Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase I (Tdp1) and topoisomerase I (Top1).

Tyrosyl-DNA phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationships. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 µM to 111 µM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean graph midpoints ranged from 0.02 to 2.34 µM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents.

Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.

We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.

Structural examination of ring-closing metathesis-derived 15-member macrocycles as Grb2 SH2 domain-binding tetrapeptide mimetics.

Ring-closing metathesis (RCM) was employed to join carboxy-terminal alkenyl glycine side chains together with vinyl- and allyl-functionality appended to the beta-methylene of amino-terminal phosphotyrosyl (pTyr) mimetics. This required the synthesis of a variety of new pTyr mimetics, including a novel aza-containing analogue. Many of the resulting 15-member macrocyclic tetrapeptide mimetics exhibited low nanomolar Grb2 SH2 domain-binding affinities in spite of the fact that differing ring junction stereochemistries and geometries of the RCM-derived double bond were employed. The finding that significant latitude exists in the structural requirements for ring closure may facilitate the development of therapeutically relevant macrocyle-based Grb2 SH2 domain-binding antagonists. The synthetic approaches used in this study may also find application to peptide mimetics directed at other biological targets.

Examination of acylated 4-aminopiperidine-4-carboxylic acid residues in the phosphotyrosyl+1 position of Grb2 SH2 domain-binding tripeptides.

A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-binding peptide to form a general platform, which was then acylated with a variety of groups to yield a library of compounds designed to explore potential binding interactions, with protein features lying below the betaD strand. The highest affinities were obtained using phenylethyl carbamate and phenylbutyrylamide functionalities.

Design and synthesis of 4-(alpha-hydroxymalonyl)phenylalanine as a new phosphotyrosyl mimetic and its use in growth factor receptor bound 2 src-homology 2 (Grb2 SH2) domain-binding peptides.

A new phosphotyrosyl mimetic 4-(alpha-hydroxymalonyl)phenylalanine and its incorporation into a Grb2 SH2 domain-binding tripeptide are presented. In whole-cell studies using malonyl ethyl ester prodrug derivatives, it was observed that the 4-(alpha-hydroxymalonyl)phenylalanyl-containing peptide exhibited greater efficacy than the nonhydroxylated 4-(malonyl)phenylalanyl-containing congener in blocking the association of Grb2 with activated erbB-2 tyrosine kinase. These results are consistent with de-esterification and at least partial intracellular decarboxylation.

Examination of phosphoryl-mimicking functionalities within a macrocyclic Grb2 SH2 domain-binding platform.

Reported herein are the design, synthesis, and Grb2 SH2 domain-binding affinities of several phosphoryl-mimicking groups displayed within the context of a conformationally constrained macrocyclic platform. With use of surface plasmon resonance techniques, single-digit nanomolar affinities were exhibited by phosphonic acid and malonyl-containing diacidic phosphoryl mimetics (for 4h and 4g, K(D) = 1.47 and 3.62 nM, respectively). Analogues containing monoacidic phosphoryl mimetics provided affinities of K(D) = 16-67 nM. Neutral phosphoryl-mimicking groups did not show appreciable binding.

Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing alpha-methylphenylalanyl based phosphotyrosyl mimetics.

Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c) or alpha-methyl-p-phosphonophenylalanine ((alpha-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal position (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. The tetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneously presents structural features of both (alpha-Me)Ppp and Ac6c residues. The current study compares the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (alpha-Me)Ppp residues when incorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptide platform. The highest binding affinity (KD = 14.8 nM) was exhibited by the (alpha-Me)Ppp-containing parent, with the corresponding Ac6c-containing peptide being nearly 2-fold less potent (KD = 23.8 nM). The lower KD value was attributable primarily to a 50% increase in off-rate. Replacement of the Ac6c residue with the tetralin-based hybrid resulted in a further 4-fold decrease in binding affinity (KD = 97.8 nM), which was the result of a further 6-fold increase in off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation of the key structural components found in (alpha-Me)Ppp into the Ac6c residue, the tetralin hybrid does enhance binding on-rate. However, net binding affinity is decreased due to an associated increase in binding off-rate. Alternatively, global conformational constraint of an (alpha-Me)Ppp-containing peptide by beta-macrocyclization did result in pronounced elevation of binding affinity, which was achieved primarily through a decrease in the binding off-rate. Mathematical fitting using a simple model that assumed a single binding site yielded an effective KD of 2.28 nM. However this did not closely approximate the data obtained. Rather, use of a complex model that assumed two binding sites resulted in a very close fit of data and provided KD values of 97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformational constraint in the pY + 1 residue proved to be deleterious, global conformational constraint through beta-macrocyclization achieved higher affinity. Similar beta-macrocyclization may potentially be extended to SH2 domain systems other than Grb2, where bend geometries are required.

Utilization of a nitrobenzoxadiazole (NBD) fluorophore in the design of a Grb2 SH2 domain-binding peptide mimetic.

Fluorescence labeling has become a general technique for studying the intracellular accumulation and localization of exogenously administered materials. Reported herein is a low nanomolar affinity Grb2 SH2 domain-binding antagonist that utilizes the environmentally-sensitive nitrobenzoxadiazole (NBD) fluorophore as a naphthyl replacement. This novel agent should serve as a useful tool to visualize the actions of this class of Grb2 SH2 domain-binding antagonists in whole cell systems.

Ring-closing metathesis of C-terminal allylglycine residues with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic as an approach to novel Grb2 SH2 domain-binding macrocycles.

Ring-closing metathesis (RCM) of peptides often requires insertion of allylglycines at the intended sites of ring juncture, which can result in the displacement of residues that are needed for biological activity. This type of side-chain deletion can be avoided by appending beta-vinyl substituents onto the parent residues at the intended sites of ring juncture, thereby effectively converting them into functionalized allylglycine equivalents. Such an approach has been previously applied in modified form to growth-factor receptor bound 2 (Grb2) SH2 domain-binding peptides by using an N-terminal beta-vinyl-functionalized phosphotyrosyl mimetic and C-terminal 2-allyl-3-aryl-1-propanamides that lacked the alpha-carboxyl portion of allylglycine residues. These C-terminal moieties involved lengthy synthesis and once prepared, required an individual total synthesis of each final macrocycle. Work reported herein significantly enhances the versatility of the original approach through the use of C-terminal allylglycine amides that can be prepared from commercially available L- and D-allylglycines and suitable amines. This methodology could be generally useful where macrocylization is desired with maintenance of functionality at a site of ring juncture.

Utilization of a common pathway for the synthesis of high affinity macrocyclic Grb2 SH2 domain-binding peptide mimetics that differ in the configuration at one ring junction.

As typified by 2-{(9S,10S,14R,18S)-18-(2-amino-2-oxoethyl)-14-[(5-methyl-1H-indol-1-yl)methyl]-8,17,20-trioxo-10-[4-(phosphonomethyl)phenyl]-7,16,19-triazaspiro[5.14]icos-11-en-9-yl}acetic acid ((14R)-1b), ring-closing methathesis-derived macrocyclic tetrapeptide mimetics have recently been reported that bind with high affinity to Grb2 SH2 domains in both extracellular and whole-cell assays. The synthetic complexity of this class of agents limits further therapeutic development. Although a significant component of this synthetic complexity arises from the presence of three stereogenic centers, C(9) (S), C(10) (S), and C(14) (R), it is unclear whether stereoselective introduction of defined configuration at C(14) is required for high-affinity binding. Reported herein is a synthetic route to these macrocycles lacking stereoselectivity in the formation of the C(14) ring junction, which is four synthetic steps shorter than the original stereoselective synthesis. Separation of C(14)-epimers obtained by this approach was achieved by preparative HPLC. Molecular-dynamics studies of ligands bound to the Grb2 SH2 domain protein indicated that the (14R)-configuration should display more-favorable interactions with the protein relative to the (14S)-epimer. Indeed, although surface-plasmon-resonance-derived binding constants to Grb2 SH2 domain protein indicated that the affinity of the (14R)-epimer (KD = 4.8 nM) is greater than that of the (14S)-epimer (KD = 11 nM), it is only marginally so. Therefore, little affinity would be lost through a non-stereoselective synthesis of the C(14)-center. Further studies are in progress to explore reduced structural complexity at the C(14)-center.