is illustrated in Figure 2 with the (4R,8S,9R)- and (4S,8R,9S)-diastereomers, but is representative of all 32 compounds examined. Like the observations made with 1–8, no distinctions or discernable trends were observed that reflect the relative or absolute stereochemistry of the chromophore.
Finally, concerned that this behavior of 1–8 may simply represent promiscuous inhibition of the protein–protein interaction by chlorofusin aggregates, the assay was conducted with chlorofusin and nutlin-1 in the presence of 0.01% Triton.22 This had no effect on the measured IC50’s, suggesting that 1 and the related analogues 2–8 are not acting as promiscuous aggregate inhibitors.
As such, the results provide a clear, but unusual depiction of the structural features of 1 required for inhibition of p53–MDM2 binding. Neither the chlorofusin cyclic peptide (9) and its simple derivatives (e.g. 10–12) or simple derivatives of the chlorofusin chromophore (e.g. 13–16) exhibit significant inhibition of this protein–protein interaction. In contrast, chlorofusin and each of its seven chromophore diastereomers exhibit effective, albeit not especially potent, inhibition of p53–MDM2 binding that was essentially independent of the chromophore relative and absolute stereochemistry. Our present studies are not able to distinguish whether this reflects the impact of a flexible linker joining the two domains of the natural product or whether it may reflect an as yet undetected reversible, covalent attachment of the chromophore driven by weak cyclic peptide binding.23
We gratefully acknowledge the financial support of the National Institutes of Health (CA41101) and the Skaggs Institute for Chemical Biology. RCC and SYL are Skaggs Fellows.
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Ryan C. Clark, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
Sang Yeul Lee, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
Inkyu Hwang, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
Mark Searcey, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK.
Dale L. Boger, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.