CXCR4 Targeted Libraries
Medicinal and Computational Chemistry Dept., ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, CA 92121 USA, Service: +1 877 ChemDiv, Tel: +1 858-794-4860, Fax: +1 858-794-4931, Email: ChemDiv@chemdiv.com
Chemokines (chemotactic/chemoattractant cytokines) are highly basic small secreted proteins consisting on average of 70-125 amino acids with molecular masses ranging from 6 to 14 kDa, which mediate their effects through binding to seven transmembrane domain (7-TMS) of the specific family of G-protein-coupled receptors (GPCR) located on target cell membrane. Initially, chemokines were recognized as chemo-attractants and activators of specific types of leucocytes in a variety of immune and inflammatory responses. Notable, over the past few years there has been a “chemokine revolution” in the realm of anti-cancer drug therapy and the great majority of authoritative scientists and clinicians in oncology-related field of science are now aware of their crucial role at all stages of neoplastic transformation and progression . Thus, tumor cells extensively express functional chemokine receptors, which can sustain proliferation, angiogenesis, survival, and promote organ specific localization of distant cancer metastases .
The chemokine receptor CXCR4 possesses multiple fundamental functions in both normal and pathologic physiology. CXCR4 is a GPCR receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1, previously SDF1-α). The interaction between CXCL12 and CXCR4 plays a critical role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), asthma and pulmonary fibrosis.
1. The pivotal role of CXCR4 chemokine receptor in cancer pathology
Unlike other chemokine receptors, CXCR4 is expressed in many normal tissues, including those of the central nervous system; while it is also commonly expressed by over 25 different tumor cells including cancers of epithelial, mesenchymal, haematopoeitic origin, etc. . For example, tumor cells from breast, prostate, pancreatic, lung and ovarian carcinomas, neuroblastoma and glioblastoma, all express CXCR4 [4-6]. This receptor was also found in acute lymphoblastic and myeloblastic leukemia, in non-Hodgkin`s lymphoma, in tumors derived from kidney, as well as in melanoma and rhabdomyosarcoma [6,7]. In other cancer cells studied, CXCR4 may be co-expressed with other CC or CXC chemokine receptors or less commonly, other receptors are present without expression of CXCR4. Several lines of evidence show that the CXCL12-CXCR4 chemokine system may also be involved in promoting tumor cell survival and growth.
For instance, in cells from adult glioblastoma and pediatric medulloblastoma, the CXCR4-CXCL12 signaling network induced chemotaxis and enhanced proliferation and survival . In several types of cancer, including glioma, melanoma, NSCLC, renal, and thyroid, CXCL12 can stimulate tumor proliferation and/or survival of CXCR4-expressing tumor cells . Production of CXCL12, both at mRNA and at protein level, has been detected in several CXCR4-expressing tumors, thus suggesting a possible autocrine or paracrine loop of growth. It was recently reported that CXCR4 is frequently expressed in human pancreatic cancer cells and that CXCL12, in addition to enhancing motility and invasion, promotes their proliferation and excites anti-apoptotic effects . Furthermore, Wang and Ma have suggested that β-catenin is a vital key intracellular factor in the CXCR4-CXCL12 axis promoting metastatic events of pancreatic cancer . It was recently reported that CXCR4 is expressed in several types of malignant brain tumors . Human breast cancer cells express CXCR4 and CCR7 . Notably, CXCR4 repression was found to effectively inhibit metastasis of breast cancer cells in experimental animal models . The specific ligands for these receptors CXCL12 and CCL21 are found at elevated levels in lymph nodes, lung, liver and bone marrow, organs to which breast tumors generally metastasize. In addition, as communicated in several previous studies, melanoma cells generally express CCR7 and CCR10 chemokine receptors , they also significantly co-express CXCR4 and CXCR3 which play a critical role in tumor growth, metastasis and tissue invasion . Leukemic and lymphoma cells also express a wide variety of chemokine receptors, including CXCR4. Recently, CXCR4 have reported to be a key effector in the formation of peritoneal carcinomatosis in gastric cancer in proliferation, migration and invasion of epithelial ovarian cancer cells [15,16]. Furthermore, there is growing in vitro and in vivo evidence that CXCR4 expression by leukemia cells allows for homing and their retention within the marrow. As such, leukemia cells appear to utilize CXCR4 to access niches that are normally restricted to progenitor cells, and thereby reside in a microenvironment that favors their growth and survival . It was also recently reported that CXCR4-CXCL12 signaling activates phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) and stimulates meningioma cell proliferation . Considering the really high diversity and infinite complexity of chemokine related signal propagation we discuss here the basic features of chemokine CXCR4-CXCL12 signaling network and disclose the main principles of how this pair works in the field of cancer growth and progression.
It should be noted that CXCR4 is not a tumor specific marker and not all cancers express this receptor. Both receptor and its specific ligand are widely expressed in normal tissues and play a fundamental role in a variety of physiological processes including fetal development, joint mobilization of haemopoietic stem cells and specific trafficking of a majority of leukocyte types [19,20].
2. Signaling via CXCR4-CXCL12 chemokine system
The chemokine receptor CXCR4 belongs to the large superfamily of GPCR receptors, and is directly involved in a number of biological processes including organogenesis, hematopoiesis, and immune response. Several recent reports highlight the high complexity of intra/extracellular signal transduction initiated by chemokine receptors, especially by CXCR4 [21,22]. In general, chemokines activate 7-TM GPCR chemokine receptors which are coupled to heterotrimeric Gαβγ protein subunit. Heterotrimeric Gproteins associate with the intracellular domains of GPCRs when in their inactive, or guanosine diphosphate (GDP)-bound, state. Upon chemokine ligand binding, the GDP is readily exchanged for guanosine triphosphate (GTP) resulting in instantaneous activation of corresponding G-protein. The active G-protein subsequently initiates dissociation of Gαβγ into its Gα and Gβγ subunits to stimulate many intracellular mediators. In contrast to other chemokine receptors, stimulation of CXCR4 can lead to prolonged activation of both mentioned subunits . Signaling via CXCR4 also enhances tyrosine phosphorylation, association of components of focal adhesion complexes such as paxillin and NF-κB activity in nuclear extracts . In a metastatic breast cancer cell line, NF-κB directly regulates the CXCR4 promoter and can upregulate expression of CXCR4, facilitating increased responses to CXCL12 . In breast cancer cell lines, CXCL12 also induces phosphorylation of FAK, Pyk2, the cytoskeletal proteins paxillin and Crk, the tyrosine phosphatase SHP2 and the adaptor protein Cbl . There is one interesting report of cross talk between the BCR/ABL oncogenic tyrosine kinase and CXCR4 signalling . In CML, BCR/ABL kinase phosphorylates, activates and disregulates proliferation and survival pathways of progenitor cells in the bone marrow. Immature leukemic cells leave the marrow and are found in large numbers in the blood and spleen. BCR/ABL strongly activates a CXCR4-dependent signaling component through the Src family tyrosine kinase, Lyn. Cross talk between BCR/ABL and CXCR4 signaling may allow the oncoprotein to couple to PI3-kinase, MAPK cascades and ‘take over’ the chemokine pathway. This could lead to disruption of chemotaxis and hence release of the transformed cells into the periphery. Undoubtly, the precise signaling mechanisms by which all chemokine receptors regulate cellular function will become evident in time, but it has proven to be difficult to link specific CCR or CXCR signaling pathways right through to a biological response such as chemotaxis, cell growth and progression.
3. The complicity of CXCR4 in cancer metastasis, angiogenesis, adhesion and invasion
Cancer metastasis results from a non-random process, in which organ selectivity by the tumor cells is highly dependent on interactions between tumor and host stromal cells as well as between cancer cell and several essential molecular factors expressed at the remote organs that eventually turn into preferred sites of metastasis formation [28,29]. In aggregate, these factors support the consecutive steps required for metastasis formation, including tumor cell adhesion to microvessel walls, extravasation into target tissue and migration. For instance, chemokine CXCL12, lysophosphatidic acid (LPA) and thrombin promote the migration and invasion of cancer cells through their cognate receptors, CXCR4, LPA1 and PAR1, respectively, enabling the cancer cells to escape from the location of the primary tumor . Notable, metastasis is a major cause of morbidity and mortality in breast cancer patients. To prevent these lethal outcomes, improved strategies to treat metastatic neoplasms are strongly needed. Blood flow and other mechanical factors influence the delivery of cancer cells to specific organs, whereas molecular interactions between the cancer cells and the new organ influence the probability that the cells will grow there. Inhibition of the growth of metastases in secondary sites offers a promising approach for effective cancer therapy. Metastases arise following the spread of cancer from a primary site and the formation of new tumor nidus in distant organs. When cancer is detected at an early stage, before it has spread, it can often be treated successfully by small molecule inhibitors of tumor growth or surgery/local irradiation, and the patient will be cured. However, when cancer is detected after it is known to have metastasized, treatments are much less successful.
Many chemokines play multiple roles in tumor growth, invasion and metastasis by inducing cellular transformation, angiogenesis, secretion of proteinases, and organ specific metastasis . As mentioned above, chemokines control the directional migration of leukocytes and it seems that mechanisms utilized for leukocyte trafficking may also be used by tumor cells. Studies on the contribution of chemokine receptors to organ specific metastasis are providing important clues about why some cancers metastasize to specific organs. Recent elegant studies have shown that tumor cells express patterns of chemokine receptors, including CXCR4, that ‘match’ chemokines that are specifically expressed in organs to which these cancers commonly metastasize .
Cancer cells migrate towards the chemoattractant gradient until reaching the site for secondary colonization. For chemokine receptor expression by a cancer cell to be advantageous a chemokine gradient is required/needs to be established and in breast, prostate and ovarian cancer, neuroblastoma, melanoma and some forms of leukemia, the respective ligand is strongly expressed at sites of tumor spread. Tumor cell migration in response to CXCR4 stimulation requires the polarization of intracellular signaling molecules that results in a leading edge that protrudes outward, coupled with contractile forces at the back and sides of the cell to propel the cell towards a chemoattractant. For example, melanoma cells express functional CCR7, CCR10 (and lower levels of CXCR4, not shown) (Fig. (1)). Melanoma cells also express specific ligands for these receptors at the two major sites of metastasis, skin and lymph nodes . Both breast cancer cells and primary breast tumors were found to express the chemokine receptors CXCR4 and CCR7 at high levels. The specific soluble ligands for these receptors CXCL12 and CCL21 are found at elevated levels in lymph nodes, lung, liver and bone marrow—organs to which breast tumors often metastasize—whereas skin tissue expresses high levels of CCL27, a soluble ligand for the CCR10 receptor [33,34]. Therefore, breast cancer cells that are taken to the lung by the blood flow would find a strong chemokine-receptor ‘match’, which would lead to chemokine mediated signal activation. By contrast, breast cancer cells taken to skin would not find such a match. Melanoma cells, however, taken to skin by the circulation (or by local invasion) would find a CCL27-CCR10 chemokine-receptor ‘match’ that would lead to the activation of chemokine-mediated pathways. These results found further support in experimental tumor models: transduction of tumor cells with CCR7 conferred improved ability to metastasize to regional lymph nodes , while CXCR4-transfected cells preferentially migrated to the lung .
Cyclooxygenase 2 (COX2) expressed in tumor and stromal cells generates prostaglandin E2 (PGE2), which binds to EP2 (pro-angiogenic factor) receptors on cancer cells and promotes tumor cell proliferation and extracellular matrix (ECM) degradation through the expression of matrix metalloproteinase 2 (MMP2) and MMP9 , a response also elicited by thrombin and CXCL12. Stimulation of mentioned GPCR receptors (CXCR4, LPA1, PAR1 and EP2) also causes increased release of vascular endothelial growth factor (VEGF), thereby promoting vascular permeability, which is important for tumor cell extravasation and tumor angiogenesis. Specifically for solid tumors, as they grow, the hypoxic condition in the tumor microenvironment results in the stabilization of hypoxiainducible factor-1 (HIF1), which upregulates CXCL12 and VEGF. Cancer cells also produce several CC and CXC chemokines, such as CCL2, CCL5, CXCL8 (interleukin 8 (IL8)) to recruit tumor associated macrophages (TAMs) and leukocytes to the tumor. These immune cells then help to promote blood vessel growth by releasing VEGF and other angiogenic factors (AF). Concomitantly, tumor or stromal inflammatory mediators that act on GPCRs, such as IL8, prostaglandin E2 (PGE2) and sphingosine-1phosphate (S1P), can also regulate the activity of MMPs that degrade the ECM, which clears a path, at the same time as endothelial cell chemotaxis, often involving the coordinated activation of a network of small GTPases such as Rho and Rac and their downstream targets by Gα13 or Gβγ when released from Gαi, paves the way for new blood vessel growth. Finally, S1P is released following the activation of sphingosine kinase activity, and functions in an autocrine and paracrine manner to cause tumor and endothelial cell proliferation and migration. Inflammatory cytokines that accumulate in the tumor milieu also stimulate the nuclear factor B (NFB)-dependent increased expression and release of IL8 from stromal and cancer cells, which promotes endothelial cell migration towards the growing tumor. Ultimately, proangiogenic GPCRs activate a network of small GTPases, Akt, and mitogen-activated protein kinase (MAPK) signaling that promotes the migration, survival and growth of endothelial cells. Several other important mediators are also implicated in cancer growth, metastasis, invasion and angiogenesis, these include HIF1α, hypoxia-inducible factor-1; IL8, interleukin 8; NFB, nuclear factor B, etc.
Clinical importance and therapeutic implications of the pivotal CXCL12-CXCR4 interaction in cancer cell migration was recently exhaustively discussed by Arya et al . In most in vitro studies with CXCR4 expressing cancer cell lines, activation of the receptor with CXCL12 stimulates specific migration of cancer cells or invasion through matrigel or through monolayers of endothelial cells, fibroblasts or bone marrow stromal cells . Furthermore, blocking CXCR4 was found to inhibit metastasis of breast cancer cells in experimental animal models. Thus, breast cancer cells migrated towards tissue extracts from these target organs and chemotaxis could be partially abrogated by neutralizing antibodies to CXCR4. Interestingly, recent study clearly showed that breast cancer metastized to the brain and it was found that ErbB2 overexpression is strongly associated with the brain metastatic phenotype . Other factors, including VEGF, MMPs and CXCR4 are also involved in the metastasis of breast cancer cell to the brain. It was additionally found that CXCR4 antagonists block both growth of primary tumor and organ-specific metastasis of head and neck cancer in xenograft mouse models . Thus, using the orthotopic SCCHN animal model, it was showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Furthermore, collected data from 600 prostate cancer patients revealed that CXCR4 protein expression was significantly elevated in localized and metastatic prostate cancer compared to normal or benign prostate tissue and CXCL12 protein levels were higher in metastatic, compared to normal, prostate tissue .
Overall these results support the concept that chemokines could direct tumor cell migration in vivo:
malignant cells bearing chemokine receptors on the cell surface would be endowed with the capability to respond to chemokine gradient and selectively migrate to specific organs where the chemokine is present. However, the process of metastasis is complex multi-step process and there are several stages at which the interaction between tumor cell chemokine receptors and their ligands could be important. It was suggested that, in addition to tumor cell movement to a gradient, chemokines play a critical role in tumor cell adhesion, invasion, survival, growth and angiogenesis.
As mentioned above, the chemokine signaling pathway via chemokine receptors can modulate many intracellular functions including expression of integrins by tumor cell which can then facilitate adhesion of cancer cells to and/or invasion through the extracellular matrix. Thus, it was demonstrated that CXCL12 stimulation of different ovarian cancer cell lines upregulates the expression of β1 integrin . For one’s turn integrin modulation correlates with highly increased tumor cell adhesion. This migration can be abrogated by a broad spectrum of MMP inhibitors . Additionally, β1 integrins have also been reported to regulate both the formation of and adhesion within, ovarian cancer spheroids . In small cell lung cancer cells (SCLC) CXCL12 stimulation induced firm adhesion to marrow stromal cells via activation of α4β1 integrin and also induced SCLC cell invasion into the extracellular matrix . In lymphocytes, the chemokines CXCL12 and CCL21 activate adhesion, mediated by LFA-1 and VLA-4, and also transendothelial migration where the small GTPase, RAP1, serves to increase the adhesive capacity of these adhesion molecules . Adhesion mechanisms can also impact on chemokine receptor expression, e.g. in non-transformed lymphocytes, activation of L-selectin (by antibody crosslinking or specific ligands) mobilizes intracellular stores of CXCR4 to increase cell surface expression .
It has long been known that chemokines induce production of proteases, such as matrix metalloproteases and urokinase-type plasminogen activator (u-PA) in tumor cells and TAM. Tumor-derived proteases can cleave the extra-cellular matrix molecules and lead to the dissolution of the basement membrane. Thus, they are important for invasion and it has been suggested that monocytes infiltrating the tumor tissue provide cancer cells with a ready-made path for invasion (countercurrent invasion theory) . A variety of proteolytic enzymes, in particular the tissue type plasminogen activator (t-PA), u-PA and the large family of matrix-metalloproteinases (MMPs) have been implicated in this degradation . The activity of these enzymes has been associated with more aggressive neoplastic behavior. For example, t-PA and u-PA and their respective receptors, annexin II and u-PAR, were demonstrated to contribute to the invasive behavior of pancreatic cancer . MMP-2 expression is increased in several tumors and strongly correlates with nodal status and tumor stage . Chemokines are potent inducers of enzymes and receptors which degrade the extracellular matrix and favor tumor invasion. In a gene expression analysis, the chemokine CCL5 specifically induced gene expression of various MMPs, especially MMP9, along with the u-PA receptor . Macrophages can produce proteases and strong evidence demonstrates that chemokines activate TAM to release MMPs in the tumor microenvironment (Fig. (1)). In particular, MMP9 derived from hematopoietic cells of host origin, has been shown to contribute to skin carcinogenesis. In addition, MMP9 has complex effects beyond matrix degradation, including promotion of angiogenesis and release of growth factors . Moreover, several chemokines and chemokine receptors, like CXCL16-CXCR6 and more common CCR5 and CXCL12CXCR4, connected to CD4+ T-cells were reported to enhance invasion and disease progression in an experimental model of skin carcinogenesis . Even if CXCL12 is a non-ELR chemokine, its activity has been implicated in neo-angiogenesis . There are also links between CXCR4 and vascular endothelial growth factor (VEGF). Thus, it was recently shown that vascularization of the gastrointestinal tract is defective in mice lacking either CXCR4 or its ligand, CXCL12 . In breast cancer cell lines, VEGF was demonstrated to have an autocrine action and induce expression of CXCR4 which promoted migration and invasion towards CXCL12 . Several other chemokines and chemokine receptors are strongly associated with key stages of tumor growth and progression, angiogenesis and metastasis, invasion and adhesion. Some of them were found to regulate the activity of many different cellular factors. For example, it was recently found that chemokines regulate MEK1/2 and AKT-related intracellular pathways play a critical role in cholangiocarcinoma cell invasion . As reported by Yang et al, inhibition of
CXCR4-mediated cyclic AMP suppression effectively suppress brain tumor growth in vivo .
4. Antagonists of CXCR4 chemokine receptor
4.1. Peptide-based inhibitors of CXCR4 activity
Undoubtedly, CXCR4 is the most actively studied chemokine receptor implicated in a wide variety
of critical vital intra/extracellular signaling pathways in normal and pathologic physiology. Basically, the CXCR4-CXCL12 signaling network was found to strongly regulate tumor cells migration in mammals and organ-specific metastatic events in different in vivo models. In addition, this ligand-receptor system manipulate cancer growth and progression, angiogenesis, adhesion and tissue invasion. For instance, interaction between CXCL12 and CXCR4 plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This specific acting was also found to be involved in number intractable diseases, including HIV infection, rheumatoid arthritis and pulmonary fibrosis. It was conjectured that this interaction may be a critical therapeutic target in numerous pathologic conditions, therefore CXCR4 antagonists have been proposed as potential drug candidates. It`s patently obvious that these findings have exciting implications in the field of cancer therapeutics, with several peptide-based and small molecule CXCR4 antagonists having been developed, which may provide clinical benefit in the therapy of cancer pathology.
The majority of CXCR4 antagonists which entered in preclinical or clinical trials are the peptide-based agents (Fig. (2)). They represent promising lead-like compounds and drug candidates mainly targeted for the treatment of HIV infection, but there are the strong evidences that they also can be effectively used in anticancer drug therapy. It was recently shown, that immunodeficient mice inoculated with CXCR4-positive human NSCLC had significantly lower organ-specific metastasis while they were injected by antibodies against CXCL12 in non-small-cell lung cancer in vivo models [61-63]. Several CXCR4 inhibitors and/or CXCL12 blockers were found to delay tumor growth and reduced tumor mass in NOD/SCID mice infested by non-Hodgkin’s lymphoma . It was also shown that in vivo neutralizing the interactions of CXCL12-CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung [6,13]. CXCR4 was also found to be highly expressed in several types of malignant brain tumors, including malignant melanoma [12,65].
Several 14-mer peptides, T-140 [[L-3-(2-naphthyl)alanine3]-T134] (1) and its structural analogues TN-14003 (2), 4F-Benzoyl-TN-14003 (3), 4F-Benzoyl-TE-14011 (4), Ac-TE-14011 (5) (Fig. (2)) were previously developed as specific CXCR4 antagonists. These compounds were initially identified as potential HIV-entry inhibitors, anti-RA and anticancer agents, particularly against chronic lymphocytic/acute lymphoblastic leukemia [66-68]. Whole series of T-140 analogues represent peptide-based antagonists composed of 14 amino acid residues acting against HIV infection through its co-receptor, CXCR4. As mentioned above, CXCR4 and its endogenous ligand CXCL12 have also been recognized to be involved in organ-specific metastasis in several types of cancers. For example, T-140 and its structurally related analogues were recently identified as anti-metastatic and anti-apoptotic agents targeted for the treatment of chronic lymphocytic leukemia and breast cancer [66,68-70]. These compounds effectively inhibited CXCL12-induced migration of human breast cancer cells (MDA-MB231), human leukemia T-cells (Sup-T1) and human umbilical vein endothelial cells at concentrations of 10-100 nM in vitro . Slow release administration of a highly potent and biostable T-140 analog, 4Fbenzoyl-TN-14003, via subcutaneous injection, led to partial, but statistically significant repression of pulmonary metastasis of MDA-MB-231 cells in SCID mice, even though no attempt was made to inhibit other potential targets such as CCR7. These results demonstrate that T-140 analogs can be efficiently utilized for effective anticancer therapy as anti-metastatic agents. Furthermore, several highly potent small molecule antagonists of CXCR4 were recently developed based on topological pharmacophore of T140 analogs .
Based on knowledge of T-140 3D-pharmacophore, several other peptide-based analogues were also developed and tested for their in vitro activity against several tumor cultures, such as lymphoblastic leukemia cells . It was recently shown, that growth and viability of chronic lymphocytic leukemia (CLL) B-cells are favored by interactions between CLL and non-tumoral accessory cells. In turn, CLL cells were also found to express a high level of CXCR4 chemokine receptor that manage leukemia cell chemotaxis and metastasis . Marrow stromal cells or nurse-like cells constitutively secrete CXCL12 thereby attracting and rescuing CLL B-cells from apoptosis in a contact-dependent fashion. It was recently reported, that CXCR4-specific antagonists (T-140 (1), TN-14003 (2) and TC-14012) strongly inhibit CXCR4-CXCL12 signaling cascades in CLL cells . Thus, T-140 and its analogues strongly inhibit chemotaxis and migration of CLL cells beneath stromal cells as well as actin polymerization. It was found that CXCL12-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), specific signal transducers and intracellular activator of transcription-3 (STAT-3) could be dramatically abolished by T-140 related CXCR4 antagonists. Furthermore, TN-14003 (2) and TC-14012 effectively block the anti-apoptotic effect of CXCL12 and stromal cell-mediated protection of CLL cells from spontaneous cellular apoptosis. As a result, treatment with CXCR4 antagonists resensitized CLL cells cultured with stromal cells to fludarabine-induced apoptosis. These findings have demonstrated that CXCR4 antagonists effectively inhibit CXCL12-induced cell migration, CXCR4-CXCL12 signaling pathway and stromal protection of CLL cells from spontaneous or fludarabine-induced apoptosis. Therefore, the CXCR4CXCL12 signaling system represents a potential and surely attractive biological target in CLL drug therapy, and it becomes clear, that small molecule CXCR4 antagonists may have hopeful activity in the treatment of patients suffering this disease.
As recently reported by Mori et al , several CXCR4 antagonists can effectively inhibit CXCL12-induced migration and tissue invasion of human pancreatic cancer cells. The authors have primarily investigated the role of CXCL12-CXCR4 network in the pancreatic cancer metastasis via cell migration and invasion, and the inhibitory effect of novel highly potent CXCR4 antagonist, TN-14003 (3), on pancreatic cancer cell metastasis. The overall expression of CXCR4 receptors was tentatively detected in six pancreatic cancer cell lines using Western blotting and immunocytochemistry assays. It was found that CXCL12 stimulated both migration and invasion of cancer cells in a dose-dependent manner. The pernicious effect of CXCL12-induced cancer metastasis was observed at the ligand concentrations of ~100 ng/ml. Observed effect was completely blocked by TN-14003 at nanomolar concentration (~100 nM). The stimulatory effect of CXCL12 on cancer cell migration and the inhibitory action of TN-14003 were mediated via the alteration in phosphorylation of MAPK kinases in the same way as T-140 related compounds repress the growth and migration of CLL cells. Thus, actin polymerization initiated by CXCL12 (100 ng/ml) resulted in significant increase of cancer metastatic events, which could be effectively reduced by TN-14003 (100 nM). Interestingly, CXCL12 enhanced cancer cell adhesion to laminin was not reversed by TN-14003. Based on these results, it was concluded that CXCL12-CXCR4 signaling pathway is involved deeply in pancreatic cancer metastasis through migration and invasion; therefore, the small molecule antagonists of CXCR4 receptor designed based on TN-14003 topological organization might be effective anti-metastatic agents targeted against pancreatic cancer.
Pharmacophore identification of specific CXCR4 inhibitor, T-140, contribute to development of novel effective anti-HIV agents with very high selectivity indexes . A polyphemusin peptide analogue, T-22 ([Tyr(5,12), Lys7]-polyphemusin II), and its shortened potent analogue, T-134 (des-[Cys(8,13), Tyr(9,12)]-[D-Lys10, Pro11, L-citrulline16]-T22 without C-terminal amide bond) have demonstrated specific binding ability and high activity toward CXCR4 chemokine receptor. Although these compounds were initially identified as anti-HIV agents, they also have been tested for their inhibitory potency against several cancer types in vitro. For instance, inhibition of CXCR4 activity by peptide antagonist, T-22, blocks metastatic implantation of CXCR4-transduced B16 (CXCR4-luc-B16) melanoma cells in lung . Thus, in vitro, CXCR4 inhibition caused by T-22 renders B16 cells susceptible to killing by antigenspecific T-cells. In vivo, T-22 synergizes with cyclophosphamide or anti-CTLA4 mAb in the treatment of established lung metastases, suggesting a novel strategy for augmenting the efficacy of immunotherapy.
Several novel highly potent inhibitors of CXCR4 receptor with promising pharmacokinetic profile were recently designed based on the naturally occurring β-hairpin peptide polyphemusin-II and optimized using classical medicinal chemistry approach . The design method involved incorporating important residues from polyphemusin II into a macrocyclic template-bound beta-hairpin mimetic. The potency and ADME properties of the tested peptidomimetics were optimized in iterative cycles using a parallel synthesis technique, resulting in the CXCR4 inhibitors POL-2438 (6) and POL-3026 (7) (Fig. (6)). Initially, the inhibitory abilities of these compounds were unambiguously confirmed in vitro in a series of HIV-1 invasion biological assays. Thus, POL-3026 showed excellent plasma stability, high selectivity for CXCR4 chemokine receptor, favorable pharmacokinetic properties in dog, therefore this compound has the sufficient potential to become a promising drug candidate for the treatment of HIV infections, cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and also can be beneficially applied in stem cell transplant therapy. Furthermore, novel small molecule CXCR4 antagonists 8 and FC131 [cyclo(D-Tyr-Arg-Arg-L-3-(2-naphthyl)alanine-Gly)] were recently found by the efficient utilization of cyclic pentapeptide libraries using a structural tuning of core tetrapeptide scaffolds. These compounds have been already tested in vitro for their activity against several tumor models [79,80]. It was also found that compound 8 is the most potent inhibitor from the synthesized pentapeptide libraries and may be particularly useful for the treatment of some cancer types.
Complex application of ligand- and mechanism-based design led to novel competitive chemokine CXCR4 antagonist, CTCE-9908 (9), promoted by Chemokine Therapeutics (Fig. (3)) . This compound represents an exciting new generation of drugs being developed that promise more targeted therapies to treat the underlying cancer while keeping healthy cells intact. CTCE-9908 is currently in phase I/II trials for the treatment of advanced breast and ovary cancers, metastatic lung cancer and metastatic prostate cancer. Furthermore, phase I development is also under way for the treatment for localized osteosarcoma and bone cancer.
CTCE-9908 directly inhibits the basic function of chemokine CXCL12-CXCR4 signaling pathway in vitro. In preclinical studies, this compound has been shown to drastically reduce cancer metastases by 50-70% and to have promising anti-angiogenic features . This compound has also been shown to dramatically inhibit spontaneous formation and progression of lung metastases by a 67% decrease in the number of visible lung nodules in mice infected by osteosarcoma . Although CTCE-9908 was primarily designed to selectively block the CXCR4 activity, there are strong evidences that this compound can also inhibit CXCR7 receptor, because chemokine ligand CXCL12 is believed to activate at least two sets of receptors, CXCR4 and CXCR7, which have been deeply implicated in cancer growth and metastasis. Of note, the results of phase I dose-escalation trial in 24 healthy patients revealed no significant toxicities associated with CTCE-9908 injections in the dosage of 0.5, 2, and 5 mg/kg of body weight. Since CTCE-9908 is not primary cytotoxic, it can be successfully utilized in combination and synergistically with chemotherapy and surgery. In 2005, the FDA assigned orphan drug designation to CTCE-9908 for the treatment of osteogenic sarcoma. Moreover, this compound is being designed to address cardiovascular and infectious diseases.
RCP-168 (10) is a novel peptide-based inhibitor of CXCR4 chemokine receptor (Fig. (3)). This compound overcomes stroma-mediated chemoresistance in chronic and acute leukemia [84-86]. As reported by Zeng etal , polypeptide RCP-168 possesses the strongest antagonistic activity against CXCL12- or stromal cell-induced chemotaxis of leukemic cells. Furthermore, RCP-168 was found to significantly reduce the binding affinity of anti-CXCR4 monoclonal antibody 12G5 to surface of CXCR4 in a concentration-dependent manner and inhibit CXCL12–induced AKT activation as well as extracellular signal-regulated kinase phosphorylation. Finally, RCP-168 greatly enhanced chemotherapy-induced apoptosis in stroma-cocultured Jurkat, primary chronic lymphocytic leukemia, and in a subset of acute myelogenous leukemia cells harboring Flt3 mutation. The same results were also obtained using the small molecule CXCR4 inhibitor AMD-3465. The combined data suggest that the CXCL12-CXCR4 interaction contributes to the resistance of leukemia cells to chemotherapy-induced apoptosis. Therefore, inhibition of these interactions by RCP-168 represents a promising and feasible strategy for targeting leukemic cells within the bone marrow microenvironment.
Protein transduction domains (PTDs), such as the TAT PTD, have been shown to deliver a wide variety of cargo in cell cultures and to treat cancer and cerebral ischemia in several preclinical models . The TAT PTD penetrate the cell membrane by a common lipid raft-dependent macropinocytosis mechanism. Consequently, PTDs resemble small-molecule therapeutics in their lack of pharmacologic tissue specificity in vivo. Two peptides, a p53-activating peptide (DV3-TATp53C’) and a cyclin-dependent kinase 2 antagonist peptide (DV3-TAT-RxL), were recently targeted against CXCR4 in multiple malignancies. Treatment of tumor cells expressing these peptides resulted in an enhancement of tumor cell-killing compared to the treatment with nontargeted parental peptides. These observations clearly show that a multi-domain approach can be effectively used to further refine and enhance the tumor selectivity of biologically active, transducible macromolecules for treatment of cancer.
4.2. Small molecule inhibitors of CXCR4 activity
A wealth of data concerning small molecule CXCR4 receptor antagonists has been generated over the last few years, as a variety of these small molecules have been tested, and the understanding of structure activity relationships has improved [88,89]. Stay aloof from a wide variety of peptide-based CXCR4 antagonists, several small molecule agents are currently in active development as promising next generation anticancer therapeutics (Fig. (4, 5)). One of the most known small molecule inhibitors of CXCR4 activity is AMD-3100 (11) (Fig. (4)) and its bismacrocyclic analogues . The bicyclam AMD3100 (originally called JM-3100) in which the two cyclam rings are connected via aromatic bridge was designed from JM-2763 and was initially identified as a promising anti-HIV agent. Now, this compound is under active development for the treatment of several cancer types.
Systemic utilization of the selective CXCR4 inhibitor AMD-3100 effectively blocked the heightened metastatic potential of CXCR4-expressing pancreatic cancer cells  and strongly inhibited growth and progression of intracranial medulloblastoma and glioblastoma and in xenograft models by increasing the cellular apoptosis and decreasing the excessive proliferation of tumor cells . Recently it was found that CXCR4 chemokine receptor is overexpressed in various glioma cell lines including glioblastoma. In cells from adult glioblastoma and pediatric medulloblastoma, CXCR4-CXCL12 signaling pathway may induce intracellular chemotaxis and enhance tumor cell proliferation, progression and survival . AMD-3100 was shown to obstinately resist these effects in vitro. If AMD-3100 was used for the treatment of mice bearing intracranial glioblastoma or medulloblastoma, tumor burden was significantly smaller in AMD-3100-treated animals. The similar effect was also obtained using the combination of AMD-3100 with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Treatment of glioblastoma multiforme cells with BCNU followed by AMD-3100 resulted in synergistic antitumor efficacy in all cells tested as well as treatment using subtherapeutic doses of BCNU in combination with AMD-3100 resulted in significant tumor regression in vivo, and this reflects both increased apoptosis and decreased proliferation following combination drug therapy .
CXCL12 factor is known to selectively increase the expression level of membrane type-2 matrix metalloproteinase (MT2-MMP), as well as against any other types, including MT-MMPs, MMP-2 or MMP-9. As communicated by Zhang et al , the CXCL12 enhanced MT2-MMP expression was effectively blocked by AMD-3100. Obtained results highlight the promising potential of AMD-3100 as an effective agent blocking the tumor tissue invasion and metastasis. Recent studies have clearly demonstrated the high ability of AMD-3100 to reduce the activation of extracellular signal-regulated kinases 1 and 2 as well as Akt kinase. These specific intracellular mediators implicated in the CXCR4related signaling downstream pathways, promote tumor cells survival, proliferation, and migration. Therefore, CXCR4 is a critical receptor promoted the progression of diverse brain malignances and provided a scientific rationale for clinical evaluation of AMD-3100 in treating both adults and children with malignant brain tumors.
It was recently identified that CXCR4 chemokine receptor is one of the key intracellular regulators promoting the growth and progression of primary melanoma . Thus, CXCL12-CXCR4 signaling system was tested towards induction of phosphorylation, proliferation, apoptosis, and migration capabilities of extracellular signal-regulated kinase-1 and -2 (Erk-1 and Erk-2). It was found that CXCL12 activated induction of both Erk-1 and Erk-2 kinases was specifically inhibited by AMD-3100 in vitro . Furthermore, AMD-3100 effectively reduced tumor growth and ascitic fluid formation in nude mice inoculated with Human gastric carcinoma cell lines (NUGC4 cells) . It was also recently shown that administration of CXCL12 and TNF-alpha increased synergistically ICC cell migration, which could be effectively suppressed by AMD-3100 . Finally, bicyclam AMD-3100 is actively pursued as a stem cell mobilizer in patients with multiple myeloma and non-Hodgkin’s lymphoma, therefore it can be effectively used in transplantation [98,99].
A series of novel highly potent inhibitors of CXCR4 activity containing the common cyclohexylamino ring as well as indole (12 and 13) and imidazole 14 fragments were recently developed by Takeda Chemical Industries as promising anticancer agents (Fig. (5)) [100,101].
Bioisosteric analogues 15-17 in which indole and imidazole fragments were replaced by phenyl and naphthalene, while carboxamide moiety was replaced by sulfonamide fragment were also synthesized and tested for their activity against several tumor cell lines .
Several novel antagonists of CXCR4 chemokine receptor were recently disclosed in patent applications. Thus, 5,6-dihydroimidazo[2,1-b][1,3]thiazoles 18-22 (Fig. (5)) developed by Novartis were described as potential compounds targeted for the treatment of transplant rejection, inflammatory and autoimmune diseases, cancer and other proliferative disorders including HIV infection .
Diimidazoles 23 and 24 (Fig. (5)) which are close topological analogs of compounds 12-17 were recently discovered by Ono corporation as promising agents for the treatment of inflammatory and immune disorders such as rheumatoid arthritis, transplant rejection, allergic diseases, HIV infection, neurological, cardiovascular and metabolic diseases . These compounds are currently entered in early phase of biological evaluation for the treatment of several cancer types. Furthermore, pyrimidine-based compounds 25 and 26 (Fig. (5)) were also recently designed by Ono and have partially described in patent application as potential antagonists for their utilization in the treatment of HIV infection, inflammatory and immune disorders such as rheumatoid arthritis, transplant rejection, allergic diseases, neurological, cardiovascular, metabolic diseases, etc. [104,105]. Thus, the exemplified compound 25 displayed an IC50 value of 1.6 nM towards the inhibition of CXCL12-CXCR4 signaling pathway in human CEM cells.
Structures of several CXCR4 antagonists are still not disclosed. These include novel small molecule inhibitors WZ-811, WZ-40 and WZ-811S which are currently entered in the early stages of preclinical studies promoted by Emory University for the treatment of different cancer types . Compound OPL-CXCL12-LPM developed by Osprey Pharmaceuticals represents a highly potent inhibitor of CXCL12-CXCR4 signaling pathway which can be effectively used for the treatment of several tumors and arthritic diseases. As communicated by the originator, this compound was extensively tested in different tissue cultures and in mouse xenograft models. Surprising, OPL-CXCL12-LPM has not exhibited detectable systemic toxicity in preliminary animal toxicology studies and the immediate goal of Osprey Pharmaceuticals is to capitalize upon proof of principle in animals and validate this compound in advanced phases of clinical trial especially for anticancer therapy. A novel small molecule antagonist of CXCR4 chemokine receptor, BKT-140, was recently developed by Biokine Therapeutics as an effective therapeutic agent. Particularly, this compound is currently entered in preclinical trials for the treatment of some cancer types .
4.3. Strategies to the rational in silico design of novel CXCR4 antagonists
Undoubtedly, high-throughput screening (HTS) of large diversity-based libraries is still a common strategy within many pharmaceutical companies for the discovery of novel chemokine receptor ligands. For example, design, synthesis, and discovery of novel CCR1, CCR4, CCR5 and CCR8 antagonists were recently described in several recent publications [107-110]. HTS technique was also successfully applied for the identification of novel small molecule antagonists of other chemokine receptors, including CXCR1 and CXCR2 . Several recent studies discussed the utilization of high-throughput technology for identification of novel highly potent antagonists of CXCR4 chemokine receptor [112,113].
Although pharmacophore hypothesis for small molecule antagonists of whole receptors belonging to CXCR family remains elusive mainly as a result of conformational flexibility inherent in the identified ligands, several specific pharmacophore models were recently described and validated. For example, pharmacophore identification of a specific CXCR4 inhibitor, T-140, led to development of novel effective anti-HIV agents with very high selectivity indexes . It was clearly identified that a common pharmacophore frame contains four indispensable amino acid residues (Arg2, Nal3, Tyr5, and Arg14). Based on this result, a series of L-citrulline (Cit)-substituted analogues of T-140 with decreased net positive charges have been synthesized and evaluated in terms of anti-HIV activity and cytotoxicity. As a result, novel effective inhibitors, TC-14003 and TC-14005, possessing higher selectivity indexes as against of T-140 have been developed. Pharmacophore hypothesis was also generated for T-140 analogue, T-22 which specifically blocks T cell-line-tropic HIV-1 infection .
A minimalistic 3D-pharmacophore model was recently developed for several other cyclopentapeptide CXCR4 antagonists . For instance, an exhaustive systematic exploration of the conformational space for a series of analogs of FC-131, a cyclopentapeptide CXCR4 antagonist, has been recently performed. By comparing the resulting low-energy conformations using different sets of atoms, specific conformational features of high/medium affinity compounds were identified. These features included the spatial arrangement of three pharmacophoric side chains as well as the orientation of a specific backbone amide bond. Together these features represent a minimalistic 3D pharmacophore model for binding of the cyclopentapeptide antagonists to CXCR4 receptor. The model enables rationalization of the experimental affinity data for this class of compounds as well as for the peptidomimetic KRH-1636.
In the last decade chemokines and chemokine receptors received a great attention as promising targets for the treatment of many solid and hematological cancers in addition to atherosclerosis, psoriasis, Alzheimer’s disease, allergy, diabetes and HIV infection, etc. In the normal tissue they play key role in several vital processes including chemotaxis of immune cells within inflammatory hearth. On the other hand, in cancer, they are implicated in a majority of pathological events controlled tumor growth and progression. Among a variety of chemokine receptors CXCR4, and its specific endogenous ligand CXCL12, were found to highly express in many types of solid tumors. This ligand-receptor pair remains one of the critical regulatory system promoting uncontrolled tumor cell growth, organ-specific metastasis, angiogenesis and tissue-invasion. Design of small molecule antagonists of tumor associated chemokine receptors, especially against CXCR4, and their clinical utilization attract growing number of industrial and academic scientists. These agents represented by derivatives of various peptide-based antagonists, including cyclic tetrapeptides and their structurally-related or topological analogues, as well as of numerous small molecule inhibitors containing different structural patterns. Biochemically, they induce growth arrest and apoptosis and/or terminal differentiation as well as the blockage of metastasis and angiogenesis in a variety of solid and hematological neoplasms in patients with advanced disease. Unfortunately, relatively modest progress in understanding pharmacology and clinical role of chemokine receptors antagonists has been made since their discovery. From this point of view, specific natural and synthetic inhibitors of chemokine activity are promising tools for dissecting role of chemokines and their receptors in both normal and aberrant biological processes. Further optimization of these molecules into clinical candidates may yield drugs with enhanced efficacy against cancers, neurodegenerative and inflammatory diseases. As briefly outlined in this paper, successful discovery of novel CXCR4 antagonists leads relies on a combination of techniques from a wide range of disciplines, including high-throughput screening, 2D/3D-pharmacophore-based design and traditional medicinal chemistry approaches.
Concept and Applications
CXCR4-targeted library design at CDL involves:
• A combined profiling methodology that provides a consensus score and decision based on various advanced computational tools:
1. Unique morphing and funneling procedures in designing novel potential CXCR4 ligands with high IP value. We apply CDL’s proprietary ChemosoftTM software and commercially available solutions from Accelrys, MOE, Daylight and other platforms.
2. Neural Network tools for target-library profiling, in particular Self-organizing Kohonen Maps, performed in SmartMining Software.
3. A molecular docking approach to focused library design.
5. Chemogenomics approach to target library design.
4. Computational-based `in silico` ADME/Tox assessment for novel compounds includes prediction of human CYP P450-mediated metabolism and toxicity as well as many pharmacokinetic parameters, such as Brain-Blood Barrier (BBB) permeability, Human Intestinal Absorption (HIA), Plasma Protein binding (PPB), Plasma half-life time (T1/2), Volume of distribution in human plasma (Vd), etc.
The fundamentals for these applications are described in a series of our recent articles on the design of exploratory small molecule chemistry for bioscreening [for related data visit ChemDiv. Inc. online source: www.chemdiv.com].
• Synthesis, biological evaluation and SAR study for the selected structures:
1. High-throughput synthesis with multiple parallel library validation. Synthetic protocols, building blocks and chemical strategies are available.
2. Library activity validation via bioscreening; SAR is implemented in the next library generation.
We practice a multi-step approach for building CXCR4-focused library:
(1) The small-molecular ligands for CXCR4 subclass (more than 400 compounds) are compiled into a unique knowledge base (reference ligand space). Among these compounds, 14 agents are currently being evaluated in advanced clinical trials or already launched in market. The knowledge base is analyzed for the pharmacophore hypotheses (Fig. 10A) and particular bioisosteric rules (Fig. 10B) used in the subsequent morphing procedures. The reference ligand space is used as a source for designing novel chemotypes via a variety of structure morphing procedures: bioisosteric, pharmacophore, 2D and 3D similarity.
(2) One of the difficult challenges in data analysis is to be able to represent whatever complexities might be intrinsic to the data in a simple and intuitive form. In order to minimize the complexity and reduce number of individual plots needed to visualize this sort of data, one must attempt to reduce the dimensionality of the representation. Several different techniques were proposed to achieve dimensionality reduction, while preserving the topology of the original space. That is, points near each other in the highdimensional space are also near each other in the low-dimensional space. Among these algorithms Kohonen-based SOM’s and Sammon maps are the most widely used techniques for dimensionality reduction with different conceptual basis.
In particular, Self-organizing Kohonen maps  belong to a class of neural networks known as competitive learning or self-organizing networks which in turn are based on unsupervised learning rule (see notes). They were originally developed to model the ability of the brain to store complex information as a reduced set of salient facts without loss of information about their interrelationships. High-dimensional data are mapped onto a two-dimensional rectangular or hexagonal lattice of neurons in such a way as to preserve the topology of the original space. This methodology has successfully been used in various medicinal chemistry applications [for review, see: 117(a-f)].
We have used this approach for compound selection and focused-library profiling. Thus, the collected training data set consisted of known 16540 drug-compounds, including 1212 CR-antagonists, was then filtered and preprocessed. Various molecular descriptors were calculated for Kohonen modeling using SmartMining software. As a result of specific selection procedure, at the output, an experimental set consisted of 7 molecular descriptors including Zagreb index, E-state indexes for the following structural fragments: >C-, -CH2-, -CH3, the number of H-bond donors, HB2 (a structural descriptor which encodes the strength of H-bond acceptors following an empirical rule) and LogP was determined. This set was then used for Kohonen map generation. The whole Self-organizing Kohonen Map of 16 thousand pharmaceutical leads and drugs generated as a result of the unsupervised learning procedure is depicted in Fig. 11.
Distribution of several GPCR-specific groups of pharmaceutical agents on the Kohonen map: (a) chemokine receptors agonists/antagonists (1,212 compounds); (b) dopamine D1-D4 agonists/antagonists (320 compounds); (c) endothelin ETA/ETB antagonists (121 compounds); (d) CCKA/B agonists/antagonists (77 compounds); (e) bradykinin agonists/antagonists (12 compounds); (f) muscarinic M1 agonists (121 compounds); (g) serotonin receptors agonists/antagonists (477 compounds); (h) tachykinin NK1/NK2 antagonists (103 compounds); (i) β-adrenoceptor agonists/antagonists (556 compounds)
Distribution of representative target-specific groups of GPCR-ligands within the Kohonen map demonstrates that most of these groups have distinct locations in specific regions of the map (Fig. 12).
As shown in Fig. 12, compounds targeted specifically on different GPCR subclasses including α/βadrenoceptors, dopamine D1-D4 receptors, tachykinin NK1/NK2, serotonin and chemokine receptors can be successfully separated within the map constructed. The distribution of six chemokine-specific groups of pharmaceutical agents within the Kohonen map is shown in Fig. 13.
We have used this model for rational compound selection. The generated virtual library has
been tested and structures were classified in different groups in accordance to the predicted activity, in particular against CXCR4 receptor (Fig. 14).
Distribution of the tested compounds (dotted line) within the Kohonen map: (a) the overlapping with GPCR agonists/antagonists area; (b) the overlapping with chemokine receptor antagonists areas; (c) the selection of compounds which can be regarded as potential agents acting against CXCR4 chemokine receptor (shaded area)
(3) A major challenge in the application of structure-based drug design methods to chemokine receptors is the paucity of structural information. Therefore, several actual studies were targeted for the chemokine receptors structure identification and binding sites determination. For instance, Wu et al  reported five independent crystal structures (3OE6) of CXCR4 bound to an antagonist small molecule IT1t (Fig. 15) and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.
A classical target-based design using molecular docking approach was recently carried out to provide a basis for the development of novel small molecule antagonists of CCR2 , CCR5 . We have effectively used these data and 3D-models listed above for generation of CXCR4-focused library and further structure optimization.
(4) During the past decade, the main paradigm in medicinal chemistry has been turning gradually from traditional receptor-specific studies and biological assays to a novel cross-receptor vision. Currently, such approach becomes increasingly applied within the whole pharmaceutical research to enhance the efficiency of modern drug discovery. Among these methods, chemogenomics comprises a special discipline targeted particularly at systematically studying the biological effect of a vast number of smallmolecular compounds on a wide spectrum of principal biological targets. This strategy is fundamentally based entirely on the paradigm originally introduced by Klabunde  who has formulated the basic principle – ‘similar receptors bind similar ligands’. So, we have used this strategy for the CR-target library design.
The chemokine superfamily includes a large number of ligands that bind to a smaller number of receptors , at that multiple chemokine ligands can bind to the same receptor and vice versa. Whereas the perceived complexity and promiscuity of receptor binding introduce an additional challenge in understanding the common mechanism of chemokine ligands binding, with respect to chemogenomics they provide a valuable starting point to investigate key interrelationships across chemokine receptor subfamily. Currently, there are more than 20 functionally signaling chemokine receptors and more than 45 corresponding chemokine ligands in humans . The chemokine ligands and receptors have been divided into several major groups based on their expression patterns and functions. Their genomic organization also provides an alternative CRs-classification. This is apparent from the phylogenetic tree presented in Fig. 16.
As shown in phylogenetic dendogram, CXCR4 is located closely to CXCR1, CXCR2 as well as CXCR3. This means primarily that these receptors possess a similar genotype and based on this observation they can be logically grouped into the common CXCR family differed genetically from CCR subclass but not significantly. Prima facie, it seems to be perfectly reasonable to investigate small molecule space around whole CXCR subclass however there are different similarity criteria which should be thoroughly considered. In addition, a binding site composition and corresponding space cavity jointly play a key role in the ligand binding process. Furthermore, the majority of ligand-receptor complexes are not static structures, they can change dynamically upon ligand binding. Thus, enforced conformational changes across the active binding site can also be achieved by ligand partial binding followed by internal cavity formation fitted appropriately for deep embedding. It is particularly interesting that there are several scientific reports highlighted the partial sequence homology (25-30%), while there is a high binding sites similarity between CCR5 and CXCR4 . In addition, a high degree of similarity was also determined for CCR5 and CCR3 . Therefore, from the chemogenomics point of view, it is of practical relevance to test the agents acting against CXCR4 also on activity towards CXCR1-3 and CCR3/5. Thus, compounds are profiled against a set of receptors and not tested against single targets. We have effectively used this modern approach and related theoretical basis for the analysis of the selected structures and their target-specific profile obtained from the biological screening outcome.
(5) A series of consecutive funneling procedures are applied to enhance the target-specific relevance of novel compounds. During this step, we address the compound’s lead-likeness (enforcing partial Rule of 3 compliance), the availability of unique R-groups, the pre-synthetic analysis of privileged templates, the IP potential, and the feasibility of high-throughput chemistry. We also consider the ADME/Tox issues (such as HIA and BBB-permeability, plasma protein binding, cytochrome P450 substrate and inhibition potential) and key physico-chemical properties (such as DMSO and water solubility, stability and ionization potential, etc.). The funneling procedures are carried out by CR-specific neural networks, fragment and property-based models. Diversity of the final selection is optimized using proprietary algorithms.
Synthesis and biological evaluation
(4) Novel CXCR4-targeted library is synthesized according to the above criteria.
(5) Compounds targeted against CXCR4 receptor are planned to be validated by bioscreening in collaboration with academic institutions.
Our strategy has proven to be efficient for generation of protein class-targeted libraries. The higher hit rate over diverse libraries, along with identification of novel active chemotypes with optimized diversity and ADME properties, has been shown in multiple studies. Using the computational approaches listed above we have compiled the focused library consisted of more than 13K small molecule compounds targeted against CXCR4 receptor. Representative set of CXCR4-biased compounds is shown below.
We provide rapid and efficient tools for follow-up chemistry on discovered hits, including single isomer chemistry, stereoselective synthesis and racemic mixture separation. Targeted library is updated quarterly based on a “cache” principle. Older scaffolds/compounds are replaced by templates resulting from our in-house development (unique chemistry, literature data, computational approaches) while the overall size of the library remains the same (ca. 12-15K compounds). As a result, the library is renewed each year, proprietary compounds comprising 50-75% of the entire set. Clients are invited to participate in the template selection process prior to launch of our synthetic effort.
1. Zlotnik, A. Int. J. Cancer, 2006, 119, 2026.
2. Ruffini, P.A.; Morandi, P.; Cabioglu, N.; Altundag, K.; Cristofanilli, M. Cancer, 2007, 109, 2392.
3. Allavena, P.; Marchesi, F.; Mantovani, A. Current Cancer Therapy Reviews, 2005, 1, 81.
4. Harvey, J.R.; Mellor, P.; Eldaly, H.; Lennard, T.W.; Kirby, J.A.; Ali, S. Clin. Cancer Res., 2007, 13, 1562.
5. Yang, Y.C.; Lee, Z.Y.; Wu, C.C.; Chen, T.C.; Chang, C.L.; Chen, C.P. Int. J. Gynecol. Cancer, 2007, 17, 676.
6. Müller, A.; Homey, B.; Soto, H.; Ge, N.; Catron, D.; Buchanan, M.E.; McClanahan, T.; Murphy, E.; Yuan, W.; Wagner, S.N.; Barrera, J.L.; Mohar, A.; Verástegui, E.; Zlotnik, A. Nature, 2001, 410, 50.
7. Balkwill, F. Nat. Rev. Cancer, 2004, 4, 540.
8. Rubin, J.B.; Kung, A.L.; Klein, R.S.; Chan, J.A.; Sun, Y.; Schmidt, K.; Kieran, M.W.; Luster, A.D.; Segal, R.A. Proc. Natl.
Acad. Sci., 2003, 100, 13513.
9. Kulbe, H.; Levinson, N.R.; Balkwill, F.; Wilson, J.L. Int. J. Dev. Biol., 2004, 48, 489.
10. Marchesi, F.; Monti, P.; Leone, E.; Zerbi, A.; Vecchi, A.; Piemonti, L.; Mantovani, A.; Allavena, P. Cancer Res., 2004, 64, 8420.
11. Wang, Z.; Ma, Q. Med. Hypotheses, 2007, 69, 816.
12. Rubin, J.B.; Kung, A.L.; Klein, R.S.; Chan, J.A.; Sun, Y.; Schmidt, K.; Kieran, M.W.; Luster, A.D.; Segal, R.A. Proc. Natl. Acad. Sci., 2003,100,13513.
13. Luker, K.E.; Luker, G.D. Cancer Lett., 2006, 238, 30.
14. Robledo, M.M.; Bartolome, R.A.; Longo, N.; Rodriguez-Frade, J.M.; Mellado, M.; Longo, I.; Van Muijen, G.N.; SanchezMateos, P.; Teixido, J. J. Biol. Chem., 2001, 276, 45098.
15. Yasumoto, K.; Koizumi, K.; Kawashima, A.; Saitoh, Y.; Arita, Y.; Shinohara, K.; Minami, T.; Nakayama, T.; Sakurai, H.; Takahashi, Y.; Yoshie, O.; Saiki, I. Cancer Res., 2006, 66, 2181.
16. Jiang, Y.P; Wu, X.H.; Xing, H.Y.; DU, X.Y. Chin. Med. J., 2007, 120, 1251.
17. Burger, J.A.; Bürkle, A. Br. J. Haematol., 2007, 137, 288.
18. Barbieri, F.; Bajetto, A.; Porcile, C.; Pattarozzi, A.; Massa, A.; Lunardi, G.; Zona, G.; Dorcaratto, A.; Ravetti, J.L.; Spaziante, R.; Schettini, G.; Florio, T. Ann. N. Y. Acad. Sci., 2006, 1090, 332.
19. Stumm, R.; Hollt, V. J. Mol. Endocrinol., 2007, 38, 377.
20. Rossi, D.; Zlotnik, A. Annu. Rev. Immunol., 2000, 18, 217.
21. Busillo, J.M.; Benovic, J.L. Biochim. Biophys. Acta., 2007, 1768, 952.
22. Johnson, Z.; Power, C.A.; Weiss, C.; Rintelen, F.; Ji, H.; Ruckle, T.; Camps, M.; Wells, T.N.C.; Schwarz, M.K.; Proudfoot, A.E.I., Rommel, C. Biochem. Soc. Trans., 2004, 32, 366.
23. Tilton, B.; Ho, L.; Oberlin, E.; Loetscher, P.; Baleux, F.; Clark-Lewis, I.; Thelen, M. J. Exp. Med., 2000, 192, 313.
24. Ganju, R.K.; Brubaker, S.A.; Meyer, J.; Dutt, P.; Yang, Y.; Qin, S.; Newman, W.; Groopman, J. E. J. Biol. Chem., 1998, 273, 23169.
25. Helbig, G.; Christopherson, K.W.2nd; Bhat-Nakshatri, P.; Kumar, S.; Kishimoto, H.; Miller, K. D.; Broxmeyer, H. E.; Nakshatri, H. J. Biol. Chem., 2003, 278, 21631.
26. Fernandis, A.Z.; Prasad, A.; Band, H.; Klosel, R.; Ganju, R.K. Oncogene, 2004, 23, 157.
27. Ptasznik, A.; Urbanowska, E.; Chinta, S.; Costa, M.A.; Katz, B.A.; Stanislaus, M.A.; Demir, G.; Linnekin, D.; Pan, Z.K.; Gewirtz, A.M. J. Exp. Med., 2002, 196, 667.
28. Zlotnik, A. Cancer Genomics Proteomics, 2007, 4, 211.
29. Ben-Baruch, A. Clin. Exp. Metastasis, 2007, in press.
30. Koizumi, K.; Hojo, S.; Akashi, T.; Yasumoto, K.; Saiki, I. Cancer Sci., 2007, 98, 1652.
31. Chambers, A.F.; Groom, A.C.; MacDonald, I.A. Nature, 2002, 2, 563.
32. Singh, S.; Sadanandam, A.; Singh, R.K. Cancer Metastasis Rev., 2007, in press.
33. Hao, L.; Zhang, C.; Qiu, Y.; Wang, L.; Luo, Y.; Jin, M.; Zhang, Y.; Guo, T.B.; Matsushima, K.; Zhang, Y. Cancer Lett., 2007, 253, 34.
34. Woo, S.U.; Bae, J.W.; Kim, C.H.; Lee, J.B.; Koo, B.W. Ann. Surg. Oncol., 2007, in press.
35. Wiley, H.E.; Gonzalez, E.B.; Maki, W.; Wu, M.T.; Hwang, S.T. J. Natl. Cancer Inst., 2001, 93, 1638.
36. Murakami, T.; Maki, W.; Cardones, A.R.; Fang, H.; Tun Kyi, A.; Nestle, F.O.; Hwang, S.T. Cancer Res., 2002, 62, 7328.
37. Karahan, N.; Guney, M.; Baspinar, S.; Oral, B.; Kapucuoglu, N.; Mungan, T. Eur. J. Gynaecol. Oncol., 2007, 28, 184.
38. Arya, M.; Ahmed, H.; Silhi, N.; Williamson, M.; Patel, H.R. Tumour Biol., 2007, 28, 123.
39. Hwang, J.H.; Chung, H.K.; Kim, D.W.; Hwang, E.S.; Suh, J.M.; Kim, H.; You, K.H.; Kwon, O.Y.; Ro, H.K.; Jo, D.Y.; Shong, M. J. Clin. Endocrinol. Metab., 2003, 88, 408.
40. Cheng, X.; Hung, M.C. Cancer Metastasis Rev., 2007, in press.
41. Yoon, Y.; Liang, Z.; Zhang, X.; Choe, M.; Zhu, A.; Cho, H.T.; Shin, D.M.; Goodman, M.M.; Chen, Z.G.; Shim, H. Cancer Res., 2007, 67, 7518.
42. Sun, Y.X.; Wang, J.; Shelburne, C.E.; Lopatin, D.E.; Chinnaiyan, A.M.; Rubin, M.A.; Pienta, K.J.; Taichman, R.S. J. Cell Biochem., 2003, 89, 462.
43. Scotton, C.J.; Wilson, J.L.; Milliken, D.; Stamp, G.; Balkwill, F.R. Cancer Res., 2001, 61, 4961.
44. Milliken, D.; Scotton, C.; Raju, S.; Balkwill, F.; Wilson, J. Clin. Cancer Res., 2002, 8, 1108.
45. Casey, R.C.; Skubitz, A.P. Clin. Exp. Metastasis, 2004, 18, 67.
46. Burger, M.; Glodek, A.; Hartmann, T.; Schmitt-Graff, A.; Silberstein, L.E.; Fujii, N.; Kipps, T.J.; Burger, J.A. Oncogene, 2003, 22, 8093.
47. Shimonaka, M.; Katagiri, K.; Nakayama, T.; Fujita, N.; Tsuruo, T.; Yoshie, O.; Kinashi, T. J. Cell Biol., 2003, 161, 417.
48. Ding, Z.; Issekutz, T.B.; Downey, G.P.; Waddell, T.K. Blood, 2003, 101, 4245.
49. Opdenakker, G.; Van Damme, J. Immunol. Today, 1992, 13, 463.
50. Van den Steen, P.E.; Dubois, B.; Nelissen, I.; Rudd, P.M.; Dwek, R.A.; Opdenakker, G. Crit. Rev. Biochem. Mol. Biol., 2002, 37, 375.
51. Nagakawa, Y.; Aoki, T.; Kasuya, K.; Tsuchida, A.; Koyanagi, Y. Pancreas, 2002, 24, 169.
52. Krecicki, T.; Zalesska-Krecicka, M.; Jelen, M.; Szkudlarek, T.; Horobiowska, M. Clin. Otolaryngol., 2001, 26, 469.
53. Locati, M.; Deuschle, U.; Massardi, M.L.; Martinez, F.O.; Sironi, M.; Sozzani, S.; Bartfai, T.; Mantovani, A. J. Immunol., 2002, 168, 3557.
54. Zheng, H.; Takahashi, H.; Murai, Y.; Cui, Z.; Nomoto, K.; Niwa, H.; Tsuneyama, K.; Takano, Y. Anticancer Res., 2006, 26, 3579.
55. Daniel, D.; Meyer-Morse, N.; Bergsland, E.K.; Dehne, K.; Coussens, L.M.; Hanahan, D. J. Exp. Med., 2003, 197, 1017.
56. Petit, I.; Jin, D.; Rafii, S. Trends Immunol., 2007, 28, 299.
57. Tachibana, K.; Hirota, S.; Iizasa, H. Nature, 1998, 393, 591.
58. Bachelder, R.E.; Wendt, M.A.; Mercurio, A.M. Cancer Res., 2002, 62, 7203.
59. Leelawat, K.; Leelawat, S.; Narong, S.; Hongeng, S. World J. Gastroenterol., 2007, 13, 1561.
60. Yang, L.; Jackson, E.; Woerner, B.M.; Perry, A.; Piwnica-Worms, D.; Rubin, J.B. Cancer Res., 2007, 67, 651.
61. Su, L.P.; Zhang, J.P.; Xu, H.B.; Chen, J.; Wang, Y.; Xiong, S.D. Zhonghua Yi Xue Za Zhi., 2005, 85, 1190.
62. Phillips, R.J.; Burdick, M.D.; Lutz, M.; Belperio, J.A.; Keane, M.P.; Strieter, R.M. Am. J. Respir. Crit. Care Med., 2003, 167, 1676.
63. Belperio, J.A.; Phillips, R.J.; Burdick, M.D.; Lutz, M.; Keane, M.; Strieter, R. Chest., 2004, 25, 156S.
64. Bertolini, F.; Dell’Agnola, C.; Mancuso, P.; Rabascio, C.; Burlini, A.; Monestiroli, S.; Gobbi, A.; Pruneri, G.; Martinelli, G.
Cancer Res., 2002, 62, 3106.
65. Zhou, Y.; Larsen, P. H.; Hao, C.; Yong, V. W. J. Biol. Chem., 2002, 277, 49481.
66. Abraham, M.; Biyder, K.; Begin, M.; Wald, H.; Weiss, I.D.; Galun, E.; Nagler, A.; Peled, A. Stem Cells., 2007, 25, 2158.
67. Burger, M.; Hartmann, T.; Krome, M.; Rawluk, J.; Tamamura, H.; Fujii, N.; Kipps, T.J.; Burger, J.A. Blood, 2005, 106, 1824.
68. Tamamura, H.; Hiramatsu, K.; Mizumoto, M.; Ueda, S.; Kusano, S.; Terakubo, S.; Akamatsu, M.; Yamamoto, N.; Trent,
J.O.; Wang, Z.; Peiper, S.C.; Nakashima, H.; Otaka, A.; Fujii, N. Org. Biomol. Chem., 2003, 1, 3663.
69. Tamamura, H.; Hori, A.; Kanzaki, N.; Hiramatsu, K.; Mizumoto, M.; Nakashima, H.; Yamamoto, N.; Otaka, A.; Fujii, N. FEBS Lett., 2003, 550, 79.
70. Liang, Z.; Wu, T.; Lou, H.; Yu, X.; Taichman, R.S.; Lau, S.K.; Nie, S.; Umbreit, J.; Shim, H. Cancer Res., 2004, 64, 4302.
71. Mellor, P.; Harvey, J.R.; Murphy, K.J.; Pye, D.; O’Boyle, G.; Lennard, T.W.; Kirby, J.A.; Ali, S. Br. J. Cancer., 2007, 97, 761.
72. Tamamura, H.; Tsutsumi, H.; Masuno, H.; Mizokami, S.; Hiramatsu, K.; Wang, Z.; Trent, J.O.; Nakashima, H.; Yamamoto, N.; Peiper, S.C.; Fujii, N. Org. Biomol. Chem., 2006, 4, 2354.
73. Juarez, J.; Bradstock, K.F.; Gottlieb, D.J.; Bendall, L.J. Leukemia, 2003, 17, 1294.
74. Barretina, J.; Juncà, J.; Llano, A.; Gutiérrez, A.; Flores, A.; Blanco, J.; Clotet, B.; Esté, J.A. Ann. Hematol., 2003, 82, 500.
75. Mori, T.; Doi, R.; Koizumi, M.; Toyoda, E.; Ito, D.; Kami, K.; Masui, T.; Fujimoto, K.; Tamamura, H.; Hiramatsu, K.; Fujii,
N.; Imamura, N. Mol. Cancer Ther., 2004, 3, 29.
76. Tamamura, H.; Omagari, A.; Oishi, S.; Kanamoto, T.; Yamamoto, N.; Peiper, S.C.; Nakashima, H.; Otaka, A.; Fujii, N. Bioorg. Med. Chem. Lett., 2000, 10, 2633.
77. Lee, C.H.; Kakinuma, T.; Wang, J.; Zhang, H.; Palmer, D.C.; Restifo, N.P.; Hwang, S.T. Mol. Cancer Ther., 2006, 5, 2592.
78. DeMarco, S.J.; Henze, H.; Lederer, A.; Moehle, K.; Mukherjee, R.; Romagnoli, B.; Robinson, J.A.; Brianza, F.; Gombert, F.O.; Lociuro, S.; Ludin, C.; Vrijbloed, J.W.; Zumbrunn, J.; Obrecht, J.P.; Obrecht, D.; Brondani, V.; Hamy, F.; Klimkait, T. Bioorg. Med. Chem., 2006, 14, 8396.
79. Tamamura, H.; Araki, T.; Ueda, S.; Wang, Z.; Oishi, S.; Esaka, A.; Trent, J.O.; Nakashima, H.; Yamamoto, N.; Peiper, S.C.; Otaka, A.; Fujii, N. J. Med. Chem., 2005, 48, 3280.
80. Tamamura, H.; Tsutsumi, H.; Masuno, H.; Fujii, N. Curr. Med. Chem., 2007, 14, 93.
81. Faber, A.; Roderburg, C.; Wein, F.; Saffrich, R.; Seckinger, A.; Horsch, K.; Diehlmann, A.; Wong, D.; Bridger, G.; Eckstein, V.; Ho, A.D.; Wagner, W. J. Biomed. Biotechnol., 2007, 2007, 26065.
82. Anderson, Y.L.; DeWitte, G.M.; Nakada, M. European Journal of Cancer, 2006, 42, 793.
83. Laverdiere, C.; Gorlick, R. Clin. Cancer Res., 2006, 12, 5254.
84. Tamamura, H.; Tsutsumi, H. Chem. Biol., 2006, 13, 8.
85. Kumar, S.; Choi, W. T.; Dong, C.Z.; Madani, N.; Tian, S.; Liu, D.; Wang, Y.; Pesavento, J.; Wang, J.; Fan, X.; Yuan, J.; Fritzsche, W.R.; An, J.; Sodroski, J.G.; Richman, D.D.; Huang, Z. Chem. Biol., 2006, 13, 69.
86. Zeng, Z.; Samudio, I.J.; Munsell, M.; An, J.; Huang, Z.; Estey, E.; Andreeff, M.; Konopleva. M. Mol. Cancer Ther., 2006, 5, 3113.
87. Snyder, E.L.; Saenz, C.C.; Denicourt, C.; Meade, B.R.; Cui, X.S.; Kaplan, I.M.; Dowdy, S.F. Cancer Res., 2005, 65, 10646.
88. Khan, A.; Greenman, J.; Archibald, S.J. Curr. Med. Chem., 2007, 14, 2257.
89. Tsutsumi, H.; Tanaka, T.; Ohashi, N.; Masuno, H.; Tamamura, H.; Hiramatsu, K.; Araki, T.; Ueda, S.; Oishi, S.; Fujii, N.
Biopolymers, 2007, 88, 279.
90. Khan, A.; Greenman, J.; Archibald, S.J. Curr. Med. Chem., 2007, 14, 2257.
91. Saur, D.; Seidler, B.; Schneider, G.; Algul, H.; Beck, R.; Senekowitsch-Schmidtke, R.; Schwaiger, M.; Schmid, R.M. Gastroenterology, 2005, 129, 1237.
92. Rubin, J.B.; Kung, A.L.; Klein, R.S.; Chan, J.A.; Sun, Y.; Schmidt, K.; Kieran, M.W.; Luster, A.D.; Segal, R.A. Proc. Natl.
Acad. Sci., 2003, 100, 13513.
93. Redjal, N.; Chan, J.A.; Segal, R.A.; Kung, A.L. Clin. Cancer Res., 2006, 12, 6765.
94. Zhang, J.; Sarkar, S.; Yong, V.W. Carcinogenesis, 2005, 26, 2069.
95. Kim, J.; Mori, T.; Chen, S.L.; Amersi, F.F.; Martinez, S.R.; Kuo, C.; Turner, R.R.; Ye, X.; Bilchik, A.J.; Morton, D.L.; Hoon, D.S. Ann. Surg., 2006, 244, 113.
96. Scala, S.; Giuliano, P.; Ascierto, P.A.; Ierano, C.; Franco, R.; Napolitano, M.; Ottaiano, A.; Lombardi, M.L.; Luongo, M.; Simeone, E.; Castiglia, D.; Mauro, F.; De Michele, I.; Calemma, R.; Botti, G.; Caraco, C.; Nicoletti, G.; Satriano, R.A.; Castello, G. Clin. Cancer Res., 2006, 12, 2427.
97. Ohira, S.; Sasaki, M.; Harada, K.; Sato, Y.; Zen, Y.; Isse, K.; Kozaka, K.; Ishikawa, A.; Oda, K.; Nimura, Y.; Nakanuma, Y. Am. J. Pathol., 2006, 168, 1155.
98. De Clercq, E. Mini Rev. Med. Chem., 2005, 5, 805.
99. Holtan, S.G.; Porrata, L.F.; Micallef, I.N.; Padley, D.J.; Inwards, D.J.; Ansell, S.A.; Johnston, P.B.; Gastineau, D.A.; Markovic, S.N. Clin. Lymphoma Myeloma., 2007, 7, 315.
100. Glickman, F.; Streiff, M.; Thoma, G.; Zerwes, H.-G. EP Patent, 2006, 1720845; WO Patent, 2005, 85219121.
101. Kamiyama, K.; Kanzaki, N.; Kawamoto, T.; Mochizuki, M.; Hasuoka, J. JP Patent, 2002, 371042.
102. Glickman, F.; Streiff, M.; Thoma, G.; Zerwes, H.-G. EP Patent, 2006, 1720845; WO Patent, 2005, 2005085219.
103. Kokubo, M.; Takaoka, Y. WO Patent, 2006, 022454.
104. Zhan, W.; Liang, Z.; Yoon, Y.; Snyder, J.; Liotta, D.; Shim, H. Proc. Am. Assoc. Cancer Res., 2006, 47, Abst 1120.
105. Ochiai, H.; Ohhata, A.; Takaoka, Y.; Shibayama, S. WO Patent, 2006, 090853.
106. Dwyer, M.; Yu, Y.; Biju, P.; Jakway, J.; Lundell, D.; Fine, J. S.; Hecker, E. A.; Aki, C. J.; Chao, J.; Chao, J.; Merritt, J.R.; Lai, G.; Wu, M.; Taveras, A.G. US Patent, 2004, 106794.
107. Naya, A.; Sagara, Y.; Ohwaki, K.; Saeki, T.; Ichikawa, D.; Iwasawa, Y.; Noguchi, K.; Ohtake, N. J. Med. Chem., 2001, 44, 1429.
108. Habashita, H.; Kokubo, M.; Hamano, S.; Hamanaka, N.; Toda, M.; Shibayama, S.; Tada, H.; Sagawa, K.; Fukushima, D.; Maeda, K.; Mitsuya, H. J. Med. Chem., 2006, 49, 4140.
109. Ghosh, S.; Elder, A.; Guo, J.; Mani, U.; Patane, M.; Carson, K.; Ye, Q.; Bennett, R.; Chi, S.; Jenkins, T.; Guan, B.; Kolbeck, R.; Smith, S.; Zhang, C.; LaRosa, G.; Jaffee, B.; Yang, H.; Eddy, P.; Lu, C.; Uttamsingh, V.; Horlick, R.; Harriman, G.; Flynn, D. J. Med. Chem., 2006, 49, 2669.
110. Purandare, A.V.; Wan, H.; Somerville, J.E.; Burke, C.; Vaccaro, W.; Yang, X.; McIntyre, K.W.; Poss, M.A. Bioorg. Med. Chem. Lett., 2007, 17, 679.
111. Busch-Petersen, J. Curr. Top Med. Chem., 2006, 6, 1345.
112. Whitcomb, J.M.; Huang, W.; Fransen, S.; Limoli, K.; Toma, J.; Wrin, T.; Chappey, C.; Kiss, L.D.; Paxinos, E.E.; Petropoulos, C.J. Antimicrob. Agents Chemother., 2007, 51, 566.
113. Zhao, Q.; Lu, H.; Schols, D.; De Clercq, E.; Jiang, S. AIDS Res. Hum. Retroviruses, 2003, 19, 947.
114. Tamamura, H.; Imai, M.; Ishihara, T.; Masuda, M.; Funakoshi, H.; Oyake, H.; Murakami, T.; Arakaki, R.; Nakashima, H.; Otaka, A.; Ibuka, T.; Waki, M.; Matsumoto, A.; Yamamoto, N.; Fujii, N. Bioorg. Med. Chem., 1998, 6, 1033.
115. Vabeno, J.; Nikiforovich, G.V.; Marshall, G.R. Biopolymers, 2006, 84, 459.
116. Kohonen, T. (1990) The self-organizing map. Proceedings of the IEEE 78, 1464-80.
117 (a) Bauknecht, H., Zell, A., Bayer, H., Levi, P., Wagener, M., Sadowski, J., and Gasteiger, J. (1996) Locating biologically active compounds in medium-sized heterogeneous datasets by topological autocorrelation vectors: dopamine and benzodiazepine agonists J Chem Inf Comp Sci 36, 1205-13.15; (b) Anzali, S., Barnickel, G., Krug, M., Sadowski, J., Wagener, M., Gasteiger, J., and Polanski, J. (1996) The comparison of geometric and electronic properties of molecular surfaces by neural networks: application to the analysis of corticosteroid binding globulin activity of steroids J Comp-Aid Mol Des 10, 521-34: (c) Brűstle, M., Beck, B., Schindler, T., King, W., Mitchell, T., and Clark, T. (2002) Descriptors, physical properties, and druglikeness J Med Chem 45, 3345-55; (d) Rabow, A. A., Shoemaker, R. H., Sausville, E. A., and Covell, D. G. (2002) Mining the National Cancer Institute’s tumor-screening database: identification of compounds with similar cellular activities J Med Chem 45, 818-40; (e) Korolev, D., Balakin, K. V., Nikolsky, Y., Kirillov, E., Ivanenkov, Y. A., Savchuk, N. P., Ivashchenko, A. A., and Nikolskaya, T. (2003) Modeling of human cytochrome P450-mediated drug metabolism using unsupervised machine learning approach J Med Chem 46, 3631-43; (f) Savchuk, N. P. (2003) In silico ADME-Tox as part of an optimization strategy Curr Drug Disc 4, 17-22.
118. Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch V, Abagyan R, Brooun A, Wells P, Bi FC, Hamel DJ, Kuhn P, Handel TM, Cherezov V, Stevens RC. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science. 2010 Nov 19;330(6007):1066-71.
119. Berkhout, T. A.; Blaney, F. E.; Bridges, A. M.; Cooper, D. G.; Forbes, I. T.; Gribble, A. D.; Groot, P. H.; Hardy, A.; Ife, R. J.; Kaur, R.; Moores, K. E.; Shillito, H.; Willetts, J.; Witherington, J. CCR2: Characterization of The Antagonist Binding Site from a Combined Receptor Modeling/Mutagenesis Approach. J. Med. Chem. 2003, 46, 4070-86.
120. (a) Fano, A.; Ritchie, D. W.; Carrieri, A. Modeling The Structural Basis of Human CCR5 Chemokine Receptor Function: from Homology Model Building and Molecular Dynamics Validation to Agonist and Antagonist Docking. J. Chem. Inf. Model. 2006, 46, 1223-35; (b) Paterlini, M. G. Structure Modeling of The Chemokine Receptor CCR5: Implications for Ligand Binding and Selectivity. Biophys J. 2002, 83, 3012-31; (c) Kellenberger, E.; Springael, J. Y.; Parmentier, M.; Hachet-Haas, M.; Galzi, J. L.; Rognan, D. Identification of Nonpeptide CCR5 Receptor Agonists by Structure-based Virtual Screening. J. Med. Chem. 2007, 50, 1294-303.
121. Klabunde, T. (2006) Chemogenomic approaches to ligand design. In Ligand design for G-protein-coupled receptors, Rognan, D., (Ed). Wiley-VCH, Weinheim.
122 (a) Zlotnik, A., and Yoshie, O. (2000) Chemokines: a new classification system and their role in immunity Immunity 12, 121-7; (b) Yoshie, O., Imai, T., and Nomiyama, H. (2001) Chemokines in immunity Adv Immunol 78, 57-110.
123. (a) Pérez-Nueno, V. I., Ritchie, D. W., Rabal, O., Pascual, R., Borrell, J. I., and Teixidó, J. (2008) Comparison of ligandbased and receptor-based virtual screening of HIV entry inhibitors for the CXCR4 and CCR5 receptors using 3D ligand shape matching and ligand-receptor docking J Chem Inf Model 48, 509-33; (b) Spencer, E. H. (2005) Development of a Structure Prediction Method for G-Protein Coupled Receptors, Thesis, California Institute of Technology.
124. Efremov, R., Truong, M. J., Darcissac, E. C., Zeng, J., Grau, O., Vergoten, G., Debard, C., Capron, A., and Bahr, G. M. (2001) Human chemokine receptors CCR5, CCR3 and CCR2B share common polarity motif in the first extracellular loop with other human G-protein coupled receptors European Journal of Biochemistry 263, 746-56.