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A new phosphotyrosyl mimetic 4-(α-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-(α-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.

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, KD = 1.47 and 3.62 nM, respectively). Analogues containing monoacidic phosphoryl mimetics provided affinities of KD = 16−67 nM. Neutral phosphoryl-mimicking groups did not show appreciable binding.

Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c) or α-methyl-p-phosphonophenylalanine ((α-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 (α-Me)Ppp and Ac6c residues. The current study compares the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (α-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 (α-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 affnity (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 (α-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 (α-Me)Ppp-containing peptide by β-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 β-macrocyclization achieved higher affinity. Similar β-macrocyclization may potentially be extended to SH2 domain systems other than Grb2, where bend geometries are required.

Macrocyclization from the phosphotyrosyl (pTyr) mimetic's β-position has previously been shown to enhance Grb2 SH2 domain-binding affinity of phosphonate-based analogues. The current study examined the effects of such macrocyclization using a dicarboxymethyl-based pTyr mimetic. In extracellular assays affinity was enhanced approximately 5-fold relative to an open-chain congener. Enhancement was also observed in whole-cell assays examining blockade of Grb2 binding to the erbB-2 protein-tyrosine kinase.

The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that represents an attractive target for anticancer therapeutic intervention. Here, a ring-closing metathesis approach is utilized to synthesize a 5-methylindolyl-containing tetrapeptide mimetic (6) that exhibits unprecedented in vitro Grb2 SH2 domain-binding affinity (Kd = 93 pM). Key to the preparation of 6 is the enantioselective synthesis of (2S)-2-(3-(5-methylindolyl)methyl)pent-4-enylamine (12) as one of two ring-closing segments.

Grb2 SH2 domains are protein-docking modules that exert important functions in both normal and pathogenic signal transduction processes. Development of synthetic Grb2 SH2 domain binding ligands is being pursued by several groups as potential new therapies for a variety of diseases, including certain cancers. In these efforts, macrocyclization has been successfully utilized to take advantage of preferential recognition by Grb2 SH2 domains of ligands in β-bend conformations. Recent examples of this approach include olefin-metathesis-derived macrocycles that employ ring closure at the β-position of key pTyr-mimicking residues. In the current study, a novel phosphatase-stable β-amino-pTyr mimetic designated “Pmpβ” was utilized to prepare variants of previously reported olefin-metathesis-derived macrocycles. An initial set of simplified cyclic peptides lacking key naphthyl side chain functionality was first synthesized to determine optimum ring size, with results indicating that a four-unit ring-closing segment was appropriate. On the basis of these findings, macrolactamization was undertaken with a more highly functionalized, naphthyl-containing γ-amino acid analogue. The resulting cyclic β-amino peptide is the first of a new class of pTyr-mimetic-containing ligands that may have utility in the development of antagonists of both Grb2 SH2 domains and other pTyr-dependent signaling systems.

Phosphotyrosyl (pTyr) residues play important roles in cellular signal transduction by facilitating recognition and binding necessary for critical protein−protein interactions, and for this reason pTyr motifs represent attractive starting points in the development of signaling antagonists. Although the pTyr phosphoryl moiety is central in these phenomena, its incorporation into signaling inhibitors is contraindicated due to enzymatic lability and limited bioavailabilty associated with phosphate esters. To address these limiations, an entire field of study has arisen devoted to the design and utilization of pTyr mimetics. This Account provides a perspective on the roles of pTyr residues in signal transduction and approaches to pTyr mimetic development.

While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an α-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the α-nitrogen of pTyr residues or from the α-position of P⁰ pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic α-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC50 value of 0.002 μM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185^erbB-2 and exhibited antimitogenic effects with submicromolar IC50 values.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202−3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN−DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg²⁺ at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg²⁺-based assays, while IN inhibition by salicylhydrazides is strictly Mn²⁺-dependent. Additionally, in target and cell-based assays, the MSHs have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerases I and II at concentrations that effectively inhibit IN. These data suggest that MSHs are selective inhibitors of HIV-1 IN and may serve as leads for antiviral therapeutics.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl β-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3‘-processing (3‘-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the “left” and “right” sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3‘-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Examination of the PTP1B inhibitory potency of an extensive series of phosphotyrosyl (pTyr) mimetics (Xxx) expressed in the EGFr-derived hexapeptide platform Ac-Asp-Ala-Asp-Xxx-Leu-amide previously led to the finding of high inhibitory potency when Xxx = 4-(phosphonodifluoromethyl)phenylalanyl (F2Pmp) (Ki = 0.2 μM) and when Xxx = 3-carboxy-4-carboxymethyloxyphenylalanyl (Ki = 3.6 μM). In the first instance, further work led from the F2Pmp-containing peptide to monomeric inhibitor, 6-(phosphonodifluoromethyl)-2-naphthoic acid (Ki = 22 μM), and to the pseudo-dipeptide mimetic, N-[6-(phosphonodifluoromethyl)-2-naphthoyl]-glutamic acid (Ki = 12 μM). In the current study, a similar approach was applied to the 3-carboxy-4-carboxymethyloxyphenylalanyl-containing peptide, which led to the preparation of monomeric 5-carboxy-6-carboxymethyloxy-2-naphthoic acid (Ki = 900 μM). However, contrary to expectations based on the aforementioned F2Pmp work, incorporation of this putative pTyr mimetic into the pseudo-dipeptide, N-[5-carboxy-6-carboxymethyloxy-2-naphthoyl]-glutamic acid, resulted in a substantial loss of binding affinity. A reevaluation of binding orientation for 5-carboxy-6-carboxymethyloxy-2-naphthoic acid was therefore undertaken, which indicated a 180° reversal of the binding orientation within the PTP1B catalytic site. In the new orientation, the naphthyl 2-carboxyl group, and not the o-carboxy carboxymethyloxy groups, mimics a phosphoryl group. Indeed, when 5-carboxy-2-naphthoic acid itself was examined at neutral pH for inhibitory potency, it was found to have Ki = 31 ± 7 μM, which is lower than parent 5-carboxy-6-carboxymethyloxy-2-naphthoic acid. In this fashion, 5-carboxy-2-naphthoic acid (or more appropriately, 6-carboxy-1-naphthoic acid) has been identified as a novel PTP1B binding motif.

One drawback frequently associated with olefin metathesis-mediated peptide macrocyclization, the loss of side chain functionality at sites of ring closure, may be circumvented by incorporation of side chain functionality within the ring-closing olefin segments. This approach is demonstrated in the preparation of a macrocyclic Grb2 SH2 domain antagonist designed as a conformationally constrained β-bend mimic.

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.