Abstract Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF “Sticky-traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Sticky-traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity. Synopsis A locally delivered, bifunctional recombinant “Sticky-trap” comprising a VEGF-trap and a heparin-binding domain (HBD) can prevent aberrant ocular angiogenesis without affecting systemic physiological VEGF-processes.

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• Bifunctional “Sticky-trap” is comprised of VEGF-trap and a carboxy-terminal HBD. • Treatment with recombinant Sticky-trap can prevent the development of abnormal ocular angiogenesis.

• Sticky-trap remains at the site of delivery; thus, it has a local antiangiogenic activity. Realtek Sound Driver Windows 7 32 Bit. • Sticky-trap applied in the eye does not compromise systemic physiological VEGF-processes, such as wound healing and kidney function.

• • Introduction Abnormal vessel formation is implicated in the pathophysiology of a number of diseases including diabetic retinopathy (DR), retinopathy of pre-maturity (ROP), the wet form of age-related macular degeneration (AMD), cancer and obesity (Carmeliet,,; Folkman, ). Inhibition of pathological angiogenesis has been shown to improve vision in patients with AMD, DR and ROP (Ferrara & Kerbel,; Gasparini et al,; Kerbel,; Mintz-Hittner et al, ), as well as help maintain metabolic homeostasis in obese individuals (Sun et al, ). Over the past decade, different approaches to inhibit VEGF signalling have been developed, such as recombinant trap proteins (VEGF-trap; Holash et al, ), monoclonal antibodies (bevacizumab; Ellis,; Ferrara et al, ) and small multi-kinase inhibitors (Wilhelm et al,; Chow & Eckhardt, ), some of which have been approved for clinical use (Grothey & Galanis,; Heath & Bicknell, ) in treating eye diseases as well as certain types of cancer. However, the aforementioned approaches may be accompanied by a number of severe side effects due to the disruption of proper vessel function in various organs (Eremina et al,,; Kamba et al,; Verheul & Pinedo,; Chen & Cleck,; Boehm et al, ).

As a consequence, the use of anti-VEGF therapies may be relatively contraindicated, particularly in certain patient populations, such as pregnant women (Petrou et al, ), patients undergoing surgery (Cortes et al, ), pre-mature infants (Hard & Hellstrom, ) and elderly patients with comorbidities, such as cardiovascular insufficiency (Chen & Cleck,; Nazer et al, ). The storage of numerous growth factors in the extracellular matrix (ECM), as well as the creation of morphogen gradients during development, is controlled by the interaction between the heparin-binding domain (HBD; a sequence of positively charged amino acids) of the growth factor and extracellular-matrix heparan sulphate proteoglycans (HSPGs). VEGF contains two regions with HBDs coded by exons 6 and 7. Alternative splicing of these exons results in distinct isoforms (Robinson & Stringer,; Ladomery et al, ).

VEGF121 is missing both exons and is therefore completely soluble. VEGF145 and VEGF165 contain exon 6 and 7, respectively, and are therefore partially soluble.

VEGF189 has both exons and is strictly retained at the secretion site (Houck et al,; Park et al, ). In order to circumvent the aforementioned disadvantages of current antiangiogenic therapies, we have developed novel biologics with properties that allow for local inhibition of angiogenesis. By genetically fusing the original VEGF-trap (Holash et al, ) to the HBDs of VEGF, we created novel VEGF-traps, referred to as Sticky-traps, engineered to possess: (i) a short systemic half-life in it's soluble form and (ii) local antiangiogenic activity via the ability to bind to the ECM thus leading to locally retained, biologically active molecules that provide prolonged angiostatic activity at the site of pathological neovascularization. Results Design and production of VEGF Sticky-traps The original VEGF-trap (Holash et al, ) is composed of IgG-like domains 2 and 3 of VEGF-R1 and VEGF-R2, respectively, fused to the constant region (Fc) of immunoglobulin IgG1 (Fig A and ). The Fc region is composed of two IgG-like domains, CH2 and CH3, and is responsible for the long serum half-life of VEGF-trap, due to its ability to bind to the FcRn receptor located on endothelial, epithelial and circulating blood cells, after which it is recycled back into the circulation instead of undergoing degradation (Ghetie & Ward, ). In order to shorten the half-life, we replaced the CH2 domain, which has been previously shown to be necessary for binding to FcRn (Mueller et al, ), with a hinge domain (H'; Glaser et al, ) and a poly-glycine-serine linker (Glaser et al,; Fig A and ), and named this molecule Short-trap. To derive Sticky-traps, we modified the Short-trap by adding the HBDs of the VEGF isoforms (HBDs; Munoz & Linhardt, ), encoded by exons 6 and 7, at the carboxy-terminus.

We also included VEGF exon 8, which is necessary for proper disulphide-bond formation of the HBD (Krilleke et al, ). In total, we generated three Sticky-traps: Sticky-trap68, Sticky-trap78 and Sticky-trap678, where the numbers indicate which VEGF exons were included (Fig A and ). Finally, we generated a “trapless” construct coding only for the short Fc (shFc), as a negative control (Fig A and ). Schematic representation and biochemical characterization of traps • • • ABasic protein structure of traps. VEGF-trap is composed of the VEGF-binding region (domain 2 and 3 of VEGFR-1 and -2, respectively) and the Fc region of IgG1 (CH2 and CH3 domains). The Sticky-traps, Sticky-trap68, Sticky-trap78 and Sticky-trap678 contain the heparin-binding domains (HBDs) encoded by exons 6 and 8, 7 and 8, and 6, 7 and 8 of vascular endothelial growth factor, respectively. See and methods for further details.

• B–DAffinity of traps to extracellular matrix (ECM). (B) Immunostaining (red signal) and Western blot analysis (on the right of the image) of traps in PC-3 cell monolayers and conditioned supernatant, respectively. Similar results are shown in for the A-673 transgenic lines. Plus (+) dox samples were collected 48 h after addition of doxycycline-containing media. Scale bar, 100 μm. (C) Binding of recombinant traps to ECM. (D) Affinity of recombinant traps to heparin-Sepharose column.

• E, FAssessment of traps ability to bind human VEGF. (E) Free VEGF levels in the conditioned of PC-3 cancer transgenic cell lines, co-cultured of wild-type PC-3 cells. Media were collected after 48 h of culture with or without the addition of doxycycline. (F) Binding affinity (KD) of recombinant traps to human VEGF 165 (VEGF-trap; 9.0 pM, Short-trap; 12.2 pM, and Sticky-trap 13.9 pM). • GInhibition of VEGF-induced human umbilical vein endothelial cells proliferation by recombinant traps.

Source data are available online for this figure. We then used genetic and recombinant protein-based approaches to characterize these new biologics. In both approaches, we combined the piggyBac transposon (Ding et al, ) transgene delivery system with a tetracycline-based ON/OFF switch (Agha-Mohammadi et al,; ) for transgene expression in a doxycycline (dox)-dependent manner. To test the biologic activity/property of the engineered novel biologics with genetic approaches, we derived transgenic cancer cell lines (PC-3, A-673 and HT-29; ). In the absence of doxycycline (dox), none of the transgenes were expressed, while in the presence of dox, all were expressed at a high level and in comparable amounts (). We also attached green fluorescent protein (EGFP) as a reporter for transgene expression. Flow cytometry for EGFP in the induced cells showed that on average 88% of the cells expressed the transgene ( and ).

To produce and purify recombinant proteins, we generated stably transfected 293 suspension cell lines using vectors allowing for expression of recombinant traps in a dox inducible manner (Li et al, ). Of the Sticky-traps, Sticky-trap678 was selected for further characterization, given its superior potency in a xenograft assay (discussed below). All of the traps formed homodimers, had the predicted molecular weight () and were glycosylated (). Molecular and biochemical characterization of Sticky-traps Immunostaining, performed on transgenic PC-3 (Fig B) and A-673 cells (), for the Fc domain showed—as expected—that shFc, VEGF-trap and Short-trap did not bind to the ECM.

However, Sticky-traps were bound and retained by the ECM components, indicating that the VEGF HBDs remained functional in these molecules. Further support for the strong affinity of Sticky-traps to the ECM was obtained from Western blot analysis, in which high levels of soluble (free) shFc, VEGF-trap and Short-trap were detected in the culture media, while Sticky-traps were hardly detectable (Fig B and ). Using recombinant proteins, we performed ECM-based and heparin column assays (Fig C and D). Sticky-trap showed a dose-dependent ability to bind to the ECM (Matrigel matrix), while VEGF-trap, Short-trap and IgG showed no affinity (Fig C). Furthermore, high NaCl concentration was required for elution of Sticky-trap, further indicating its strong affinity for the ECM (Fig D).

Sticky-traps also retained their ability to bind VEGF, as confirmed by a competitive ELISA that detects only free VEGF (Maynard et al,; Tissot van Patot et al, ). After addition of dox to the media of transgenic cell lines, both Short-trap and Sticky-traps, similar to VEGF-trap, blocked the appearance of free VEGF in the conditioned media ().

Binding was also shown to be extracellular as VEGF was still completely sequestered from the supernatant when a 1:1 mix of wild-type and transgenic PC-3 cells was assayed (Fig E). We used equilibrium binding assays to determine the binding affinity of recombinant traps for VEGF. Various amounts of traps were incubated with VEGF165, and the free, unbound VEGF165 was measured using a sensitive immunoassay. All traps showed strong affinity for VEGF with similar KD (VEGF-trap; 9.0 pM, Short-trap; 12.2 pM, and Sticky-trap 13.9 pM; Fig F). Finally, as a functional assay, we used human umbilical vein endothelial cell (HUVEC) to examine the ability of recombinant traps to inhibit VEGF-induced proliferation and inhibit VEGFR-2 Y1175 phosphorylation. Various amounts of traps were pre-incubated with fixed amounts of VEGF and added to HUVEC cell cultures.

All traps were able to efficiently block VEGF-induced proliferation in a dose-dependent manner (Fig G; percentage inhibition compared to vehicle only), as well as inhibit VEGFR2 phosphorylation at Y1175 (). The enhanced ability of Sticky-trap to inhibit HUVEC proliferation in doses higher than 10 pM is presumably attributed to the fact that both Sticky-trap and VEGF have the same HBD. Therefore, it is possible that Sticky-trap can affect the bioavailability of VEGF by competing for available binding sites in the ECM, as well as compromise the ability of VEGF to bind to neuropilin-1, which is mediated through exons 7 and 8 of the HBD, and is required for VEGFR-2 activation (Parker et al, ). Pharmacokinetics and systemic distribution In order to characterize the in vivo pharmacokinetics of Sticky-trap, after subcutaneous injection, we used an ELISA assay to measure their concentration in the serum. As expected, high amount of VEGF-trap was detected ( C max = 10 μg/ml; AUC = 24 μg × days/ml), while both Short-trap and Sticky-trap had an AUC that was around 400-fold lower ( C max = 0.10 μg/ml; AUC = 0.065 μg × days/ml and C max = 0.10 μg/ml; AUC = 0.06 μg × days/ml, respectively; Fig A), and were practically undetectable 24–48 h after injection.

Pharmacokinetic profile and tissue distribution of traps • • • ATraps (100 μg) were injected subcutaneously into C57BL/6J mice, and serum levels were estimated using an ELISA assay. Error bars represent s.e.m. • BSerum levels of VEGF at various time points after subcutaneous injection of traps (100 μg) into C57BL/6J mice. Error bars represent s.e.m.

( n = 5; *** P. Characterization of recombinant traps in vivo antiangiogenic activity in xenograft models • • • ATumour growth kinetics of HT-29 xenografts. Treatment (30 μg per tumour, intratumorally injected; two-times per week) initiated 1 week after tumour implantation, once tumours reached an average size 100 mm 3. Error bars represent s.e.m. ( n = 12; * P 0.05).

Similarly, a longer time period was required for the scab to fall off from the wounds on animals bearing tumours expressing VEGF-trap compared to control animals and those expressing Sticky-traps (Fig B, D and E; P 0.05). Wound-healing assay and detection of regressed vessels in the trachea • • • AWounds were generated at day 8 in the neck area of mice bearing xenografts of transgenic A-673 expressing either VEGF-trap or Sticky-traps. Wound size and spontaneous fall of the scab were monitored daily. • B–EAt day 20, a different group of animals was euthanized and the trachea was dissected (A, asterisk). Serum was collected at day 0, 8 and 20 (A, arrows; ).

(B and C) Wound size at day 4 compared to day 0 (* P 0.05, one-way ANOVA). (B, D and E) Spontaneous scab fall (** P 0.05, one-way ANOVA; n = 9 for wild-type, n = 10 for VEGF-trap, and n = 13 for Sticky-traps; n = 4 for Sticky-trap68, n = 4 for Sticky-trap78, n = 5 for Sticky-trap678). (D) H&E staining of representative wounds. Dashed line indicates the wound area, and asterisk the scab.

Scale bars, 500 μm (top row) and 50 μm (bottom row). • F, GImmunostaining of tracheal vessels for CD-31 (yellow) and type-IV collagen (red). Arrows indicate regressed (ghost) vessels, that is, vessels of type-IV collagen immunoreactivity devoid of CD31 immunoreactivity (*** P = 0.0008 for w.t. Versus VEGF-trap and P = 0.0009 for Sticky-trap678 versus VEGF-trap, # P >0.05, one-way ANOVA; n = 4 for w.t., n = 5 for VEGF-trap and n = 5 for Sticky-trap678). In a second model, we also measured systemic VEGF suppression by examining tracheal micro-vessels (Kamba et al, ) by whole-mount immunostaining with antibodies recognizing endothelial cells (anti-CD31) and their basement membrane (anticollagen IV).

Functioning vessels are double positive, while vessel regression leads to areas positive for collagen IV and negative for CD31 (called empty sleeves; Kamba et al, ). Animals with tumours expressing Sticky-trap678 and non-tumour-bearing controls had very few empty sleeves and the majority of the vessels were double positive (Fig F and G; P >0.05). In contrast, animals with tumours expressing the VEGF-trap showed a fivefold increase in empty sleeve formation (Fig F and G; P. Subcutaneous injection of traps in neonatal pups • • • Immunostaining of kidneys for vessels (CD31) and perfusion (lectin). Yellow arrows; perfused glomeruli, white arrowheads; partially perfused glomeruli. Scale bars, 50 μm. • Quantification of lectin perfusion (*** P.

Biodistribution of traps in the eye • • • AImmunostaining of cross sections of mouse eyes, intravitreally injected with the traps (10 μg) and dissected either 2 or 12 days post-injection. Scale bar, 500 μm. • B, CBinding of traps in the ciliary body (B), and inner-limiting membrane (C) of the eye. Scale bar, 50 μm. More detailed immunostaining analysis is shown in. L, lens; ilm, inner-limiting membrane; cb, ciliary body; gcl, ganglion cell layer. • DTraps' serum levels 2, 6 and 24 h post-intravitreal injections.

( n = 4; ** P. In vivo characterization of trap activity in the mouse model of oxygen-induced retinopathy (OIR) • • • A–HPups were exposed to hyperoxia for 5 days, P7-P12, and traps (2.5 μg) were injected intravitreally at P12, once the mice were returned to normoxia. Eyes were dissected either 5 or 9 days post-injection, at P17 (A–D) and P21 (E–H), respectively. (B and F) Whole-mount immunostaining of retinas for neovascular tuft formation (lectin-positive signal, red pseudocolour) and persisting vaso-obliteration (yellow pseudocolour). (C and G) Area of tuft formation at P17 and P21, respectively ( n = 7–8; *** P. Vascular leakage quantified by Evans Blue dye accumulation in the retina of mice exposed to hyperoxia • • n = 8 for vehicle, VEGF-trap and Sticky-trap; n = 3 for normoxia; *P. The paper explained Problem Excessive production of vascular endothelial growth factor (VEGF-A) leads to abnormal retinal angiogenesis in patients with DR and in newborns suffering of ROP.

If left untreated, both conditions often lead to blindness. Current clinical therapies aimed at inhibiting VEGF have shown some success. However, systemic VEGF inhibition has detrimental side effects, such as kidney toxicity, impaired wound healing, highly elevated blood pressure, gastrointestinal perforation, haemorrhage, thrombosis, reversible posterior leukoencephalopathy, cardiac impairment and endocrine dysfunction. Infants—in which organogenesis is still active—are even more critically affected. Results In this study, we developed a novel inhibitor of VEGF, referred to as VEGF Sticky-trap.

It comprised of three components: (a) the VEGF-trap region (b) a modified Fc region that ensures short serum half-life and (c) a HBD that allows Sticky-trap to remain at the place of injection. Using a mouse model of DR and ROP, we proved Sticky-trap to be highly effective.

In contrast to currently available treatments, once injected intravitreally, Sticky-trap does not exit into the circulation. Instead, it binds the ECM and remains in the eye for a prolonged period of time. Based on these findings, we investigated and showed that local Sticky-trap treatments do not affect distant wound healing, neither do they compromise the kidney vasculature. Impact This is the first report of a local acting angiogenesis inhibitor that can immediately be translated into the clinic as an effective, and safe treatment of DR and ROP. This is of particular importance for elderly patients and pre-mature infants requiring VEGF suppression and who are especially sensitive to potential side effects of such therapies. Ancillary Article Information.

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Abstract Amongst the chemokine signalling axes involved in cancer, chemokine CXCL12 acting on chemokine receptor CXCR4 is particularly significant since it orchestrates migration of cancer cells in a tissue-specific metastatic process. High CXCR4 tumour expression is associated with poor prognosis of lung, brain, CNS, blood and breast cancers. We have identified a new class of small molecule CXCR4 antagonists based on the use of computational modelling studies in concert with experimental determination of in vitro activity against CXCL12-induced intracellular calcium mobilisation, proliferation and chemotaxis. Molecular modelling proved to be a useful tool in rationalising our observed potencies, as well as informing the direction of the synthetic efforts aimed at producing more potent compounds.

Citation: Vinader V, Ahmet DS, Ahmed MS, Patterson LH, Afarinkia K (2013) Discovery and Computer Aided Potency Optimization of a Novel Class of Small Molecule CXCR4 Antagonists. PLoS ONE 8(10): e78744.

Editor: Robyn Klein, Washington University, United States of America Received: August 15, 2013; Accepted: September 22, 2013; Published: October 18, 2013 Copyright: © 2013 Vinader et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors thank Yorkshire Cancer Research for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Kamyar Afarinkia is a PLOS ONE Editorial Board member.

This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Introduction Chemokines play a multifaceted role in the biology of the cell[,]. They elicit their biological effects by binding to their cognate cell surface receptors.

This binding initiates a number of intracellular secondary message cascades which account for the diverse biological role emanating from this signalling axis. It is therefore not surprising that disregulation in the chemokine signalling is implicated in the pathophysiology of many diseases and conditions, ranging from inflammatory[,] and autoimmune[] diseases, to pain[-], infection[,], and in particular, cancer[-]. Amongst the chemokine signalling axes involved in cancer, chemokine CXCL12, acting on chemokine receptor CXCR4 is particularly significant. CXCR4 is widely detected in human cancers of epithelial, mesenchymal and haematopoietic origin[].

Its ligand, CXCL12 is abundant in liver, bone and brain, which are the common sites of metastasis for cancers of these organs and tissues[]. This observation has led to the hypothesis that the CXCL12/CXCR4 axis orchestrates a site-specific metastatic process[,]. The involvement of the CXCR4/CXCL12 axis in promoting cancer is widely reported, both generally [,-] and for specific cancers such as lung[-], brain[], CNS[], blood[], and breast[,], including breast-to-bone and breast-to-brain metastases[-]. Furthermore, the therapeutic benefit of CXCR4 modulation in cancer is extensively demonstrated in the literature, using both neutralising antibodies and siRNA-mediated knockdown of the receptor in preclinical metastatic tumour models[-]. Peptide antagonists of CXCR4, such as TN14003[] and CTCE-9908[], () are shown to be antimetastatic in animal preclinical models.

For example, CTCE-9908 retards tumour growth in a prostate mouse model[], inhibits both primary breast tumour growth and metastasis[-], particularly to bone[,], and enhances the efficacy of anti-VEGF mAb (DC101) treatment or docetaxel in a mouse model[]. Of course, peptide based CXCR4 antagonists are difficult to deliver orally, a route that could be favoured for treatment of cancer metastasis that require repeat dosing especially in an outpatient setting. However, following positive results from these in vivo studies, CTCE-9908 is reported to have progressed to the clinic[]. Structures of TN14003, AMD3100, AMD3465, AMD11070, and GSK812397. Non-peptide CXCR4 antagonists generally fall into various chemotypes () but their promise as antimetastatic agents remains unfulfilled.

Although, the small molecule CXCR4 antagonist AMD3100 (Plerixafor) is used clinically in conjunction with granulocyte colony-stimulating factor (G-CSF) to improve harvesting of hematopoietic stem cells prior to autologous transplantation[,]. Furthermore, GSK812397[], and AMD11070[], have anti-HIV activity, the latter with clinical potential.

In view of the significant role that CXCR4 activation plays in cancer and other diseases, identification of novel small molecule antagonists, which would have an appropriate profile for clinical progression, has gathered pace in recent years[]. Here, we report the identification of ICT5040 (1) a new CXCR4 antagonist chemotype, identified through in silico screening.

We show that this in silico hit, although chemically distinct from it, has a similar functional activity to AMD3100, a benchmark CXCR4 antagonist. Furthermore, we report the first phase of a computationally driven potency optimisation, supported by a robust and reliable in silico model.

Cell culture Human breast adenocarcinoma cell line, MDA-MB-231, and human glioblastoma cell line U87-MG were obtained from the European Collection of Cell Cultures (ECACC; Health Protection Agency, Salisbury, UK) and maintained as monolayers in RPMI-1640 supplemented with 10% (v/v) fetal calf serum, 1mM sodium pyruvate and 2 mM L-glutamine (Sigma-Aldrich, Dorset, UK). Cells were grown in 75cm 2 culture flasks in an atmosphere of 5% CO 2 at 37 °C and harvested in a solution of trypsin-EDTA at the logarithmic growth phase. All cell lines were used at low-passage. Flow cytometry Expression of CXCR4 on the surface of MDA-MB-231 and U87-MG cell lines was determined using the FlowCellect Chemokine Receptor CXCR4 Surface Expression Identification and Quantification Kit (Millipore, Watford, UK), and processed as per the manufacturer’s instructions. Flow cytometry analysis was performed using a FACS-Calibur flow cytometer (BD Biosciences; San Jose, CA, USA). The data was analyzed using the CellQuest software (BD Biosciences). This data is included in the file S2.

Calcium mobilisation assay 4 x 10 4 U87-MG cells were seeded into each well of a 0.1% gelatine-coated 96-well black-wall microtiter plate. After 24 h, the growth medium was replaced with 100µl of the dye loading solution (Molecular Probes TM Fluo-4 NW (no wash), Invitrogen F36206). The plates were incubated at 37 °C for 30 minutes and at room temperature for an additional 30 minutes.

20μL of a given concentration of the antagonist in medium, or plain medium as control, was added to each well and the plate was incubated at 37 °C for 15 minutes and at room temperature for an additional 30 minutes. The plate was transferred into a Fluoroskan Ascent FL instrument (Thermo Scientific) and the fluorescence in response to the addition of 20 μl CXCL12 (R&D Systems, Oxford, UK, product number 350-NS) (10ng/ml in the well, 12.4 nM final concentration) was measured at room temperature (Ex 485 nm, Em 538 nm). IC 50 is calculated as the concentration of the antagonist required to half the maximal response to CXCL12. Data is presented as the mean ±SE of at least 3 independent experiments.

Cell proliferation assay U87-MG cells were cultured to a density of 1 x 10 4 cells/ml in RPMI-1640 containing 10% FCS and treated with either a given concentration of the antagonist or no antagonist (control) for an hour. CXCL12 (100ng/ml final concentration) was added, and the cells were transferred to five 96-well tissue culture plates which were the incubated at 37 °C. The number of cells in the plates were counted using MTT assay at days 0-4 as follows: the culture medium was removed and replaced with 200µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5mg/ml stock, diluted in complete RPMI-1640 to 0.5 mg/ml). The culture medium containing MTT was removed after incubation for 4 hours. Following the addition of 150 μl of DMSO per well, the optical density (OD) of each well was measured at 540nm using a Thermo Multiskan EX microplate reader. Data is presented as the mean ±SD of at least 3 independent experiments.

Agarose spot assay [] A 0.5% solution was prepared by adding agarose (Ultrapure TM low-melting agarose; Invitrogen, Paisley, UK) to sterile PBS and heating the mixture until all agarose particles were dissolved. The agarose solution was then cooled to 40 °C. To this solution was added either lyophilized CXCL12 reconstituted to a final stock concentration of 12.5 µM in sterile PBS containing 0.1% Bovine Serum Albumin (BSA) to produce a final concentration of 125 nM CXCL12, or PBS (as control). Two independent drops (10µl) of CXCL12/agarose solution (maintained at 40 °C) and two control spots were pipetted onto the base of a sterile 20 mm diameter glass-bottomed cell culture dish (MatTek Corporation, MA, USA). The dish was then cooled for 5 minutes at 4 °C to allow the agarose spot to solidify. MDA-MB-231 cells (1.7 x10 5 cells, 1 ml) in RPMI-1640 medium containing 10% FCS in the presence or absence of different concentrations of antagonists, were incubated at 37°C, 5% CO 2 for 1 hour, then added to the petri dishes and incubated for a further 4 hours, to allow cells to adhere.

The media was replaced with RPMI-1640 medium containing 0.1% FCS and the corresponding concentration of antagonists, or control, and the dish incubated overnight. Images of the area under the agarose were acquired using a Nikon Coolpix X5000 digital camera attached to a Nikon Eclipse TEZ000-U inverted microscope. The number of invading cells under the agarose spot was quantified using ImageJ software. The values reported herein are averaged of three independent dishes (6 readings in total). Boyden chamber assay 600µl of a solution of CXCL12 (100ng/ml) in RPMI-1640 containing no FCS was placed in the lower compartments of the 24 well plate; 150µl of cell suspension (6.7 x 10 5 cells/ml) in RPMI-1640 containing no FCS were placed in the upper compartment of the transwell inserts. When CXCR4 antagonists were evaluated, the desired compounds were incubated with the cells for 1hr before seeding in the upper compartment.

The upper and lower compartments are separated by a 6.5mm polycarbonate filter with a pore diameter of 8µm (Corning, Sigma product number CLS3422) coated with 50µg/ml collagen suspension. After 16 hr, the cells that had not migrated to the lower chamber were scraped off with a cotton bud. The filters were fixed with 70% ethanol and the cells stained with haematoxylin. The stained transwell membranes were cut, mounted onto microscope slides and analysed under microscope for the number of migrated cells. Six non-overlapping fields were analysed using ImageJ software to count average number of migrated cells. Modelling studies leading to the identification of ICT5040 Like other chemokine receptors, CXCR4 belongs to the rhodopsin-like (class A) G-protein-coupled 7-transmembrane helical domain (GPCR-7TM) superfamily.

Since the report of the first crystal structure of a member of this family over ten years ago, homology modelling and virtual screening has been extensively and successfully used to identify molecules that bind with GPCR receptors, including CXCR4[]. Since then, a number of other GPCR crystal structures have been reported, confirming and further justifying a role for computational modelling in computer assisted drug design[]. Our work was initiated before a crystal structure for CXCR4 was available[]. We started this study by constructing and validating a homology model based on bovine rhodopsin GPCR-7TM (pdb code 1U19) as a template (). This homology model was subsequently shown to be in good agreement with the published crystal structure of CXCR4[], particularly for the binding pocket which we used for virtual screening (). A view of the residues surrounding the binding pocket of CXCR4.

The top 100 virtual hits predicted from their binding affinity were screened for their effect on decreasing CXCL12 induced intracellular calcium mobilisation (calcium flux assay) at a single concentration (50 µM) that we assigned to be the maximum concentration defining an actual hit. At this concentration, eight compounds showed >20% reduction in calcium mobilisation. For these eight compounds, we determined IC 50 values from full dose-response curves. ICT5040, 1 () was revealed as the most potent (IC 50 = 3.8 ± 0.4 µM). The known CXCR4 antagonist AMD3100 produced an IC 50 = 0.8 ± 0.3 µM in this assay which is in agreement for the value previously reported in the literature (IC 50 = 0.6 µM)[]. In addition, ICT5040 (1) demonstrated a reduction in CXCL12-induced proliferation and migration.

A small library of structural analogues of ICT5040 (1), compounds 2- 5, was synthesised (), and we showed that they also reduced CXCL12-induced intracellular calcium mobilisation (). This suggested that the pyridyl-oxadiazole biaryl system pharmacophore can tolerate modification, while maintaining activity. Therefore, we decided to carry out a series of chemical modifications in order to identify more potent structural variants. Compound pIC 50 ΔG (kcal/mol) Compound pIC 50 ΔG (kcal/mol) AMD3100 6.10 N/A 13. Initial chemical modifications to ICT5040 In the first phase of this investigation, we considered three structural variations to ICT5040 (1) in order to improve its activity: modification to the (i) sulphur substituent; (ii) pyridyl core; and (iii) substituents at positions 2- and 6- of the pyridine-ring.

Using the general synthetic route outlined (), we prepared compounds 6- 10a in which the sulphur substituent was modified. However testing these compounds in the calcium flux assay showed that larger substituents were all significantly weaker antagonists, suggesting the steric bulk of the substituent adversely affects binding efficiency. The thiol compound 10a was also inactive although it is most likely that this compound tautomerises to 10b ().

Therefore, we decided to maintain the methyl substituent on the sulphur atom throughout further optimisation efforts. Compounds 11-13. Subsequently, we explored modification to the 6-position of the pyridine ring. Starting from commercially available 6-hydroxynicotinic acid, we prepared compounds 14- 17 () and 18-19 (). Compound 14, 15, 18 and 19 were relatively inactive, but in contrast, compound 16 and 17 had a similar potency to compound 1 (). We concluded that an oxygen or nitrogen atom at the 6-position of the pyridine reduces potency. However, this loss of activity could be compensated for if the oxygen or nitrogen atom is substituted with a chain that contains a polar group.

Binding poses for: (a) compound 1; (b) compound 20; (c) compound 22; (d) compound 25. The analysis of the binding mode of ICT5040 within this model reveals that the S-Me substituent of the oxadiazole ring is in proximity of His281 (TMVII). According to our model, replacement of the S-Me with larger groups introduces both steric congestion and lipophilicity near the imidazole ring in the side chain of this residue. This is consistent with the experimental observation that compounds 6- 9, in which the sulphur atom is substituted with larger lipophilic substituents, are less potent than 1.

The exact role of His281 residue in activation of the CXCR4 is not known, although site directed mutagenesis studies have shown that its mutation reduced the affinity of CXCR4 for AMD3465[,]. Based on our experimental observations, we can speculate however that sterically bulky lipophilic groups on the sulphur atom, adversely influence the ability of this residue to interact with any adjacent polar/acidic residue(s) which may be significant in the binding to the receptor. Further analysis of the binding mode of ICT5040 within the model also shows an interaction between the pyridine ring of the molecule as a H bond acceptor from the NH of the indole ring in Trp94. This observation rationalises the loss of activity observed in compound 11 where the pyridine ring is substituted with a benzene ring. The loss of activity observed in compound 12 and 13 can also be explained as the basicity of the ring nitrogen in pyrimidine and pyrazine are weaker than that of pyridine and hence there is weaker binding to the indole NH on Trp94. A similar argument can also rationalise why the introduction of a methoxy group at the 6-position reduced the potency. 2-Methoxy pyridine is less basic than pyridine, due to the strong inductive effect of highly electronegative oxygen atom[].

So we also expect that compound 14 is less basic than ICT5040, compound 1. Indeed docking of these molecules inside the model afforded consistently poor calculated binding affinities. The observation that potency can be restored by the introduction of a hydrogen bonding group in the side chain at the pyridyl ring’s position 6 (e.g.

Comparing compounds 14 and 16) can also be rationalised by our modelling. Whilst both Tyr115 and Glu288 are near enough to be candidates for an interaction with the side chain in compound 16 and 17, docking studies confirmed that an interaction with the former is more likely, as it gave a higher calculated affinity. Overall, the calculated binding affinity of compound 16 within the receptor was consistent with that observed for compound 1, which agrees with the similar potencies in the calcium flux assay observed for compounds 1 and 16 (). Whilst molecular modelling proved to be a useful tool in rationalising our observed potencies, it also proved valuable in informing the direction of the synthetic efforts aimed at producing compounds with improved potency. Based on our docking studies, we sought appropriate modifications to the pyridine’s 6-substituents to enable interactions with either Tyr115 or Glu288 residues.

An aminomethylene substituent at the 6- position, e.g. Compound 20 can provide the required interaction with Glu288 (). Furthermore, additional substituents on the nitrogen can provide interactions with other significant residues such as Tyr115 and Asp171 both of which are known to be important to binding to small molecule antagonists of CXCR4[]. Compound 20 and 21 were prepared as outlined (). Esterification of pyridine-2,5-dicarboxylic acid followed by treatment of the bis-ester with NaBH 4 resulted in selective reduction of the 2-carboxylate group[].

Carboxylic esters are usually resistant to reduction by NaBH 4 and the selective reduction of the 2-carboxylate group presumably arises because the nitrogen atom of the pyridyl core chelates to a borane molecule, which is generated in situ during the reaction, delivering a hydride to the adjacent carboxyl group at position 2. The carboxyl group at position 5- remains unaffected with this reducing agent. The hydroxyl group is then transformed to an N-Boc protected amine. This was then transformed to compound 20 using the sequence of reactions already shown, followed by removal of the Boc group with trifluoroacetic acid.

Compound 21 was prepared in a similar fashion (). Preparation of compounds 22-24. From modelling studies, we also postulated that the introduction of an appropriate substituent at position 2- of the pyridyl ring which can interact with Asp97 (), would increase the binding affinity between the molecule and the receptor, and thus improve inhibition. Compound 23 was synthesised as outlined (). Treatment of 2-methylnicotinic acid with 2 eq LDA followed by reaction with an alkylating agent led to chain extension at position 2- of pyridine[]. The presence of a carboxylic acid was found to be crucial as the reaction with the methyl ester failed. The product of the alkylation reaction was then transformed to the compound 25 using the sequence of reactions shown ().

As predicted by modelling studies, compound 25 was a more potent antagonist than compound 1. Preparation of compound 25. During the computational investigations, we used the free energy of binding between molecules and the receptor to guide optimisation of ICT5040. Of course, the binding between any small molecule and the GPCR receptor is a complex, multistep process.

However, this binding free energy provides a good approximation of the affinity between the small molecule and the CXCR4 receptor, and a good measure of the ease by which a small molecule can competitively block the access to the receptor by the natural ligand, CXCL12. In fact, we found a good relationship between compound potency (as measured by pIC 50 in a calcium flux assay) and the calculated free energy of its binding to the receptor (). Pharmacological characterisation of antagonists We showed that ICT5040, compound 1, reduces CXCL12-induced intracellular calcium mobilisation in U87 glioma cells. Calcium mobilisation is a feature of all activated GPCRs and generally speaking a reliable method to assess relative potency of receptor antagonists. This raises the issue of chemokine receptor selectivity. To this effect we showed that ICT5040, compound 1, does not target activation of other GPCR’s. Whilst compound 1 reduces CXCL12-induced intracellular calcium mobilisation in U87 glioma cells, it did not inhibit calcium mobilisation in the same cell line mediated by CCL21 on its cognate receptor CCR7, or fMLF on its cognate receptor, FPR-1.

CCR7 and FPR-1 are two related GPCR chemotactic receptors which we had previously shown are expressed on U87 glioma cells (data not shown). ICT5040, compound 1, also reduces CXCL12-induced proliferation of U87-glioma cells in a concentration dependent manner () consistent with the involvement of chemotactic axes in glioma proliferation[,]. We first showed that this receptor is expressed on the human glioma U87-MG cells (see file S2), and that the cell proliferation rates increase in response to CXCL12, the ligand for CXCR4 (). This increase in proliferation is specifically decreased by our small molecule CXCR4 antagonist. Whilst, ICT5040 itself has no effect on cell proliferation rates (). At a range of concentrations from 1-100 µM, ICT5040 abrogates CXCL12-induced cell proliferation. Interestingly, the antiproliferative potency of ICT5040 was similar to that of AMD3100 even though in the calcium mobilisation assay, ICT5040 potency was somewhat less.

ICT5040, compound 1, also significantly reduces CXCL12-induced migration of U87 cells as measured by the two chamber assay () and agarose-spot assay[] (). Images of U87 cells at the interface of an agarose spot and their subsequent migration into the agarose. The edge of the agarose spot is highlighted by the dotted white line. The extent of migration is dependent on the presence of CXCL12 ligand whereas cell migration is reduced in the presence of CXCR4 inhibitors (bar = 0.1 mm): (a) control (no CXCL12); (b) 125 nM CXCL12; (c) 125 nM CXCL12 and 200 µM AMD3100; (d) 125 nM CXCL12 and 200 µM ICT5040. (e) Reduction in the migration of cells by ICT5040, AMD3100 and CXCR4 mAb.

See methods for experimental details. Compound 20 is a soluble analogue of ICT5040 which is thirty-fold more potent in inhibiting intracellular calcium mobilisation. Hence we investigated the effect of compound 20 in CXCL12-induced proliferation and migration of U87 cells ( and ).

Whilst compound 20 did show better retardation of migration compared to ICT5040, it was less effective at inhibiting proliferation of U87 cells than ICT5040. Studies in vivo will ascertain whether the anti-migratory effects of compound 20 are a more important indicator of potential benefit in retarding tumour progression than a direct anti-proliferative effect.

Conclusions We have identified and synthesised a new class of small molecule CXCR4 antagonists based on the use of computational modelling in concert with experimental determination of in vitro activity. We have shown that chemotypes based on ICT5040, specifically inhibit CXCL12 induced intracellular calcium mobilisation, cell proliferation and migration, with a potency comparable to AMD3100, which is considered a benchmark small molecule antagonist of CXCR4.

Furthermore, we have carried out computational modelling led syntheses to afford compounds with improved anti-migratory potency. There is a clinical need for potent anti-metastatic CXCR4-antagonists with improved profile over existing ones[], particularly, for frequent dosing required for persistent blockade of CXCR4-induced tumour metastasis. New CXCR4 antagonist chemotypes, like the one described here, provide an opportunity to discover agents that could meet these criteria. In this regard, our computational modelling is proved to be a valuable tool to identify more potent analogues as CXCR-4 antagonists.