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Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non–small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. Mol Cancer Ther; 9(3); 706–17

The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg88 to Ala88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression. [Mol Cancer Ther 2009;8(5):1363–77]

Activation of Akt (protein kinase B), a Ser/Thr protein kinase that promotes cell survival, has been linked to tumorigenesis. Akt is activated by phosphorylation after binding of its pleckstrin homology (PH) domain to plasma membrane phosphatidyl-myo-inositol-3-phosphates, formed by phosphoinositide-3-kinase. We report a novel strategy to inhibit Akt activation based on the use of D-3-deoxy-phosphatidyl-myo-inositols (DPIs) that cannot be phosphorylated on the 3-position of the myo-inositol ring. We have studied the DPIs, DPI 1-[(R)-2,3-bis(hexadecanoyloxy)propyl hydrogen phosphate], its ether lipid derivative DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (DPIEL), and its carbonate derivative DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl carbonate]. We demonstrate in platelet-derived growth factor-stimulated mouse NIH3T3 cells that the DPIs bind to the PH domain of Akt, trapping it in the cytoplasm and thus preventing Akt activation. DPIEL did not inhibit myristylated-Akt, a constitutively active membrane-bound Akt expressed in NIH3T3 cells, and cell growth was not inhibited, unlike in wild-type NIH3T3 cells. Molecular modeling and docking studies show that DPIEL binds with much higher affinity to Akt's PH domain as compared with DPI and DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl carbonate]. This study shows that the DPIs are a novel class of growth inhibitory agents with a novel mechanism of action through binding to the PH domain of Akt and inhibition of Akt activation.

Epicutaneous (topical) therapy with molecularly targeted agents is an attractive modality for treatment of basal cell carcinoma (BCC), early UVB induced squamous cell carcinoma (SCC) and cutaneous metastatic disease (CMD) associated with metastatic breast cancer. There is evidence that Akt and PDKP1 play complimentary yet independent roles in PI3K pathway signaling associated with driving the growth of BCC, UVB induced SCC, and breast CMD. Both Akt and PDPK1 possess a pleckstrin homology (PH) domain, a highly conserved three-dimensional superfold with a high affinity for binding phosphatidylinositol-3-phosphates, causing Akt and PDPK1 to translocation to the plasma membrane where Akt is activated. Through reiterative molecular docking and structure refinement using a proprietary computational platform, we have identified PHT-427 as an agent that binds to the PH domains of Akt and PDPK1, inhibiting their activity. PHT-427 has antitumor activity when administered orally but importantly also following epicutaneous administration. We evaluated the antitumor potential of epicutaneous PHT-427 against CMD in an intradermal breast xenograft model and in an early UVB induced skin cancer model in mice. For the CMD investigation MCF-7 human breast cancer (mutant PIK3CA) was injected intradermally in the flank of female nu/nu mice. Groups of 5 mice were treated twice a day for 10 days with vehicle or 0.1ml PH-427 (50 mg/mL) applied to a 1 cm patch of skin over the tumor. The mice were euthanized 4 hr after the last application and blood, tumor and overlying skin were removed for analyzes. Topical PHT-427 produced 89% inhibition of tumor growth (3/5 no measurable tumor) with no change in body weight or skin toxicity and inhibited targets in skin and tumor. Levels of PHT-427 by HPL-MS were detected in the skin at the site of application, in tumor and in plasma. To evaluate the activity PHT-427 in early UVB induced skin cancer female SKH-1 hairless mice were exposed to UVB irradiation to induce Akt and the production of skin tumors. The mice were treated epicutaneously with PHT-427 or vehicle 3 times a week for 4 weeks at which point animals were sacrificed and skin, tumors and blood were obtained. In this study topical PHT-427 significantly reduced tumor incidence, tumor burden and tumor multiplicity. Following treatment with PHT-427 80% of mice were tumor free and those with tumors averaged fewer than 1 per mouse and those were < 1mm in size while 59% of untreated group had tumors, with 1.6 tumors per mouse and the majority > 1mm in size. There was no change in body weight and no apparent skin toxicity. Thus, topical application of PHT-427 can deliver active drug to skin and tumor, inhibiting AKT and PDPK1, both of which drive the PI3K pathway important in UVB induced SCC and breast CMD, with significant inhibition of tumor growth without adverse effects on normal skin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A213.

Non-melanoma skin cancers (NMSC) are a significant and increasing health problem in the United States that occur primarily on sun-exposed areas of the body and are strongly associated with chronic sun exposure. Recent advances in our understanding of the molecular basis of carcinogenesis have lead to the identification of potential molecular targets that might be used to prevent the progression of early skin cancer. AKT (protein kinase B), a pleckstrin homology (PH) lipid binding domain and a serine/threonine kinase containing protein is a key component of the phophatidylinositol-3-kinase (PtdIns3-K) cell survival signaling pathway which is activated in skin cancers. In this study, we describe the effects of a novel inhibitor of AKT (PH427) that binds to the PH domain of AKT thus preventing its binding to PtIns-(3,4,5)P3 at the plasma membrane and subsequent activation. PH427 inhibits AKT activity at low micromolar concentrations in HaCat human keratinocytes and HaCat-II,4, a ras transformed human keratinocyte cell line. We demonstrate that PH427 is able to prevent UVB-induced AKT activation and expression in both cell lines. PH427 also induced apoptosis in both HaCat and HaCat II,4 keratinocytes. Moreover, the compound is lipophilic and penetrates the skin in SKH-1 mice when applied topically. Short term in vitro studies showed that PH427 prevents UVB-induced AKT activation and UVB-induced signaling, as measured by p70S6 Kinase and p-GSK3-activation. Long-term in vivo studies utilizing SKH-1 mice have also shown significant reduction of UVB-induced skin tumors when PH427 is topically applied when compared to acetone-only control or PH427 alone. Additionally, drug treated mice exhibit reduced levels of phospho-Ser473AKT in the epidermal layers. We conclude that the topical application of PH427 may be beneficial for preventing NMSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A242.

The pleckstrin homology (PH) domain is a highly conserved three dimensional superfold found in many high valued pharmaceutical target proteins. While for the majority of PH domain proteins PtdIns binding is weak and non-specific, a subclass of approximately 40 PH domain containing proteins show high affinity for phosphatidylinositol-3-phosphates (PtdIns-3-P) causing their translocation to the plasma membrane and/or activation. Akt and PDKP1 are two such PH domain containing serine threonine kinase members of the PtdIns-3-kinase signaling pathway that plays a critical role in activating survival and anti-apoptotic signaling in cancer cells. This pathway is constitutively activated through mutation, overexpression and loss of the tumor suppressor PTEN in many different cancer types. There is emerging evidence that Akt and PDKP1 play complimentary yet independent roles in signaling by the pathway. Attempts to develop ATP catalytic site inhibitors of Akt and PDKP1 have generally resulted in toxic agents because of inhibition of off-target kinase family members. Through a process of reiterative molecular docking and structure refinement using a proprietary computational platform, and measurement of binding affinities by surface plasmon resonance (SPR) spectroscopy, we have developed inhibitors of these proteins that bind to the PH domain. We report here the development of PHT-427 a compound that binds to the PH domain of AKT and PDKP1. To develop PHT-427 an initial pharmacophore that bound to the PH domain PtdIns binding pocket was first identified. During the docking/modeling we identified a channel whithin the PH domains of both AKT and PDPK1 that could accommodate the binding of an alkyl chain. A series of analogs with C-4 to C-16 alkyl chain length were therefore synthesized and PHT-427 (C-12 chain) was found to bind with the highest affinity to the PH domains of both PDPK1 and AKT. PHT-427 gives a transient inhibition of AKT signaling in cells and tumor xenografts and a longer lasting inhibition of PDPK1 signaling. This inhibition correlated with decreased activation of downstream signaling targets for both proteins. PHT-427 given ias an oral formulation on a twice daily schedule inhibited the growth rate of human tumor xenografts in immunodeficient mice with up to an 80% inhibition in the most sensitive tumors and showed greater activity than analogs with C4, C6 or C8 alkyl chains. Tumors with a K-Ras mutation were less sensitive to PHT-427 as it has been seen for other inhibitors of the PtdIns-3-K signaling pathway. Combination antitumor studies showed that PHT-427 has greater than additive activity with paclitaxel in breast cancer and with erlotinib in non small cell lung cancer. There was minimal toxicity of PH-427 at doses that gave antitumor activity. PHT-427 given twice daily for 5 days caused no weight loss or change in blood chemistry. In summary we have identified a novel PH domain binding inhibitor of PDPK1/AKT signaling that has minimal toxicity and antitumor activity against human tumor xenografts.. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B139.