Shuxing Zhang-MS-MRMC

Send Orders for Reprints to [email protected] Mini-Reviews in Medicinal Chemistry, 2016, 16, 000-000

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Past, Present, and Future of Targeting Ras for Cancer Therapies Zhi Tan and Shuxing Zhang* Integrated Molecular Discovery Laboratory, Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Abstract: For decades, mutant Ras (mut-Ras) proteins have been identified as drivers of multiple cancers including pancreatic, lung, and colon cancers. However, targeting this oncogene has been challenging and no Ras inhibitors are on the market to date. Lately several candidates targeting the downstream pathways of Ras signaling, including PI3K and Raf, were approved for cancer treatment. However, they do not present promising therapeutic effects on patients harboring Ras mutations. Recently, a variety of compounds have been reported to impair the activity of Ras, and these exciting discoveries reignite the hope for development of novel drugs targeting mut-Ras. In this article, we will review the progress made in this field and the current state-of-the-art technologies to develop Ras inhibitors. Also we will discuss the future direction of targeting Ras.

Keywords: ?????????????????????. INTRODUCTION Ras is a protein family of small GTPases which regulates a variety of biological processes through the switch of binding to GDP or GTP. GDP-bound Ras, the inactive form, is activated by a guanine nucleotide exchange factor (GEF), which induces the release of GDP and allows GTP to bind (Fig. 1). After binding to GTP, Ras undergoes marked conformational changes and then recruits its effectors including B-Raf, PI3K, RALGDS and PLCε (Fig. 1). As a consequence, Ras is “switched on” and able to relay the signal from cell surface to cytoplasm, which plays a significant role in controlling multiple vital cellular processes such as differentiation, survival, and proliferation [2]. The activated Ras serves its function as a GTPase to cleave the terminal phosphate of the GTP and convert it to GDP. Although Ras proteins have high binding affinities to GDP and GTP, the intrinsic enzymatic activity is relatively low. However, with catalysis of GTPase-Activating Proteins (GAP), this reaction will be significantly accelerated, up to 10,000 fold. In humans, three ubiquitously expressed Ras genes encode four proteins including HRas, NRas, KRas 4A, and KRas4B. These ~21 Kda proteins are highly homologous, and their accurate regulation is critical to maintain the homeostasis normal cell and organism functions. For instance, KRas knockout mice are embryonic lethal due to liver defects and anemia [3]. In 1982, for the first time, Ras was identified mutationally activated in cancer cell lines [4]. Subsequently, intensive whole genome sequencing of human *Address correspondence to this author at the Integrated Molecular Discovery Laboratory, Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Tel: (713) 7452958; Fax: (713) 794-5577; E-mail: [email protected] 1389-5575/16 $58.00+.00

genome in different cancer types reveals that Ras proteins are frequently mutated in more than 30% of all the cancer types [5]. Moreover, Ras proteins have the highest mutation rates in pancreatic (97.7%), colon (52.2%), multiple myeoma (42.6%), and lung (32.2%) cancers [6]. Intriguingly, most of the mutations of Ras occur in three codons: G12, G13 and Q61. Specifically, around 80% KRas mutations are found in codon 12 compared to NRAS mutations that are more likely involve codon 61 (60%) and codon 12 (30%) [7]. HRas mutations preferentially occur in codon 12 and codon 61 [5a, 8]. Transgenic and genetic engineered mouse models revealed that mutant Ras proteins are sufficient to drive the initiation of multiple types of cancers in targeted tissues or in whole body [9]. These evidences show that Ras oncogenes play significant roles in early onset of cancers. Moreover, considerable experiment evidence suggests Ras oncogenes are critical in tumor maintenance in different cancer types. In Ras-mutant cancer cell lines, RNA interferences have been shown to slow down the tumor growth both in vitro and in vivo [10]. Similar phenomenon has also been observed in animal models [11]. These studies made Ras oncoproteins widely accepted as promising drug targets. RAS STRUCTURE-FUNCTION All the four isoforms of Ras proteins share 95% amino acid sequence identity and have similar 3D structures. They consist of two major structural components: the G domain and C-terminal domain. The G domain is also known as the catalytic domain and highly homologous among different isoforms. The G domain is the region carrying the function of GDP/GTP binding and GTP hydrolysis. It is composed of five G motifs, three of which are important to the GTP binding and hydrolysis [1]: (a) the phosphate binding loop (p-loop) comprised of AA 10-17, binds to the beta phosphate © 2016 Bentham Science Publishers

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Mini-Reviews in Medicinal Chemistry, 2016, Vol. 16, No. 0

Tan and Zhang

translational modifications contributing to the sub-locations of Ras proteins inside cells. One of the most important posttranslational modifications of Ras proteins is farnesylation. All of the four Ras isoforms are terminated with a “CAAX” sequence, where C is cytosine; A is usually an aliphatic amino acid; and X can be any residue. After Ras proteins are translated in the cytosol, they are farnesylated on the cysteine residue in the “CAAX box” and acquire the capability of translocating to the cell membrane to serve their functions. This modification is controlled by the farnesyltransferase Ras converting enzyme 1 [17]. Detailed mechanisms of the CAAX box processing will be discussed in the later sections.

Fig. (1). Top: Ras cycles of binding to GTP and GDP regulated by GAP and GEF; Bottom: GEFs (orange), GAPs (green), and effectors (purple) of Ras proteins.

of GDP or GTP; (b) switch I, consisting of AA 25-40, binds to the terminal phosphate of GTP but no contact with GDP; (c) switch II, including AA 57-75, in which the Q61 is the critical residue participating in the hydrolysis of GTP to GDP through activation of a catalytic water molecule (Fig. 2). The frequent mutations on the residues 12, 13, and 61 lock the Ras protein in the active state which renders the hydrolysis of GTP, though in different mechanisms. Mutations in G12 and G13 inhibit the formation of van der Waals bonds between GAP and Ras resulting in incorrect orientation of the catalytic residue Q61 [12]; on the other hand, mutations in the residue Q61 interferes with the correct coordination of a water molecule which is required to attack the γ-phosphate [13] of GTP. With these mutations, the downstream signaling of Ras are constitutively active, even without the binding of growth factors to the cell membrane [14]. The C-terminus of Ras, referred to the hypervariable region (HVR), consists of the last 23 amino acids, and it differentiates among different isoforms [15]. This region can be further divided into two parts: the linker region and the membrane-interacting lipid anchor; both are important in the plasma membrane tethering [16]. More importantly, the Cterminal domain is also the region with the major post-

Fig. (2). Structure of KRas complexed with GDP. The secondary structure of KRas is in the rainbow color cartoon, and the GDP is in magenta sticks. The red x represents the critical water molecule involved in KRas activation

STRATEGIES OF TARGETING RAS For more than two decades after realizing the transforming ability of Ras, scientists have attempted to develop compounds that bind to the proteins. However, the journey was disappointing and led to the impression that Ras proteins are “undruggable targets”. Concurrently researchers began to look for alternative strategies to solve the puzzle. Targeting the farnesylation is one of the new approaches. Many studies have been published on targeting the posttranslational modification of Ras proteins and multiple inhibitors were developed [17, 18]. At the same time, other approaches, including targeting GEF and Ras-Effector interactions, have been extensively explored. Also, due to the development of new computational methods and imaging techniques, multiple compounds have been reported directly bind to the Ras proteins. These new approaches have led to more understandings and insights about the signaling pathway. In this review, we will discuss the compounds that have been developed to date (Table 1), and we will also deliberate on the failures/successes as well as the future of targeting Ras for cancer therapy.

Past, Present, and Future of Targeting Ras for Cancer Therapies

DIRECTLY TARGETING RAS To date it remains a challenge to directly target Ras proteins. There are several reasons. First, as aforementioned, Ras proteins have very high binding affinities to GDP/GTP (at picomolar level) [6]. This fact, along with the high concentration of GDP/GTP in cells, makes it very difficult for inhibitors to compete with GDP/GTP. Second, the binding of GDP/GTP actually further stabilizes the RasGDP/GTP complex [16a]. Third, according to the crystal structures, it seems that there is no accessible pocket on the surface of Ras proteins. Although some potential pockets have been reported using computational methods, none of them could allow small molecules to bind tightly [19]. Despite these limitations, a few compounds have been identified to directly bind to Ras proteins in the last several decades. In 1997, Dr. Taveras and colleagues identified a compound, named SCH53239 (Table 1), which was initially designed to compete with GDP and bind the nucleotide binding site [20]. However, through the Scatchard analysis, SCH53239 and its water soluble analogue SCH54292 (Table 1) were found bound to a hydrophobic pocket primarily within the switch II, instead of the GDP binding site. In a nutshell, SCH54292 binds to Ras proteins, actually without interfering with the GDP binding. Although these two compounds did not make into the clinic, they provide the proof-of-principle that small molecules can be identified to directly bind Ras proteins [20]. In 2005, by using molecular modeling techniques, Peri et al. reported bicyclic analogues that are able to inhibit nucleotide exchange through binding to a similar pocket of SCH54292. Though not very potent, two of these bicyclic analogues show inhibition of Ras signaling both in cells and animal models [21]. Ras proteins can also be directed to restore the GTP hydrolysis in the mutant Ras proteins. As known, the mutations in Ras proteins impair the GTPase activity and prevent Ras from turning into inactive state even in the existence of GAP. In 1999, Ahmadian and his colleagues identified diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) (Table 1), a GTP analogue, which could covalently bind to the mutant Ras proteins and facilitate the GTP hydrolysis [22]. It was reported that DABP-GTP is hydrolyzed more efficiently by mutant Ras, especially the G12 mutant, than that by the wild type Ras proteins. Moreover, it does not damage the function of Ras as a molecular switch. The discovery of DABP-GTP demonstrated that it is possible to specifically target the active site of mutant Ras proteins. Along the same line, but more than 14 years later, Shokat’s group from UCSF using a disulphide-fragmentbased screening identified several small molecules (Table 1) irreversibly inhibit the G12C mutant KRas protein. These compounds selectively bind to a pocket majorly composed of switch II residues in the G12C mutant KRas without interfering with the wild type isoform [23]. Intriguingly, this pocket is not apparent in other published Ras structures. The authors demonstrated that two mechanisms may explain the impairment of the mutant KRas functions by these compounds: (1) Upon binding of these compounds, Ras prefers binding to GDP rather than GTP. As a consequence, the number of active form Ras proteins decreases and the


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inactive form Ras proteins increase; (2) Binding of the compounds diminish the interactions between Ras and effectors. In cellular studies, these compounds produced various effects on cells carrying G12C mutants but have no effect on cells with wild-type Ras proteins, consistent with the biochemical binding results. The compounds also impaired the functions of mutant KRas proteins in lung cancer with in vivo experiments. Another compound, SML-8-73-1 (SML) (Table 1), was also reported to irreversibly bind to the G12C mutant KRas protein [24]. This agent forms a covalent bond with the mutant cysteine. Unlike the compounds identified by Ostrem et al. [23], SML competes with GDP/GTP by directly binding to the guanine nucleotide binding pocket [24]. The binding of SML mimics the conformational change of Ras proteins induced by GDP binding. Even when they are in high concentrations, GDP and GTP can be replaced by this compound. Due to the existence of the negative charged phosphate groups, SML cannot enter the cells. In order to increase its cellular uptake, the beta phosphate group is modified and caged (SML-10-70-1). Unfortunately, this new compounds, although able to pass cell membrane, does not show significant improvement of efficacies in cells harboring KRas mutations [25]. INHIBITION OF THE RAS EXPRESSION LEVEL Another approach to targeting Ras is by inhibiting the expression of Ras proteins. As early as in 1996, Chen and his colleagues identified antisense oligodeoxynucleotides (ODNs) called ISIS-2503 which was found to selectively suppress HRas mRNA and protein expression levels in cells and xenograft mouse models [26]. In Phase I clinical trials it was also well tolerated in patients [27], however failed to improve tumor regression or patient survival rate in multiple phase II clinical trials [28]. Also, combinational therapy with gemcitabine does not improve survival rate or induce tumor regression in advanced pancreatic cancer, lung cancer and metastatic breast cancer [5a, 29]. In addition, it is possible to inhibit Ras expression by using RNAi, and it can selectively suppress mutant Ras expression while spare the wild-type isoform. Multiple groups have confirmed that knockdown of mutant Ras can inhibit tumor growth both in cell lines and in animal models of multiple types of cancers including ovarian cancer, colon cancers, lung cancers and pancreatic cancers [10a, 30]. Compared to ODNs, the RNAi techniques have the advantage of specifically inactivating mutant Ras oncogenes. Moreover, RNAi seems to be more potent than antisense ODNs [31]. However, its delivery to tumor masses is still a challenge and thus there is a long way to practically employ this technology to treat mutant Ras-driven cancers [10a]. DISRUPTION OF RAS PROTEIN LOCALIZATION Ras proteins can also be directly targeted through inhibition of their processing. There are at least two ways to achieve this: The first is to inhibit Ras translocation to the cell membrane using farnesyl transferase inhibitors (FTIs) or geranylgeranyltransferase inhibitors (GGTIs). As is discussed, in order to allow Ras to translocate to cell

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Table 1.

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Compounds Targeting Ras oncogenes.

Name

Structure

Mechanism

Activity

Phase

Reference

Prevent hydrolysis of GTP

IC50=0.5µM

Preclinical

Taveras, A. G., et al. (1997).


IC50=0.5µM

Preclinical

Taveras, A. G., et al. (1997).


IC50=57.3 µM

Preclinical

Peri, F., et al. (2005).

Restore GTP hydrolysis in mutant Ras

KD=5.4nM

Preclinical

Ahmadian, M. R., et al. (1999).

Irreversibly bind to KRasG12C

EC50=0.32µM

Preclinical

Ostrem, J. M., et al. (2013).

NH2

H N O N

N

N O

O

SCH-53239

S N

O HN

OH

H O

H N

H

SCH-54292

O

O

S

O

O

N

H

O O O H O H

O

O

O

Bicyclic analogue

OH

O

NH HN

O O

N

NH 2 O H N

DABP-GTP

P

O O

OH O

P

N

O O

P

OH

O

O

OH

H

H

OH

H

H

C

NH

N

NH2

H

O I

H N

KRASG12C

N

Inhibitor

N Cl

OH O



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(Table 1) Contd….

Name

Structure

Mechanism

Activity

Phase

Reference

Form covalent bond with cysteine

NA

Preclinical

Hunter, J. C., et al. (2014). "

Preclinical

Lim, S. M., et al. (2014).

H 2N NH

NH O

N

OH

SML-8-73-1

O

O

-

P O

N

O

P

O

-

HO

O

O

N

O O

C H2 H 2N NH O

N NH

HO

O

N

N HO

-O

O O

O P

HN

SML-10-70-1

O

P O

Form covalent EC50=26.6-47.6 µM bond with cysteine

O

O O

ISIS 2503

oligodeoxynucleotides N

Antisense inhibitor of HRas

NA

Phae II

Chen, G., et al. (1996).

PLK1 inhibitor

IC50=0.83 nM

Phase II

Luo, J., et al. (2009).

Farnesyltransferase inhibitor

IC50=7.9 nM

Phase III

End, D.W., et al. (2001).

Phase III

Liu, M., et al. (1998).

O O

N

N H

O

N

BI-2356

N H

N

N

N

O

N

N

Tipffarnib

NH2

Cl Cl

Br NH 2 N

N N

O O

Lonafarnib (SCH66336)

Br

Cl

IC50(KRas)=5.2 nM; Farnesyltransferase IC50(NRas)=2.8 nM; inhibitor IC50(HRas)=1.9 nM;

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Tan and Zhang

(Table 1) Contd….

Name

Structure

Mechanism

Activity

Phase

Reference

Prenylated protein methyltransferase inhibitor

Ki=2.6 µM

Phase II

Rotblat, B., et al. (2008)

Inhibit the KRasPDEδ interaction

KD=165nM

Preclinical

Zimmermann, G., et al. (2013).

Inhibit Sos mediated Ras activation

KD=190 µM

Preclinical

Sun, Q., et al. (2012).

Inhibit Sos mediated Ras activation

EC50=15.8 µM

Preclinical

Maurer, T., et al. (2012).

Inhibit Ras-Raf interaction

IC50= 210 µM

Preclinical

Waldmann, H., et al. (2004)

Block Ras-effector interaction

Ki=46 µM

Preclinical

Shima, F., et al. (2013)

S

Salirasib

OH

O N

N

PDEδ Inhibitor

H N O

Kras-Sos interaction inhibitor

N

N H NH2

N H

H N

RasSosinteractio n inhibitor (DCAI) H 2N

O

S

H

Sulindac

O F OH

O 2N

Kobe0065

Cl

H N

CF3

H N N H O

NO2

membrane and serve its function, the CAAX located in the termini of Ras proteins need to be farnesylated. This process involves three enzymes: the farnesyltransferase to add a farnesyl group to the cysteine; the Ras converting CAAX endopeptidase 1 (RCE1) to remove the AAX amino acids; the Isoprenylcysteine Carboxyl Methyltransferase (ICMT) to methyl esterifies the carboxyl group of the farnesyl cysteine. Currently most studies in this area are focused on the farnesyltransferase enzyme. Since 1990s, preclinical studies have shown that FTIs are able to inhibit tumor growth in different tumor cell lines and xenograft mouse models [32].

Tipffarnib and Lonafarnib are two FTIs that are in Phase II and Phase III clinical trials (Table 1). An interesting observation is that FTIs showed better effects in HRas-driven cancer cells rather than KRas-driven cancer cells [33]. Some studies indicated that an alternative enzyme exists in KRas driven cancer cells for CAAX prenylation, namely GGTase [34]. Thus in such patients, it may be more effective to combine GGTIs with FTIs for therapy [35]. It is an unfortunate that few FTIs have shown promising therapeutic effect in most of the clinical trials


[36]. Further studies identified that the inhibition of tumor growth effect induced by FTIs in some cases does not depend on the Ras mutation status, suggesting that the possibility of other targets for these FTIs in tumor cells and human organisms. Identification of these potential targets may help understand the complex mechanisms of how the FTIs inhibit the tumor growth. Another challenge hampering the usage of FTIs in clinic is lacking of appropriate biomarkers to measure patients’ response to the FTase activity. Therefore, discovery of these biomarkers will also be an urgent task in the development of FTIs to treat cancer patients harboring Ras mutations. The Ras proteins can also be dislodged from the cell membrane using farnesylcysterine mimetics which can compete with Ras for binding to the Ras escort proteins galectin 1 and galectin 3. Rotblat and co-worker reported that Salirasib is able to prevent the interaction between galectin 1 and HRas-GTP [37]. In the same study, the authors demonstrated the small molecule inhibits the Ras transformation both in vitro and in vivo. However, a later study showed that this compound does not induce partial or complete responses in patients with KRas mutations [38]. However, some recent studies reported that LIMK1/2 inhibitors synergize the activity of Salirasib, suggesting that synthetic lethality may be a promising strategy for further studies in Ras mutant-driven cancers [39] which will be discussed later in this review. In 2013, Zimmermann et al. reported a novel approach to interfere with Ras localization through disruption of interactions between KRas and Prenyl-binding protein (PDEδ) [40]. PDEδ is responsible for maintaining the correct organization of KRas4B. Small molecules (Table 1) were identified to bind the farnesyl-binding pocket of PDEδ. In the same study, the compounds inhibit the downstream signaling of Ras in a dose dependent manner, and slowed down the transformation of KRas-driven tumor cells and tumor growth in xenograft mouse models. This suggests that the farnesyl binding pocket in PDEδ protein may be a promising target for mutant KRas-driven cancer treatment. TARGETING SYNTHETIC LETHAL COMPONENTS Synthetic lethality is a concept first coined by Theodore Dobzhansky in Drosophila in 1946 [41], which refers to the phenomenon that two non-lethal simultaneous mutations can lead to cell death. For a long time since it was realized to be challenging to directly target Ras, scientists began to change the therapeutic strategies for mutant Ras-driven cancers. Synthetic lethality is one of these new approaches. Many groups have been trying to identify synthetic lethal components in Ras mutated cancers using RNAi high throughput screening. In 2009 Luo et al. identified PLK1, a gene aberrantly up-regulated in the mutant KRas cells, as a synthetic lethal target in the KRas mutant driven colorectal cancer. Later they developed an inhibitor of PLK1and showed that the inhibition slowed down the tumor growth and improved the survival rate of mice with mutant KRas [42]. Unfortunately, this inhibitor failed in Phase II clinical trials due to lacking of enough efficacies.


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TARGETING RAS-GEF INTERACTIONS Due to the high binding affinity of GDP/GTP to Ras, Ras-specific GEFs, which contain a Cdc25-homoogy catalytic domain and a Ras exchanger motif (REM), are required in the process of GDP release [43]. In humans, there are three major families of GEFs: Ras guanine nucleotide releasing factor (RasGRF), Ras guanine nucleotide releasing protein (RasGRP) and Son-of-Sevenless (Sos) proteins. Among these different types of GEFs, RasGRP and RasGRF are tissue-specifically expressed while Sos proteins are universally expressed in humans [44]. During the reaction, a motif of Cdc25 domain is inserted into the Mg2+ binding site which is located between switch I and switch II. As a consequence, the nucleotide binding pocket is open and allows GDP to release into cytoplasm [45]. Then GTP enters the binding pocket of Ras and activates the protein. Though serving similar functions, the regulations of different GEFs vary. Sos requires allosteric activation through a bridge between REM and Cdc25 domain [46]. However, Cdc25 domain of RasGRF1 does not require this activation [47]. The structural basis leading to the different Cdc25 activation regulations is yet to be clear. A fragment-based screening identified a series of compounds (Table 1) which block the binding between KRas and Sos [18b]. Crystal structures show that the compounds bind to a hydrophobic pocket which is occupied by Y71 of Sos. Upon optimization, the analogues are able to inhibit nucleotide exchange up to 70-80% at a concentration of 1mM. Other groups also reported compounds that inhibit the Sos-mediated nucleotide exchange [48]. Moreover, a detailed kinetics analysis was performed using HRas as a model, and it helped identification of the novel binding pockets of Ras-Sos interaction [49]. Besides Sos, other Rasspecific GEFs are also considered as potential drug targets. For example, RasGRP1, another Ras-specific GEF, has been reported to be involved in squamous cell carcinoma and melanoma [50]. A structural analysis of this protein was conducted and the detailed mechanism of RasGRP1 activation is clarified [51]. Unlike Sos, RasGRP1 is autoinhibited by a Cdc25-EF linker and activated through recruitment to the cell membrane by the C1 domain of RasGRP1. As more and more understanding of this protein is obtained, it becomes possible to identify small molecules targeting RasGRP1. TARGETING RAS-EFFECTOR INTERACTIONS Once mutations occur, Ras proteins become constitutively active and relay the signal to downstream pathways through binding to different effectors including PI3K and Raf. These interactions, as partially illustrated in (Fig. 3), have been reported in many studies and they are required in tumor development and progression [52]. Biological studies also demonstrated that disruption of these interactions are able to prevent Ras dependent tumor initiation [53] and induce tumor regression [52c]. Among these, the Ras-Raf interaction (Fig. 3) is considered as the most promising for small molecule development. Sulindac and its derivatives (Table 1) were discovered in 2004 and they are able to disrupt Ras-Raf interaction in concentration from 30-200µM,

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and some of these compounds also showed inhibition of MAPK phosphorylation [54]. The Ras-Raf interaction was also reported to be disrupted by MCP and derivatives [55]. According to this study, MCP can also revert Ras-dependent transformation phenotypes. Intriguingly, another non-stroidal anti-inflammatory drug, called NS398, is also able to block Ras-cRaf interaction [56]. This compound impairs the recruitment of c-Raf to the cell membrane and disrupts the Ras/c-Raf interaction, but not directly inhibit c-Raf or other Ras downstream enzymes, according to the in vitro kinase assays. As a consequence, it increases the expression levels of multiple MAPK, which are the downstream kinases of the Ras-Raf pathway.

Tan and Zhang

computer-assisted similarity search, the authors found another compound, named Kobe2062, which is active to inhibit Ras/effector interactions. Moreover, these two compounds showed interference of binding of Ras to other effectors including RalGDS and PI3K. They demonstrated inhibition of proliferation in HRas G12V cancer cells and slowed down the tumor growth in xenograft mouse models of SW480 cells as well. Recently we embarked on the study of T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1) as part of our effort of targeting the pleckstrin homology domain (PH domain) proteins for therapeutics development. TIAM1 was first mentioned in an in vitro screening in 1994 and it was reported to induce invasiveness phenotype of Tlymphoma cells [58]. It is a protein with a length of 1,591 amino acids, consisting of two PH domains (one N-terminal PH domain and one C-terminal PH domain), a PDZ domain, and a Ras-binding domain. Through binding to Ras, TIAM1 is a GEF protein and activates Rac1 [59]. Thereby, TIAM1 is a direct effector of Ras and relays the stimulated signal to downstream pathways through activation of Rac1. Intriguingly, TIAM1 was frequently reported, especially in recent years, overexpressed in multiple cancers, suggesting its critical role in tumor development and metastasis [60]. It was also reported to be a prognostic marker of cancer patients [61]. Moreover, inhibition of TIAM1 using siRNA or microRNA has shown encouraging results by many groups [62]. Also, TIAM1 siRNA showed synergistic inhibition effect of tumor growth when in combination with sorafenib [63]. Additionally, several compounds have been identified to interfere with the binding of TIAM1 to Rac1 [64], and thus inhibiting tumor cell growth in cell lines, but only with moderate activities [64d, 65]. These discoveries highly suggest that TIAM1 is an important effector of Ras oncoproteins and may be a promising target for cancer therapy. In recent years our group has conducted intensive studies of this protein. In particular, we developed a novel approach by targeting the C-terminal PH domain of TIAM1 and identified a set of diverse small molecule inhibitors. These inhibitors demonstrated promising activities in vitro and exhibited significant anti-tumor and anti-metastasis effects in animal studies (unpublished results). Our strategies of targeting PH domains for cancer therapeutics development have been intensively discussed in our previous publications [66]. TARGETING RAS DIMERIZATION AND NANOCLUSTER

Fig. (3). Top: Interactions between Ras and its effectors (adapted from [1]); Bottom: Zoomed-in view of interaction between Ras and Raf (PDB 4G0N), and the magenta represents the interacting residues.

In 2012, a group from Japan screened 40,000 compounds in silico targeting a surface pocket of MRas and identified a compound, Kobe0065 (Table 1), that inhibits MRas interactions with its partnering proteins [57]. Using a

Ras dimers were first mentioned in 1988 [67]. By using radiation inactivation techniques, the authors indicated the existence of Ras oligomers, more likely dimers, in cells. However, no more evidence was reported to manifest the significance and function of Ras dimers for more than a decade since then. Until 2000, Inouye and his colleagues revealed the existence of Ras dimers on the cell membrane and moreover, found that the dimerization is required, although not sufficient, for Raf activation [68]. After that, by using different imaging techniques, more and more reports from different groups showed the importance of Ras dimers. Through analyzing crystal structures containing Ras


proteins, Guldenhaupt et al. uncovered that Ras forms dimers in at least 50 out of 71 structures [69]. Also, the formations of these dimers are confirmed by Forster resonance energy transfer (FRET) experiments. Moreover, the authors showed that the dimers stabilize a perpendicular orientation of Ras proteins on the cell membrane and the interface of Ras dimers is composed of a loop between β2 and β3 together with alpha-helices 4 and 5. In 2014, another group confirmed the formation of H-Ras dimers on cell membrane by using the single molecular tracking (SMT) microscopy and Photon Counting Histogram (PCH) spectroscopy techniques [70]. Also, the authors reported that the residue Y64 is critical for the maintenance of the dimerization since a mutant Y64A is able to abolish the dimer formation. Similar to HRas and NRas, it seems that KRas also form dimers on the cell membrane and it has become a new strategy of targeting Ras proteins [71]. The next important issue is to clarify whether these dimers are homodimers or heterodimers [72]. These dimers may also further form transient nanoclusters [73], which may be critical for activation of Ras-dependent signaling [74]. Other proteins may also participate in the Ras nanocluster [75]. We recently reported that knockdown of Connector Enhancer of Kinase Suppressor of Ras (CNKSR1) showed tumor growth inhibition in KRas mutant cancers cells in a siRNA screen [76]. CNKSR1 co-localizes with KRas on the cell membrane as part of the Ras nanocluster which is critical for Ras to serve its functions including recruitment of effectors to the cell membrane. In KRas mutant cancers, KRas seems to mediate the cell cycle through CNK1, but the underlying mechanism is unknown. We found that deletion of CNKSR1 is able to arrest cancer cells harboring KRas mutations in the G1 phase of the cell cycles. At this moment multiple compounds targeting the PH domain of CNKSR1 have been identified to interfere with the membrane localization of the protein and they exhibited promising inhibitory effect on tumor cell proliferation. More studies are undergoing to investigate the activities of these compounds in animal studies. Additionally, the GRB2Associated Binding Protein 1 (GAB1), a scaffolding protein known to play a critical role in recruitment of GEFs and effectors of Ras, may also be part of the Ras nanoclusters [77]. Multiple reports from different groups have confirmed that GAB1 is crucial in Ras activation and signaling [78]. Using high-throughput in silico screening, our group successfully identified a handful of small molecular inhibitors targeting the PH domain of GAB1 with promising binding affinities and cancer cell killing activities, in particular in triple negative breast cancer (TNBC) cell lines [66c]. Further in vivo study of these compounds is also ongoing. We expect that the mechanism of Ras nanocluster formation will be a critical determinant of its functions. Further efforts to study the nanoclusters through joint efforts of computational and experimental approaches will finally help reveal the structural and functional properties of Ras and this will provide mechanistic explanations on how Ras proteins are involved in signaling in both normal physiological and disease processes. This will further provide proof-of-principles to target Ras for drug discovery and development.


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FUTURE PERSPECTIVES Since Ras was realized frequently mutated in cancer patients (30% in all cancer types) [79], it has become an attractive target of drug development both in academia and industry. In the past decades, intensive studies have been conducted by many groups all over the world on this intriguing target and the mystery is being unveiled as we know more and more about this protein. With such renewed enthusiasm, the US National cancer Institute (NCI) established a Ras Mega-project in 2013, with a “simple” aim: to find therapies for patients with RAS mutations [80]. So far, several FTIs successfully entered clinical trials but eventually failed due to insufficient efficacies. Recently novel strategies have emerged. However, the major issues of the current agents that targeting Ras pathways like B-RAF are the rapid drug resistance and low efficacy [81]. One of the primary reasons leading to resistance stems from the switch role of Ras in the signaling network [81a]. The Ras signaling is more complex and redundant than it was ever expected. In addition, it is evident that knockdown of mutant Ras is not sufficient to inhibit the tumor growth [82]. As a consequence, it will be critical to distinguish the Rasdependent tumors and Ras-independent tumors. In order to achieve the expected therapeutic effect, alternative strategies, such as targeting downstream effectors or Ras nanoclusters, may be more promising in certain patient populations. Currently, with burgeoning of drug polypharmacology and multi-targeting design [83], it is possible to target multiple Ras pathway components simultaneously. We anticipate that with a concerted effort of targeting Ras, there is no doubt that many new technologies and strategies will be developed, but we should also be cautiously optimistic because targeting Ras is a tough task. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS We thank OpenEye Scientific Software and ChemAxon for providing us free academic licenses. Special thanks to the Texas Advanced Computing Center (TACC) and the University of Texas M.D. Anderson Cancer Center for HPC resources. S.Z. is also partially supported by NIH 5R01CA138702, NSF CHE-1411859, and CPRIT DP150086 and RP130389. REFERENCES [1] [2] [3] [4] [5]

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Received: ????????, 2015

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Accepted: ???????????, 2015