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Background: Geranylgeranyltransferase I (GGTase I) catalyzes geranylgeranylation, a modification required for the function of many oncogenic RAS-related proteins. GGTI-2418 is a peptidomimetic small molecule inhibitor of GGTase I., Objective: The aim of this study was to establish the maximum tolerated dose of GGTI-2418 in patients with advanced solid tumors., Patients and Methods: This was a phase I, open-label, dose-escalation study conducted in two US centers (University of Pennsylvania and Indiana University) in adults with treatment-refractory advanced solid tumors. An accelerated dose-escalation schema was used across eight dose levels, from 120 to 2060 mg/m 2 , administered on days 1-5 of each 21-day cycle., Results: Fourteen patients were enrolled in the dose-escalation cohort. No dose-limiting toxicities were observed, and 2060 mg/m 2 was determined to be the maximum tolerated dose. The only potential drug-related grade 3 or 4 toxicities were elevated bilirubin and alkaline phosphatase in a single patient with concurrent malignant biliary obstruction. No objective responses were observed. Four of thirteen evaluable patients had stable disease for up to 6.7 months. The study was terminated prior to dose expansion based on a sponsor decision. Pharmacokinetic analysis demonstrated a mean terminal half-life of 1.1 h., Conclusions: GGTI2418 was safe and tolerable at all tested dose levels with some evidence of disease stability. Due to rapid elimination, dosing of GGTI2418 in this study may have been inadequate to achieve optimal inhibition of its target, GGTase I.

The growth, metastasis, and chemotherapy resistance of pancreatic ductal adenocarcinoma (PDAC) is characterized by the activation and growth of tumor-initiating cells in distant organs that have stem-like properties. Thus, inhibiting growth of these cells may prevent PDAC growth and metastases. We have demonstrated that δ-tocotrienol, a natural form of vitamin E (VEDT), is bioactive against cancer, delays progression, and prevents metastases in transgenic mouse models of PDAC. In this report, we provide the first evidence that VEDT selectively inhibits PDAC stem-like cells. VEDT inhibited the viability, survival, self-renewal, and expression of Oct4 and Sox2 transcription factors in 3 models of PDAC stem-like cells. In addition, VEDT inhibited the migration, invasion, and several biomarkers of epithelial-to-mesenchymal transition and angiogenesis in PDAC cells and tumors. These processes are critical for tumor metastases. Furthermore, in the L3.6pl orthotopic model of PDAC metastases, VEDT significantly inhibited growth and metastases of these cells. Finally, in an orthotopic xenograft model of human PDAC stem-like cells, we showed that VEDT significantly retarded the growth and metastases of gemcitabine-resistant PDAC human stem-like cells. Because VEDT has been shown to be safe and to reach bioactive levels in humans, this work supports investigating VEDT for chemoprevention of PDAC metastases.

Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.

The proteasome inhibitor bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, acts covalently, and is associated with undesired side effects. Therefore, noncovalent inhibitors that are less toxic and more effective against solid tumors are desirable. Structure activity relationship studies led to the discovery of PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nm) over trypsin-like and peptidylglutamyl peptide hydrolyzing (IC50 values >100 μm) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Mass spectrometry and dialysis studies demonstrate that PI-1840 is a noncovalent and rapidly reversible CT-L inhibitor. In intact cancer cells, PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax, and IκB-α, inhibits survival pathways and viability, and induces apoptosis. Furthermore, PI-1840 sensitizes human cancer cells to the mdm2/p53 disruptor, nutlin, and to the pan-Bcl-2 antagonist BH3-M6. Finally, in vivo, PI-1840 but not bortezomib suppresses the growth in nude mice of human breast tumor xenografts. These results warrant further evaluation of a noncovalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors.

ROCK1 and ROCK2 mediate important processes such as cell migration, invasion and metastasis, making them good targets for the development of antitumor agents. Recently, using a fragment-based approach and X-ray crystallography, we reported on the design and synthesis of novel Rho-kinase inhibitors (RKIs). Here, we selected a pair of RKIs, the closely related structural analogs RKI-18 (potent; IC50 values of 397 nM (ROCK1) and 349 nM (ROCK2)) and RKI-11 (weak/inactive; IC50 values of 38 μM (ROCK1) and 45 μM (ROCK2)), as chemical probes and determined their effects on cytoskeleton organization, signaling, apoptosis, anchorage-dependent and independent growth, migration and invasion. RKI-18 but not RKI-11 suppresses potently the phosphorylation of the ROCK substrate myosin light chain 2 (MLC2) in intact human breast, lung, colon and prostate cancer cells. Furthermore, RKI-18 is highly selective at decreasing the levels of P-MLC2 over those of P-Akt, P-S6 and P-Erk ½. RKI-18 suppresses ROCK-mediated actin fiber formation, following stimulation with LPA as well as p21-activated kinase (PAK)-mediated lamellipodia and filopodia formation following bradykinin or PDGF stimulation. Furthermore, RKI-18 but not RKI-11 inhibits migration, invasion and anchorage-independent growth of human breast cancer cells. The fact that the active ROCK inhibitor RKI-18, but not the inactive closely related structural analog RKI-11 is effective at suppressing malignant transformation suggests that inhibition of ROCK with RKI-18 results in preventing migration, invasion and anchorage-independent growth. The potential of this class of RKIs as anti-tumor agents warrants further advanced preclinical studies.

Pancreatic cancer is one of the most lethal diseases with no effective treatment. Previously, we have shown that FAK is overexpressed in pancreatic cancer and plays a key role in cancer cell survival and proliferation. FAK has been shown to interact with growth factor receptors including cMET and IGF-1R. As a novel therapeutic approach, we targeted the protein interaction of FAK with growth factor receptors to block tumor growth, alter signaling pathways and sensitize cells to chemotherapy. We have selected a small molecule compound (INT2-31) that decreases phosphorylation of AKT via disrupting interaction of FAK with cMET and IGF-1R. Our results demonstrate that interaction of a small molecule compound with FAK decreases phosphorylation of FAK Y397 while increasing FAK Y407 phosphorylation, without inhibiting the kinase activity of FAK and dramatically reduces downstream signaling to AKT. Our lead compound, INT2-31, demonstrates significant inhibition of tumor cell growth in two orthotopic models of pancreatic cancer. In addition, INT2-31 increases sensitivity to gemcitabine chemotherapy in a direct fresh biopsy xenograft model of pancreatic cancer growth.

The Rho-associated kinases ROCK1 and ROCK2 are critical for cancer cell migration and invasion, suggesting they may be useful therapeutic targets. In this study, we describe the discovery and development of RKI-1447, a potent small molecule inhibitor of ROCK1 and ROCK2. Crystal structures of the RKI-1447/ROCK1 complex revealed that RKI-1447 is a Type I kinase inhibitor that binds the ATP binding site through interactions with the hinge region and the DFG motif. RKI-1447 suppressed phosphorylation of the ROCK substrates MLC-2 and MYPT-1 in human cancer cells, but had no effect on the phosphorylation levels of the AKT, MEK, and S6 kinase at concentrations as high as 10 μmol/L. RKI-1447 was also highly selective at inhibiting ROCK-mediated cytoskeleton re-organization (actin stress fiber formation) following LPA stimulation, but does not affect PAK-meditated lamellipodia and filopodia formation following PDGF and Bradykinin stimulation, respectively. RKI-1447 inhibited migration, invasion and anchorage-independent tumor growth of breast cancer cells. In contrast, RKI-1313, a much weaker analog in vitro, had little effect on the phosphorylation levels of ROCK substrates, migration, invasion or anchorage-independent growth. Finally, RKI-1447 was highly effective at inhibiting the outgrowth of mammary tumors in a transgenic mouse model. In summary, our findings establish RKI-1447 as a potent and selective ROCK inhibitor with significant anti-invasive and antitumor activities and offer a preclinical proof-of-concept that justify further examination of RKI-1447 suitability as a potential clinical candidate., (©2012 AACR.)

Screening efforts led to the identification of PI-8182 (1), an inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Compound 1 contains a hydronaphthoquinone pharmacophore with a thioglycolic acid side chain at position 2 and thiophene sulfonamide at position 4. An efficient synthetic route to the hydronaphthoquinone sulfonamide scaffold was developed, and compound 1 was synthesized in-house to confirm the structure and activity (IC(50) = 3.0 ± 1.6 μM [n = 25]). Novel hydronaphthoquinone derivatives of 1 were designed, synthesized, and evaluated as proteasome inhibitors. The structure-activity relationship (SAR) guided synthesis of more than 170 derivatives revealed that the thioglycolic acid side chain is required and the carboxylic acid group of this side chain is critical to the CT-L inhibitory activity of compound 1. Furthermore, replacement of the carboxylic acid with carboxylic acid isosteres such as tetrazole or triazole greatly improves potency. Compounds with a thio-tetrazole or thio-triazole side chain in position 2, where the thiophene was replaced by hydrophobic aryl moieties, were the most active compounds with up to 20-fold greater CT-L inhibition than compound 1 (compounds 15e, 15f, 15h, 15j, IC(50) values around 200 nM, and compound 29, IC(50) = 150 nM). The synthetic iterations described here not only led to improving potency in vitro but also resulted in the identification of compounds that are more active such as 39 (IC(50) = 0.44 to 1.01 μM) than 1 (IC(50) = 3.54 to 7.22 μM) at inhibiting the proteasome CT-L activity in intact breast cancer cells. Treatment with 39 also resulted in the accumulation of ubiquitinated cellular proteins and inhibition of tumor cell proliferation of breast cancer cells. The hit 1 and its analogue 39 inhibited proteasome CT-L activity irreversibly.

A potent class of anticancer, human farnesyltransferase (hFTase) inhibitors has been identified by "piggy-backing" on potent, antimalarial inhibitors of Plasmodium falciparum farnesyltransferase (PfFTase). On the basis of a 4-fold substituted ethylenediamine scaffold, the inhibitors are structurally simple and readily derivatized, facilitating the extensive structure-activity relationship (SAR) study reported herein. Our most potent inhibitor is compound 1f, which exhibited an in vitro hFTase IC(50) value of 25 nM and a whole cell H-Ras processing IC(50) value of 90 nM. Moreover, it is noteworthy that several of our inhibitors proved highly selective for hFTase (up to 333-fold) over the related prenyltransferase enzyme geranylgeranyltransferase-I (GGTase-I). A crystal structure of inhibitor 1a co-crystallized with farnesyl pyrophosphate (FPP) in the active site of rat FTase illustrates that the para-benzonitrile moiety of 1a is stabilized by a π-π stacking interaction with the Y361β residue, suggesting a structural explanation for the observed importance of this component of our inhibitors.

Protein tyrosine phosphatases (PTPs) are a diverse family of enzymes encoded by 107 genes in the human genome. Together with protein tyrosine kinases (PTKs), PTPs regulate various cellular activities essential for the initiation and maintenance of malignant phenotypes. While PTK inhibitors are now used routinely for cancer treatment, the PTP inhibitor development field is still in the discovery phase. In this article, the suitability of targeting PTPs for novel anticancer drug discovery is discussed. Examples are presented for PTPs that have been targeted for anticancer drug discovery as well as potential new PTP targets for novel anticancer drug discovery.

The Akt pathway is frequently hyperactivated in human cancer and functions as a cardinal nodal point for transducing extracellular and intracellular oncogenic signals and, thus, presents an exciting target for molecular therapeutics. Here we report the identification of a small molecule Akt/protein kinase B inhibitor, API-1. Although API-1 is neither an ATP competitor nor substrate mimetic, it binds to pleckstrin homology domain of Akt and blocks Akt membrane translocation. Furthermore, API-1 treatment of cancer cells results in inhibition of the kinase activities and phosphorylation levels of the three members of the Akt family. In contrast, API-1 had no effects on the activities of the upstream Akt activators, phosphatidylinositol 3-kinase, phosphatidylinositol-dependent kinase-1, and mTORC2. Notably, the kinase activity and phosphorylation (e.g. Thr(P)(308) and Ser(P)(473)) levels of constitutively active Akt, including a naturally occurring mutant AKT1-E17K, were inhibited by API-1. API-1 is selective for Akt and does not inhibit the activation of protein kinase C, serum and glucocorticoid-inducible kinase, protein kinase A, STAT3, ERK1/2, or JNK. The inhibition of Akt by API-1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor constitutively activated Akt. Furthermore, API-1 inhibited tumor growth in nude mice of human cancer cells in which Akt is elevated but not of those cancer cells in which it is not. These data indicate that API-1 directly inhibits Akt through binding to the Akt pleckstrin homology domain and blocking Akt membrane translocation and that API-1 has anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express hyperactivated Akt.

Numerous proteins controlling cell cycle progression, apoptosis and angiogenesis are degraded by the ubiquitin/proteasome system, which has become the subject for intense investigations for cancer therapeutics. Therefore, we used in silico and experimental approaches to screen compounds from the NCI chemical libraries for inhibitors against the chymotrypsin-like (CT-L) activity of the proteasome and discovered PI-083. Molecular docking indicates that PI-083 interacts with the Thr21, Gly47 and Ala49 residues of the beta5 subunit and Asp114 of the beta6 subunit of the proteasome. PI-083 inhibits CT-L activity and cell proliferation and induces apoptosis selectively in cancer cells (ovarian T80-Hras, pancreatic C7-Kras and breast MCF-7) as compared to their normal/immortalized counterparts (T80, C7 and MCF-10A, respectively). In contrast, Bortezomib, the only proteasome inhibitor approved by the Food and Drug Administration (FDA), did not exhibit this selectivity for cancer over non-transformed cells. In addition, in all cancer cells tested, including Multiple Myeloma (MM), breast, pancreatic, ovarian, lung, prostate cancer cell lines as well as fresh MM cells from patients, PI-083 required less time than Bortezomib to induce its antitumor effects. Furthermore, in nude mouse xenografts in vivo, PI-083, but not Bortezomib, suppressed the growth of human breast and lung tumors. Finally, following in vivo treatment of mice, PI-083 inhibited tumor, but not hepatic liver CT-L activity, whereas Bortezomib inhibited both tumor and liver CT-L activities. These results suggest that PI-083 is more selective for cancer cells and may have broader antitumor activity and therefore warrants further advanced preclinical studies.

Protein farnesyltransferase (FTase) catalyzes an essential posttranslational lipid modification of more than 60 proteins involved in intracellular signal transduction networks. FTase inhibitors have emerged as a significant target for development of anticancer therapeutics and, more recently, for the treatment of parasitic diseases caused by protozoan pathogens, including malaria (Plasmodium falciparum). We present the X-ray crystallographic structures of complexes of mammalian FTase with five inhibitors based on an ethylenediamine scaffold, two of which exhibit over 1000-fold selective inhibition of P. falciparum FTase. These structures reveal the dominant determinants in both the inhibitor and enzyme that control binding and selectivity. Comparison to a homology model constructed for the P. falciparum FTase suggests opportunities for further improving selectivity of a new generation of antimalarial inhibitors.

Akt has emerged as a critical target for the development of anti-cancer therapies. It has been found to be amplified, overexpressed, or constitutively activated in numerous human malignancies with oncogenesis derived from the simultaneous promotion of cell survival and suppression of apoptosis. A valuable alternative to the more common ATP-mimetic based chemotherapies is a substrate-mimetic approach, which has the potential advantage of inherent specificity of the substrate-binding pocket. In this paper we present the development of high affinity non-peptidic, substrate-mimetic inhibitors based on the minimum GSK3beta substrate sequence. Optimization of initial peptidic leads resulted in the development of several classes of small molecule inhibitors, which have comparable potency to the initial peptidomimetics, while eliminating the remaining amino acid residues. We have identified the first non-peptidic substrate-mimetic lead inhibitors of Akt 29a-b, which have affinities of 17 and 12 microM, respectively. This strategy has potential to provide a useful set of molecular probes to assist in the validation of Akt as a potential target for anti-cancer drug design.

S3I-201 (NSC 74859) is a chemical probe inhibitor of Stat3 activity, which was identified from the National Cancer Institute chemical libraries by using structure-based virtual screening with a computer model of the Stat3 SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the Stat3beta homodimer. S3I-201 inhibits Stat3.Stat3 complex formation and Stat3 DNA-binding and transcriptional activities. Furthermore, S3I-201 inhibits growth and induces apoptosis preferentially in tumor cells that contain persistently activated Stat3. Constitutively dimerized and active Stat3C and Stat3 SH2 domain rescue tumor cells from S3I-201-induced apoptosis. Finally, S3I-201 inhibits the expression of the Stat3-regulated genes encoding cyclin D1, Bcl-xL, and survivin and inhibits the growth of human breast tumors in vivo. These findings strongly suggest that the antitumor activity of S3I-201 is mediated in part through inhibition of aberrant Stat3 activation and provide the proof-of-concept for the potential clinical use of Stat3 inhibitors such as S3I-201 in tumors harboring aberrant Stat3.

In this study, we synthesized some natural and semisynthetic prenyloxyphenylpropanoids (e.g., coumarins and cinnamic acid derivatives) and we assessed their in vitro inhibitory activity against farnesyl transferase (FTase) and geranylgeranyl transferase I (GGTase I). No compound was an effective inhibitor of FTase, while farnesyloxycinnamic acids were shown to selectively inhibit GGTase I with IC(50) values ranging from 28 to 39 microM.

Amplification, overexpression, and elevated activation of Akt have been detected in many human malignancies making it an important target for cancer therapy. The Akt substrate-binding site offers a large number of potential interactions to an appropriately designed small molecule and can form the basis for the development of selective inhibitors. Here, we report the progression of GSK3beta substrate-mimetic inhibitors towards the development of a potent, small molecule substrate-mimetic inhibitor of Akt.

Constitutive activation of the JAK/STAT3 pathway is a major contributor to oncogenesis. In the present study, structure-activity relationship (SAR) studies with five cucurbitacin (Cuc) analogs, A, B, E, I, and Q, led to the discovery of Cuc Q, which inhibits the activation of STAT3 but not JAK2; Cuc A which inhibits JAK2 but not STAT3 activation; and Cuc B, E, and I, which inhibit the activation of both. Furthermore, these SAR studies demonstrated that conversion of the C3 carbonyl of the cucurbitacins to a hydroxyl results in loss of anti-JAK2 activity, whereas addition of a hydroxyl group to C11 of the cucurbitacins results in loss of anti-STAT3 activity. Cuc Q inhibits selectively the activation of STAT3 and induces apoptosis without inhibition of JAK2, Src, Akt, Erk, or JNK activation. Furthermore, Cuc Q induces apoptosis more potently in human and murine tumors that contain constitutively activated STAT3 (i.e., A549, MDA-MB-435, and v-Src/NIH 3T3) as compared to those that do not (i.e., H-Ras/NIH 3T3, MDA-MB-453, and NIH 3T3 cells). Finally, in a nude mouse tumor xenograft model, Cuc Q, but not Cuc A, suppresses tumor growth indicating that JAK2 inhibition is not sufficient to inhibit tumor growth and suggesting that the ability of Cuc Q to inhibit tumor growth is related to its anti-STAT3 activity. These studies further validate STAT3 as a drug discovery target and provide evidence that pharmacological agents that can selectively reduce the P-STAT3 levels in human cancer cells result in tumor apoptosis and growth inhibition.

The farnesyltransferase inhibitors (FTIs) were designed to inhibit the post-translational processing of Ras proteins, which are mutated in 30% of all human cancers. Recent studies suggest, however, that the target of FTIs may be a protein other than Ras, and that these agents may be more appropriately used to treat tumors with activated wild-type ras signaling. Preliminary results from several phase II and phase III studies have been reported. The FTIs fail to show significant single-agent activity in non-small cell lung cancer, small cell lung cancer, pancreatic cancer, refractory colorectal cancer, and bladder cancer. Activity has been shown in hematologic malignancies (acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome), breast cancer, and glioma. Several combination studies of FTIs and standard cytotoxic agents are ongoing.

Farnesyltransferase (FTase) is a major molecular target for the development of novel anticancer drugs that are designed to disrupt the aberrant signal transduction circuitry of tumor cells. The discovery more than a decade ago that farnesylation of Ras, one of the most prevalent oncoproteins in human cancers, is required for its cancer-causing activity was the initial stimulus to target FTase. However, it is now clear that yet to be identified farnesylated proteins other than Ras also play a pivotal role as targets for FTase inhibitors (FTIs). In this review, important mechanistic issues on the antiproliferative, pro-apoptotic and anti-angiogenic activities of FTIs, with an emphasis on potential FTI targets, will be discussed. Important clinical issues associated with translational aspects of hypotheses-driven clinical trials will also be discussed.

Constitutively activated, tyrosine-phosphorylated signal transducer and activator of transcription (STAT) 3 plays a pivotal role in human tumor malignancy. To discover disrupters of aberrant STAT3 signaling pathways as novel anticancer drugs, we developed a phosphotyrosine STAT3 cytoblot. Using this high throughput 96-well plate assay, we identified JSI-124 (cucurbitacin I) from the National Cancer Institute Diversity Set. JSI-124 suppressed the levels of phosphotyrosine STAT3 in v-Src-transformed NIH 3T3 cells and human cancer cells potently (IC(50) value of 500 nM in the human lung adenocarcinoma A549) and rapidly (complete inhibition within 1-2 h). The suppression of phosphotyrosine STAT3 levels resulted in the inhibition of STAT3 DNA binding and STAT3-mediated but not serum response element-mediated gene transcription. JSI-124 also decreased the levels of tyrosine-phosphorylated Janus kinase (JAK) but not those of Src. JSI-124 was highly selective for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways such as those mediated by Akt, extracellular signal-regulated kinase 1/2, or c-Jun NH(2)-terminal kinase. Finally, JSI-124 (1 mg/kg/day) potently inhibited the growth in nude mice of A549 tumors, v-Src-transformed NIH 3T3 tumors, and the human breast carcinoma MDA-MB-468, all of which express high levels of constitutively activated STAT3, but it did not affect the growth of oncogenic Ras-transformed NIH 3T3 tumors that are STAT3 independent or of the human lung adenocarcinoma Calu-1, which has barely detectable levels of phosphotyrosine STAT3. JSI-124 also inhibited tumor growth and significantly increased survival of immunologically competent mice bearing murine melanoma with constitutively activated STAT3. These results give strong support for pharmacologically targeting the JAK/STAT3 signaling pathway for anticancer drug discovery.

This paper describes recent progress in the design, synthesis and biological evaluation of inhibitors for the enzyme protein farnesyltransferase (PFTase). This enzyme plays a critical role in the post-translational modification of a range of different intracellular proteins. In particular, PFTase attaches a farnesyl group to the GTPase Ras whose oncogenically mutated form is found in over 30% of human cancers. As a result PFTase inhibitors have been developed as potential cancer therapeutic drugs either by rational design based on the structure of the CAAX carboxyl terminus of Ras or random screening of chemical libraries or natural products. Some of these inhibitors show remarkable inhibition potency against PFTase at subnanomolar concentrations and >1000-fold selectivity compared to the related enzyme geranylgeranyltransferase-I. Certain of these compounds are highly effective at blocking the growth of human tumors in animal models and are now undergoing clinical trials. However, several issues in the research remain unsolved, including the mechanism by which PFTase inhibitors suppress tumor growth. Although it has been established that PFTase inhibitors block prenylation of Ras in vitro, the results in wholecells and animal studies suggest the possibility that proteins other than Ras are affected.

In 1990, more than 10 years after the discovery that the low molecular weight GTPase Ras is a major contributor to human cancer, farnesylation, a lipid posttranslational modification required for the cancer-causing activity of Ras, emerged as a major target for the development of novel anticancer agents. However, it took only 5 years from 1993, when the first farnesyltransferase inhibitors (FTIs) were reported, to 1998 when results from the first phase I clinical trials were described. This rapid progress was due to the demonstration of outstanding antitumor activity and lack of toxicity of FTIs in preclinical models. Although, many FTIs are currently in phase H and at least one is in phase III clinical trial, the mechanism of FTI antitumor activity is not known. In this review a brief summary of the development of FTIs as antitumor agents will be given. The focus of the review will be on important mechanistic and bench-to-bedside translational issues. Among the issues that will be addressed are: evidence for and against inhibition of the prenylation of Ras and RhoB proteins in the mechanism of action of FTIs; implications of the alternative prenylation of K-Ras by geranylgeranyl-transferase I (when FTase is inhibited) in cancer therapy; GGTase I inhibitors (GGTIs) as antitumor agents; effects of FTIs and GGTIs on cell cycle machinery and progression and potential mechanisms by which FTIs and GGTIs induce apoptosis in human cancer cells. A thorough discussion about bench-to-bedside issues relating to hypothesis-driven clinical trials with proof-of-principle in man will also be included. This section will cover issues relating to whether the biochemical target (FTase) is inhibited and the level of inhibition of FTase required for clinical response; are signaling pathways such as H-Ras/PI3K/Akt and/or K-Ras/Raf/MEK/Erk relevant biological readouts?; is Ras (particularly N-Ras and H-Ras) mutation status a good predictor of clinical response?; in phase I trials should effective biological dose, not maximally tolerated dose, be used to determine phase II dose?; and finally, in phase II/III trials what are the most appropriate clinical end points for anti-signaling molecules such as FTIs? Parts of this topic have been recently reviewed (Sebti and Hamilton, 2000c).

This review describes our recent efforts in the development of novel therapies for cancer. Our primary approach is to design synthetic agents that antagonize the function of growth factors that are critically involved in oncogenesis and angiogenesis. We achieve this by designing synthetic molecules that can recognize the exterior surface of the growth factor and so block the interaction with its receptor tyrosine kinase. A key step is the construction of synthetic agents that contain a large (> 400A2) and functionalized surface area to recognize a complementary surface on the target growth factor. In the course of this work we have discovered a molecule, GFB-111, that binds to PDGF, prevents it from binding to its receptor tyrosine kinase, blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases and DNA synthesis. The binding affinity for PDGF is high (IC50=250 nM) and selective over EGF, IGF-1, aFGF, bFGF and HRGbeta. In nude mouse models GFB-111 also shows significant inhibition of tumor growth and angiogenesis.

The fact that proteins such as Ras, Rac and RhoA require farnesylation or geranylgeranylation to induce malignant transformation prompted many investigators to develop farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) inhibitors (FTIs and GGTIs, respectively) as novel anticancer drugs. Although FTIs have been shown to antagonise oncogenic signalling, reverse malignant transformation, inhibit human tumour growth in nude mice and induce tumour regression in transgenic mice without any signs of toxicity, their mechanism of action is not known. This review will focus on important mechanistic issues as well as bench to bedside translational issues. These will include the relevance to cancer therapy of the alternative geranylgeranylation of K-Ras when FTase is inhibited; a thorough discussion about evidence for and against the involvement of inhibition of prenylation of Ras and RhoB in the mechanism of FTIs' antitumour activity as well as effects of FTIs and GGTIs on the cell cycle machinery and the dynamics of bipolar spindle formation and chromosome alignment during mitosis. Bench to bedside issues relating to the design of hypothesis-driven clinical trials with biochemical correlates for proof-of-concept in man will also be discussed. This will include Phase I issues such as determining maximally tolerated dose (MTD) versus effective biological dose (EBD), as well as whether Phase II trials are still needed for clinical evaluations of anti-signalling agents. Other questions that will be addressed include: what levels of inhibition of FTase activity are required for tumour response in Phase II clinical evaluations? What FTase substrates are most relevant as biochemical correlates? Are signalling pathways such as H-Ras/PI3K/Akt and K-Ras/Raf/MEK/Erk significant biological readouts? Does Ras mutation status predict response? What are appropriate clinical end-points for FTI Phase II trials? For this latter important question, time to tumour progression, median survival, percentage of patients that progress, clinical benefits and improvement in quality of life will all be discussed.

We have designed a molecule, GFB-111, that binds to platelet-derived growth factor (PDGF), prevents it from binding to its receptor tyrosine kinase, and blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases, and DNA synthesis. GFB-111 is highly potent (IC50 = 250 nM) and selective for PDGF over EGF, IGF-1, aFGF, bFGF, and HRGbeta (IC50 values > 100 microM), but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 microM. GFB-111 treatment of nude mice bearing human tumors resulted in significant inhibition of tumor growth and angiogenesis. The results demonstrate the feasibility of designing novel growth factor-binding molecules with potent anticancer and antiangiogenic activity.

The Ras protein undergoes a series of post-translational modifications at the C-terminal CAAX motif, which culminates with the anchoring of p21 Ras to the plasma membrane where it relays growth regulatory signals from receptor tyrosine kinases to various pathways of cell signal transduction. FTI-276 is a CAAX peptidomimetic of the carboxyl terminal of Ras proteins. Pharmacokinetic analysis of FTI-276 in A/J mice with a time-release pellet system showed a dose of 50 mg/kg body wt achieved an average serum level of 1.68 microg/ml for up to 30 days following implantation. In the present study, 4 week old A/J mice were initiated with a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (100 mg/kg), and monitored for 18 weeks. Mice were grouped for daily delivery (time-release pellet) of 50 mg/kg of FTI-276 for 30 days (n = 12) and the control group (n = 12). Analysis of tumors from time-release pellet treated animals showed a 60% reduction in tumor multiplicity and a 42% reduction in tumor incidence. Moreover, FTI-276 treatment resulted in a significant reduction in tumor volume (approximately 58%). Mutation analysis of the lung tumors from both treatment groups revealed that most of the tumors harbored mutations in the codon 12 of K-ras and there is no significant difference in the incidence and types of mutations between tumors from the treated and control animals. This is the first demonstration of chemotherapeutic efficacy of a synthetic CAAX peptidomimetic farnesyltransferase inhibitor in a primary lung tumor model.

Ras malignant transformation requires posttranslational modification by farnesyltransferase (FTase). Here we report on the design and antitumor activity, in monotherapy as well as in combination therapy with cytotoxic agents, of a novel class of non-thiol-containing peptidomimetic inhibitors of FTase and the closely related family member geranylgeranyltransferase I (GGTase I). The non-thiol-containing FTI-2148 is highly selective for FTase (IC50, 1.4 nM) over GGTase I (IC50, 1700 nM), whereas GGTI-2154 is highly selective for GGTase I (21 nM) over FTase (IC50, 5600 nM). In whole cells, the corresponding methylester prodrug FTI-2153 is >3000-fold more potent at inhibiting H-Ras (IC50, 10 nM) than Rap1A processing, whereas GGTI-2166 is over 100-fold more selective at inhibiting Rap1A (IC50, 300 nM) over H-Ras processing. Furthermore, FTI-2153 was highly effective at suppressing oncogenic H-Ras constitutive activation of mitogen-activated protein kinase and human tumor growth in soft agar. FTI-2148 suppressed the growth of the human lung adenocarcinoma A-549 cells in nude mice by 33, 67, and 91% in a dose-dependent manner. Combination therapy of FTI-2148 with either cisplatin, gemcitabine, or Taxol resulted in a greater antitumor efficacy than monotherapy. GGTI-2154 in similar antitumor efficacy experiments is less potent than FTI-2148 and inhibits tumor growth by 9, 27, and 46%. Combination therapy of GGTI-2154 with cisplatin, gemcitabine, or Taxol is also more effective. Finally, FTI-2148 and GGTI-2154 are 30- and 33-fold more selective and 30- and 16-fold more potent in whole cells than our previously reported thiol-containing FTI-276 and GGTI-297, respectively. Thus, our results demonstrate that this highly potent and selective novel class of non-thiol-containing peptidomimetics inhibits human tumor growth in whole animals and that combination therapy with cytotoxic agents is more beneficial than monotherapy.

The geranylgeranyltransferase I inhibitor GGTI-298 has recently been shown to arrest human tumor cells in the G1 phase of the cell cycle, induce apoptosis, and inhibit tumor growth in nude mice. In the present manuscript, we provide a possible mechanism by which GGTI-298 mediates its tumor growth arrest. Treatment of the human lung carcinoma cell line Calu-1 with GGTI-298 results in inhibition of the phosphorylation of retinoblastoma protein, a critical step for G1/S transition. The kinase activities of two G1/S cyclin-dependent kinases, CDK2 and CDK4, are inhibited in Calu-1 cells treated with GGTI-298. Furthermore, GGTI-298 has little effect on the expression levels of CDK2, CDK4, CDK6, cyclins D1 and E, but decreases the levels of cyclin A. GGTI-298 increases the levels of the cyclin-dependent kinase inhibitors p21 and p15 and had little effect on those of p27 and p16. Most interesting is the ability of GGTI-298 to induce partner switching for several CDK inhibitors. GGTI-298 promotes binding of p21 and p27 to CDK2 while decreasing their binding to CDK6. Reversal of partner switching and G1 block was observed after removal of GGTI-298. Furthermore, GGTI-298 treatment results in an increased binding of p15 to CDK4, which is paralleled with decreased binding to p27. The results demonstrate that the GGTI-298-mediated G1 block in Calu-1 cells involves increased expression and partner switching of CDK inhibitors resulting in inhibition of CDK2 and CDK4, and retinoblastoma protein phosphorylation.

The cancer-causing activity of Ras requires the prenylation of a cysteine fourth from its carboxyl terminus. Rational design of peptidomimetics of the carboxyl terminal tetrapeptide prenylation site on Ras resulted in pharmacological agents capable of inhibiting Ras processing, selectively antagonizing oncogenic signaling and suppressing human tumor growth in mouse models without side effects. This mini-review describes the efforts of several groups to design, synthesize and evaluate the biological activities of farnesyltransferase and geranylgeranyltransferase I inhibitors. Among the important issues that will be discussed are the mechanism of action of these inhibitors and the potential mechanisms of resistance to inhibition of K-Ras farnesylation.

Oncogenic H-Ras requires farnesylation for its transforming activity. Lovastatin inhibits both protein farnesylation and geranylgeranylation by decreasing cellular pools of farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), respectively. Use of lovastatin as a chemotherapeutic agent has been precluded by its significant cytotoxic effects. In this report, we describe a novel approach utilizing a combination of lovastatin and geranylgeraniol (GGOH) to potentiate the ability of lovastatin to block oncogenic H-Ras signaling and concomitantly rescue lovastatin toxicity. GGOH co-treatment with lovastatin enhances inhibition of oncogenic H-Ras processing and constitutive activation of mitogen-activated protein kinase (MAPK), and preserves the processing of geranylgeranyltransferase (GGTase) I and GGTase II protein substrates. Moreover, co-treatment with GGOH significantly (15-fold) attenuates the cytotoxic effects of lovastatin as well as prevents lovastatin-induced cell rounding. These results demonstrate that GGOH potentiates the anti-oncogenic/anti-signaling activity of lovastatin while antagonizing its cytotoxicity. These opposing effects are due to a GGOH metabolite that serves simultaneously as a potent inhibitor for farneslyltransferase as well as a substrate for GGTases I and II.

The currently understood function for Ras in signal transduction is in mediating the transmission of signals from external growth factors to the cell nucleus. Mutated forms of this GTP-binding protein are found in 30% of human cancers with particularly high prevalence in colon and pancreatic carcinomas. These mutations destroy the GTPase activity of Ras and cause the protein to be locked in its active, GTP bound form. As a result, the signaling pathways are activated, leading to uncontrolled tumor growth. Ras function in signaling requires its association with the plasma membrane. This is achieved by posttranslational farnesylation of a cysteine residue present as part of the CA1A2X carboxyl terminal tetrapeptide of all Ras proteins. The enzyme that recognizes and farnesylates the CA1A2X sequence, Ras farnesyltransferase (FTase), has become an important target for the design of inhibitors that might be interesting as antitumor agents. Several approaches have been taken in the search for in vivo active inhibitors of farnesyltransferase. These include the identification of natural products such as the chaetomellic and zaragozic acids that mimic farnesylpyrophosphate, bisubstrate transition state analogs combining elements of the farnesyl and tetrapeptide substrates and peptidomimetics that reproduce features of the carboxyl terminal tetrapeptide CA1A2X sequence. This last group of compounds has been most successful in showing highly potent inhibition of FTase and selective blocking of Ras processing in a range of Ras transformed tumor cell lines at concentrations as low as 10 nM. Certain peptidomimetics will also block tumor growth in various mouse models, with apparently few toxic side effects. These results suggest that farnesyltransferase inhibitors hold considerable promise as anticancer drugs in the clinic.

Farnesylation of the oncoprotein Ras is required for its cancer-causing activity. We have designed farnesyltransferase inhibitor (FTI)-276, a tetrapeptide mimetic of the carboxyl terminus of K-Ras4B, as a highly potent and selective inhibitor of Ras farnesylation in vitro and in vivo. FTI-276 blocked the growth in nude mice of a human lung carcinoma that expresses the two most prevalent genetic alterations in human cancers (K-Ras oncogenic mutation and deletion in the tumor suppressor gene p53). In contrast, FTI-276 did not inhibit tumor growth of a human lung carcinoma that harbors no Ras mutations. Furthermore, FTI-276 inhibited oncogenic signaling and tumor growth of NIH 3T3 cells transformed with the ras but not the raf oncogene. Inhibition of tumor growth in vivo was dose dependent and correlated with inhibition of Ras processing in tumors in vivo. The work described here identifies FTI-276 as a highly selective suppressor of Ras-dependent oncogenicity and suggests that a broad spectrum of human cancers with aberrant Ras function could benefit from farnesyltransferase inhibitor treatment.

We have synthesized fluoromycin (FLM), a novel fluorescein-labeled derivative of talisomycin S10b (TLM S10b), and used it to evaluate cellular drug accumulation and distribution in bleomycin (BLM)-sensitive and -resistant cell lines. The fluorescence intensity of FLM was 300- to 400-fold greater than that of BLM A2, TLM S10b, or the lipophilic BLM analogue, liblomycin. FLM possessed an antiproliferative potency similar to liblomycin in BLM-sensitive human A-253 squamous carcinoma cells but was less potent than BLM A2 or TLM S10b. C-10E cells, a clone of A-253 cells with 40- to 50-fold resistance to BLM A2 and TLM S10b, were 50-fold resistant to FLM. A partially revertant cell population (C-10E ND) regained sensitivity to BLM A2, TLM S10b, and FLM. FLM like BLM cleaved pGEM-3Z plasmid DNA in vitro in a concentration-dependent manner. Flow cytometric analysis of FLM content in C-10E and C-10E ND cell lines showed 4-fold and 2-fold lower fluorescence intensity, respectively, compared with A-253 cells. Similar results were seen by fluorescence spectrophotometry with cell extracts. Fluorescence microscopy indicated heterogeneous distribution among A-253 cells with at least 50% of the cells exhibiting marked nuclear fluorescence localization. In contrast, C-10E cells displayed lower cellular fluorescence and predominantly cytoplasmic localization. C-10E ND cells exhibited a mixed population of nuclear and cytoplasmic vesicular localization with fluorescence levels that were intermediate between A-253 and C-10E cells. Thus, BLM-resistant cells have reduced levels of FLM and appear to have a lower nuclear:cytoplasmic ratio of FLM. FLM may be useful in studying the intracellular fate of BLM-like drugs as well as providing a tool to detect and isolate BLM-resistant cells.