Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proofof-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.T he human transcription factor protein p53 induces cell-cycle arrest and apoptosis in response to DNA damage and cellular stress and thereby plays a critical role in protecting cells from malignant transformation (1, 2). Inactivation of this guardian of the genome either by deletion or mutation or through overexpression of inhibitory proteins is the most common defect in human cancers (1, 2). Cancers that overexpress the inhibitory proteins MDM2 and MDMX also possess wild-type p53 (p53WT), and thus pharmacological disruption of the interactions between p53 and MDM2 and MDMX offers the opportunity to restore p53-dependent cell-cycle arrest and apoptosis in this important class of tumors (3-6).MDM2 negatively regulates p53 function through multiple mechanisms, including direct binding that masks the p53 transactivation domain, impairing nuclear import of the p53 protein, and ubiquitination and proteasomal degradation of the p53 protein (6, 7). Consequently, aberrant MDM2 overexpression and gene amplification contribute to accelerated cancer development and growth (1, 8). The other negative regulator, MDMX, possesses a similar p53-binding activity and also effectively inhibits p53 transcriptional activity. Amplification of MDMX is seen in many tumors, including melanoma, breast, head and neck, hepatocellular, and retinoblastoma, and, interestingly, amplification of MDMX appears to correlate with both p53WT status and an absence of MDM2 amplification (6, 9, 10). MDMX does not have the intrinsic E3 ubiquitin ligase activity of MDM2 and cannot affect p53 stability, but MDM2/MDMX heterodimers can increase ubiquitin ligase activity relative to the MDM2 monomer. Given these functional differences, MDM2 and MDMX are each unable to compensate for the loss of the other, and they regulate nonoverlapping fu...

ABSTRACTa-Melanocyte-stimulating hormone (a-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of a-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4 (methionine) and 7(phenylalanine) a-Melanotropin (a-MSH, a-melanocyte-stimulating hormone) is a tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-ArgTrp-Gly-Lys-Pro-Val-NH2) that is synthesized and secreted by the pars intermedia of the vertebrate pituitary (1). The amino acid residues that are important in the expression of melanotropic activity have been elucidated through systematic structure-function investigations of a-MSH and a-MSH fragments on amphibian melanophores (2, 3) and, to a lesser extent, on mammalian melanoma cells (4-6). Very little information is available, however, regarding the stereochemical and conformational correlates of biological activity in either of these two biological systems.Earlier reports have shown that heat-alkali treatment of crude or purified preparations of naturally occurring a-MSH produces a partially racemized product with altered activity on amphibian melanophores both in vivo and in vitro. Such changes in biological effects have been discussed in terms of "potentiation," "prolongation," and "retardation" (7-12). Although the precise biochemical mechanism by which these unusual biological properties were produced is unknown, it appeared possible that synthetic stereostructural tailoring of a-MSH might produce an analogue that would also possess these properties. Utilizing a high-resolution gas chromatographic method to localize and quantitate specific sites of racemization within the primary sequences of peptides, we obtained additional evidence which suggested that stereochemical substitution at position 7 (replacement of L-phenylalanine by D-phenylalanine) of a-MSH or [Nle4]-a-MSH would provide an analogue with the desired biological properties. Previous investigations have shown that [Nle4]-a-MSH is more potent than a-MSH on both amphibian melanophores (2, 6) and on stimulating melanoma adenylate cyclase (6, 13), and it is also resistant to inactivation by chloramine-T (14, 15), an oxidant used in peptide iodination. Because heat-alkali treatment of this analogue also resulted in "potentiation," "prolongation," and "retardation," it was clear that alteration of the methionine residue was not a requirement for the expression of these properties. Thus, it was decided to retain the benefits of the norleucine substitution in position 4 in the synthesis of the "definitive" peptide.We report here the synthesis of [Nle4, D-Phe7]-a-MSH and present data demonstrating its unique biological properties. These include prolonged biological activity, enhanced potency relative to a-MSH in a number of biological systems, and resistance to degradation by serum enzymes. The biological properties of this analogue provide...

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR,68..,,, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR, 9ss. (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affmity binding and catalysis. The K. value for EGFR,85_.,, decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.The tyrosine phosphorylation "status" of a cell is maintained by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) (1, 2). The first PTPase to be purified and sequenced was a 35-kDa protein (PTP 1B) from human placenta (3, 4). PTP1B was shown to share amino acid sequence homology with the cytoplasmic domain of the leukocyte cell surface glycoprotein CD45 (5). CD45 was subsequently shown to have tyrosine phosphatase activity (6). This discovery drew attention to the possible biological role of the PTPases in controlling signal transduction within the cell. Isolation and characterization of cDNA by low stringency hybridization and polymerase chain reaction demonstrated that PTPases constitute a large diversified family of catalysts that can be divided into two structurally distinct groups. One group generally has an extracellular domain, transmembrane spanning region, as well as two duplicated cytoplasmic PTPase domains. The other group of PTPases corresponds to the intracellular family of enzymes that have a single PTPase domain (2).Many PTPases have been cloned but limited information is available about their functions, mechanisms of catalysis, or substrate specificities. One of the central questions in protein phosphorylation is how kinases and phosphatases distinguish the diversity of substrates that they encounter in the cell. In the case of protein kinases, the amino acid sequence surrounding the phosphorylation site plays a crucial role in determining substrate specificity (7,8). For example, the cAMP-dependent protein kinase has a strong preference for phosphorylation of Ser residues that are located two or three residues to the COOH-terminal side of basic amino acids (most commonly Arg). Tyr...

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.

The minimal sequence required for biological activity of alpha-MSH (alpha-melanotropin, alpha-melanocyte stimulating hormone) was determined in the frog (Rana pipiens) skin bioassay. The sequence required to elicit measurable biological activity was the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2), which was about 6 orders of magnitude less potent than the native tridecapeptide. Smaller fragments of this sequence (Ac-His-Phe-NH2, Ac-Phe-Arg-NH2, Ac-His-Phe-Arg-NH2) were devoid of melanotropic activity at concentrations as high as 10(-4) M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2 (Ac-alpha-MSH7-10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2 (Ac-alpha-MSH11-13-NH2). We prepared a series of fragment analogues of alpha-MSH in an attempt to determine the contribution of each individual amino acid to the biological activity of the native hormone. The minimal potency of Ac-alpha-MSH6-9-NH2 could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-alpha-MSH6-10-NH2 (Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-alpha-MSH5-10-NH2, which was only slightly more potent than Ac-alpha-MSH6-10-NH2, indicating that position 5 contributes little to the biological potency of alpha-MSH in this assay. Addition of methionine to the N-terminus of Ac-alpha-MSH5-10-NH2 resulted in the heptapeptide, Ac-alpha-MSH4-10-NH2, which was only about 4-fold more potent than Ac-alpha-MSH5-10-NH2. Addition of lysine and proline to the C-terminal of the Ac-alpha-MSH4-10-NH2 sequence yielded the peptide, Ac-alpha-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-alpha-MSH4-10-NH2. This peptide was only about 6-fold less potent than alpha-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-alpha-MSH4-10-NH2 was about 4 times more potent than Ac-alpha-MSH4-10-NH2. Ac-[Nle4]-alpha-MSH4-11-NH2 also was about 4 times more potent than Ac-alpha-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of alpha-MSH on the frog skin melanocyte receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The structural requirements of substrates for two recombinant protein tyrosine phosphatases (PTPases) are probed using various-sized synthetic phosphotyrosine (pY)-containing peptides corresponding to the autophosphorylation site in EGF receptor (EGFR) at Y992. The peptide EGFR988-998 (DADEpYLIPQQG) is chosen as a template due to its favorable kinetic constants. The contribution of individual amino acids on both sides of pY to binding and catalysis was assessed by kinetic analysis using a continuous, spectrophotometric assay. For both Yersinia PTPase and a soluble recombinant mammalian PTPase of 323 amino acid residues (rat PTP1), efficient binding and catalysis required six amino acids including the pY residue, i.e., four residues N-terminal to pY and one residue C-terminal to pY. Thus, PTPase substrate specificity is primarily dictated by residues to the N-terminal side of pY. The pY moiety and the rest of the peptide interact with PTPases in a cooperative manner. The presence of pY in the peptide substrate is necessary but not sufficient for high-affinity binding, since phosphotyrosine and other simple aryl phosphates exhibit weak binding, and dephosphorylated peptides do not bind to PTPases. Two variations on the pY moiety are also examined in order to assess their utility in PTPase inhibitor design. It is demonstrated that the thiophosphoryl analog in which one of the phosphate oxygens is replaced by sulfur can be hydrolyzed by PTPases, whereas the phosphonomethylphenylalanine analog in which the tyrosyl oxygen is replaced by a CH2 group is a competitive and nonhydrolyzable inhibitor, with Ki values of 18.6 and 10.2 microM, respectively, for the Yersinia PTPase and the rat PTP1.

The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-A resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu psi [CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has Ki less than 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178 degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.