A benzenesulfonamide derivative as a novel PET radioligand for CXCR4.

CXCR4 is involved in various diseases such as inflammation, tumor growth, and cancer metastasis through the interaction with its natural endogenous ligand, chemokine CXCL12. In an effort to develop imaging probes for CXCR4, we developed a novel small molecule CXCR4-targeted PET agent (compound 5) by combining our established benzenesulfonamide scaffold with a labeling component by virtue of click chemistry. 5 shows nanomolar affinity (IC50 = 6.9 nM) against a known CXCR4 antagonist (TN14003) and inhibits more than 65% chemotaxis at 10 nM in vitro assays. Radiofluorinated compound 5 ([18F]5) demonstrates a competitive cellular uptake against CXCL12 in a dose-dependent manner. Further, microPET images of [18F]5 exhibits preferential accumulation of radioactivity in the lesions of λ-carrageenan-induced paw edema, human head and neck cancer orthotopic xenograft, and metastatic lung cancer of each mouse model.

Synthesis and evaluation of 2,5-diamino and 2,5-dianilinomethyl pyridine analogues as potential CXCR4 antagonists.

CXCR4 and its cognate ligand CXCL12 has been linked to various pathways such as cancer metastasis, inflammation, HIV-1 proliferation, and auto-immune diseases. Small molecules have shown potential as CXCR4 inhibitors and modulators, and therefore can mitigate diseases related to the CXCR4-CXCL12 pathway. We have designed and synthesized a series of 2,5-diamino and 2,5-dianilinomethyl pyridine derivatives as potential CXCR4 antagonists. Thirteen compounds have an effective concentration (EC) of 100 nM or less in a binding affinity assay and nine of these have at least 75% inhibition of invasion in Matrigel binding assay. Compounds 3l, 7f, 7j, and 7p show a minimal reduction in inflammation when carrageenan paw edema test is conducted. Overall, these compounds show potential as CXCR4 antagonist.

Synthesis and evaluation of 2,5 and 2,6 pyridine-based CXCR4 inhibitors.

Targeting the interaction between G-Protein Coupled Receptor, CXCR4, and its natural ligand CXCL12 is a leading strategy to mitigate cancer metastasis and reduce inflammation. Several pyridine-based compounds modeled after known small molecule CXCR4 antagonists, AMD3100 and WZ811, were synthesized. Nine hit compounds were identified. These compounds showed lower binding concentrations than AMD3100 (1000nM) and six of the nine compounds had an effective concentration (EC) less than or equal to WZ811 (10nM). Two of the hit compounds (2g and 2w) inhibited invasion of metastatic cells at a higher rate than AMD3100 (62%). Compounds 2g and 2w also inhibit inflammation in the same range as WZ811 in the paw edema test at 40% reduction in inflammation. These preliminary results are the promising foundation of a new class of pyridine-based CXCR4 antagonists.

Early prediction of response to Vorinostat in an orthotopic rat glioma model.

Glioblastoma is the most common primary brain tumor and is uniformly fatal despite aggressive surgical and adjuvant therapy. As survival is short, it is critical to determine the value of therapy early on in treatment. Improved early predictive assessment would allow neuro-oncologists to personalize and adjust or change treatment sooner to maximize the use of efficacious therapy. During carcinogenesis, tumor suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. Suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza) is an orally active, potent inhibitor of histone deacetylase (HDAC) activity. A major shortcoming of the use of HDAC inhibitors in the treatment of patients with brain tumors is the lack of reliable biomarkers to predict and determine response. Histological evaluation may reflect tumor viability following treatment, but is an invasive procedure and impractical for glioblastoma. Another problem is that response to SAHA therapy is associated with tumor redifferentiation and cytostasis rather than tumor size reduction, thus limiting the use of traditional imaging methods. A noninvasive method to assess drug delivery and efficacy is needed. Here, we investigated whether changes in (1)H MRS metabolites could render reliable biomarkers for an early response to SAHA treatment in an orthotopic animal model for glioma. Untreated tumors exhibited significantly elevated alanine and lactate levels and reduced inositol, N-acetylaspartate and creatine levels, typical changes reported in glioblastoma relative to normal brain tissues. The (1)H MRS-detectable metabolites of SAHA-treated tumors were restored to those of normal-like brain tissues. In addition, reduced inositol and N-acetylaspartate were found to be potential biomarkers for mood alteration and depression, which may also be alleviated with SAHA treatment. Our study suggests that (1)H MRS can provide reliable metabolic biomarkers at the earliest stage of SAHA treatment to predict the therapeutic response.

Discovery of novel aminopiperidinyl amide CXCR4 modulators through virtual screening and rational drug design.

The C-X-C chemokine receptor type 4 (CXCR4) is a potential therapeutic target for HIV infection, metastatic cancer, and inflammatory autoimmune diseases. In this study, we screened the ZINC chemical database for novel CXCR4 modulators through a series of in silico guided processes. After evaluating the screened compounds for their binding affinities to CXCR4 and inhibitory activities against the chemoattractant CXCL12, we identified a hit compound (ZINC 72372983) showing 100 nM affinity and 69% chemotaxis inhibition at the same concentration (100 nM). To increase the potency of our hit compound, we explored the protein-ligand interactions at an atomic level using molecular dynamics simulation which enabled us to design and synthesize a novel compound (Z7R) with nanomolar affinity (IC50 = 1.25 nM) and improved chemotaxis inhibition (78.5%). Z7R displays promising anti-inflammatory activity (50%) in a mouse edema model by blocking CXCR4-expressed leukocytes, being supported by our immunohistochemistry study.

Amide-sulfamide modulators as effective anti-tumor metastatic agents targeting CXCR4/CXCL12 axis.

Breast cancer is the most frequently diagnosed malignancy and the second common cause of death in women worldwide. High mortality in breast cancer is frequently associated with metastatic progression rather than the primary tumor itself. It has been recently identified that the CXCR4/CXCL12 axis plays a pivotal role in breast cancer metastasis, especially in directing metastatic cancer cells to CXCL12-riched organs and tissues. Herein, taking the amide-sulfamide as the lead structure, the second-round structural modifications to the sulfamide structure were performed to obtain more active CXCR4 modulators against tumor metastasis. Both in vivo and in vitro experiments illustrated that compound IIIe possessed potent CXCR4 binding affinity, excellent anti-metastatic and anti-angiogenetic activity against breast cancer. More importantly, in a mouse breast cancer lung metastasis model, compound IIIe exerted a significant inhibitory effect on breast cancer metastasis. Taken together, all these positive results demonstrated that developing of CXCR4 modulators is a promising strategy to mediate breast cancer metastasis.

CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models.

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes to the lymph nodes and lungs. We have generated previously an orthotopic mouse model for head and neck metastasis and did in vivo selection of SCCHN cells through four rounds of serial metastases. A subpopulation of 686LN cells with high metastatic potential (686LN-Ms) was isolated. When the highly metastatic cells were compared with their low metastatic parental cells (686LN-Ps), we found that CXC chemokine receptor-4 (CXCR4) mRNA levels were significantly higher in the 686LN-Ms cells than the 686LN-Ps cells. Interestingly, the metastatic subclones had lost epithelial morphology and acquired mesenchymal features, which were maintained during cell expansion in vitro. This was featured by decreased E-cadherin and involucrin and increased vimentin and integrin beta(1). These results imply that CXCR4 and epithelial-mesenchymal transition markers can be potential biomarkers to identify the subpopulation of cells with high metastatic potential. Using the orthotopic SCCHN animal model, we showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Because the reduction of metastasis seen in the treated group could have resulted from 2-fold reduction in primary tumor size compared with that in the control group, we examined the effects of the CXCR4 antagonist in an experimental metastatic animal model in which 686LN-Ms cells were i.v. injected. 686LN-Ms cells failed to metastasize in the CXCR4 antagonist-treated group, whereas they metastasized to the lungs in the control group. Our data indicate that CXCR4 is an important target to inhibit tumor progression in SCCHN.

Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based derivatives as CXCR4 inhibitors.

The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD). Blocking this interaction could potentially reduce inflammatory symptoms in IBD patients. In this work, several thiophene-based and furan-based compounds modeled after AMD3100 and WZ811-two known antagonists that interrupt the CXCR4-CXCL12 interaction-were synthesized and analyzed. Fifteen hit compounds were identified; these compounds exhibited effective concentrations (EC) lower than 1000 nM (AMD3100) and inhibited invasion of metastatic cells by at least 45%. Selected compounds (2d, 2j, 8a) that inhibited metastatic invasion at a higher rate than WZ811 (62%) were submitted for a carrageenan inflammation test, where both 8a and 2j reduced inflammation in the same range as WZ811 (40%) but did not reduce inflammation more than 40%. Select compounds were also modeled in silico to show key residue interactions. These preliminary results with furan-based and thiophene-based analogues contribute to the new class on heterocyclic aromatic-based CXCR4 antagonists.

Development of CXCR4 modulators based on the lead compound RB-108.

The CXCR4/CXCL12 axis plays prominent roles in tumor metastasis and inflammation. CXCR4 has been shown to be involved in a variety of inflammation-related diseases. Therefore, CXCR4 is a promising potential target to develop novel anti-inflammatory agents. Taking our previously discovered CXCR4 modulator RB-108 as the lead compound, a series of derivatives were synthesized structurally modifying and optimizing the amide and sulfamide side chains. The derivatives successfully maintained potent CXCR4 binding affinity. Furthermore, compounds IIb, IIc, IIIg, IIIj, and IIIm were all efficacious in inhibiting the invasion of CXCR4-positive cells, displaying a much more potent effect than the lead compound RB-108. Notably, compound IIIm significantly decreased carrageenan-induced swollen volume and paw thickness in a mouse paw edema model. More importantly, IIIm exhibited satisfying PK profiles with a half-life of 4.77 h in an SD rat model. In summary, we have developed compound IIIm as a new candidate for further investigation based on the lead compound RB-108.

Assessing Treatment Response of Glioblastoma to an HDAC Inhibitor Using Whole-Brain Spectroscopic MRI.

Histone deacetylases regulate a wide variety of cellular functions and have been implicated in redifferentiation of various tumors. Histone deacetylase inhibitors (HDACi) are potential pharmacologic agents to improve outcomes for patients with gliomas. We assessed the therapeutic efficacy of belinostat (PXD-101), an HDACi with blood-brain barrier permeability. Belinostat was first tested in an orthotopic rat glioma model to assess in vivo tumoricidal effect. Our results showed that belinostat was effective in reducing tumor volume in the orthotopic rat glioma model in a dose-dependent manner. We also tested the antidepression activity of belinostat in 2 animal models of depression and found it to be effective. Furthermore, we confirmed that myo-inositol levels improved by belinostat treatment in vitro. In a human pilot study, it was observed that belinostat in combination with chemoradiation may delay initial recurrence of disease. Excitingly, belinostat significantly improved depressive symptoms in patients with glioblastoma compared with control subjects. Finally, spectroscopic magnetic resonance imaging of 2 patient cases from this pilot study are presented to indicate how spectroscopic magnetic resonance imaging can be used to monitor metabolite response and assess treatment effect on whole brain. This study highlights the potential of belinostat to be a synergistic therapeutic agent in the treatment of gliomas.

Anti-inflammatory hybrids of secondary amines and amide-sulfamide derivatives.

The CXCR4/CXCL12 chemokine axis can chemotactically accumulate inflammatory cells to local tissues and regulate the release of inflammatory factors. Developing novel CXCR4 modulators may provide a desirable strategy to control the development of inflammation. A series of novel hybrids were designed by integrating the key pharmacophores of three CXCR4 modulators. The majority of compounds displayed potent CXCR4 binding affinity. Compound 7a exhibited 1000-fold greater affinity than AMD3100 and significantly inhibited invasion of CXCR4-positive tumor cells. Additionally, compound 7a blocked mice ear inflammation by 67% and suppressed the accumulation of inflammatory cells in an in vivo mouse ear edema evaluation. Western blot analyses revealed that 7a inhibited the CXCR4/CXCL12-mediated phosphorylation of Akt and p44 in a dose-dependent manner. Moreover, compound 7a had no observable cytotoxicity and displayed a favorable plasma stability in our preliminary pharmacokinetic study. These results confirmed that this is a feasible method to develop CXCR4 modulators for the regulation and reduction of inflammation.

Silencing of CXCR4 blocks breast cancer metastasis.

RNA interference technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. For the first time in cancer research, we show that direct injection of a pool of naked small interfering RNA (siRNA) duplexes can prevent tumorigenesis in an animal model, suggesting a novel preventive and therapeutic strategy for cancer management. As a model system, we used siRNA duplexes of CXCR4 to block breast cancer metastasis. Here, we show that blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis.

Novel anti-inflammatory agents targeting CXCR4: Design, synthesis, biological evaluation and preliminary pharmacokinetic study.

CXCR4 plays a crucial role in the inflammatory disease process, providing an attractive means for drug targeting. A series of novel amide-sulfamide derivatives were designed, synthesized and comprehensively evaluated. This new scaffold exhibited much more potent CXCR4 inhibitory activity, with more than 70% of the compounds showed notably better binding affinity than the reference drug AMD3100 in the binding assay. Additionally, in the Matrigel invasion assay, most of our compounds significantly blocked the tumor cell invasion, demonstrating superior efficacy compared to AMD3100. Furthermore, compound IIj blocked mice ear inflammation by 75% and attenuated ear edema and damage substantially in an in vivo model of inflammation. Western blot analyses revealed that CXCR4 modulator IIj significantly blocked CXCR4/CXCL12-mediated phosphorylation of Akt. Moreover, compound IIj had no observable cytotoxicity and displayed a favourable plasma stability in our preliminary pharmacokinetic study. The preliminary structure-activity relationships were also summarized. In short, this novel amide-sulfamide scaffold exhibited potent CXCR4 inhibitory activity both in vitro and in vivo. These results also confirmed that developing modulators targeting CXCR4 provides an exciting avenue for treatment of inflammation.

Development of CXCR4 modulators by virtual HTS of a novel amide-sulfamide compound library.

CXCR4 plays a crucial role in recruitment of inflammatory cells to inflammation sites at the beginning of the disease process. Modulating CXCR4 functions presents a new avenue for anti-inflammatory strategies. However, using CXCR4 antagonists for a long term usage presents potential serious side effect due to their stem cell mobilizing property. We have been developing partial CXCR4 antagonists without such property. A new computer-aided drug design program, the FRESH workflow, was used for anti-CXCR4 lead compound discovery and optimization, which coupled both compound library building and CXCR4 docking screens in one campaign. Based on the designed parent framework, 30 prioritized amide-sulfamide structures were obtained after systemic filtering and docking screening. Twelve compounds were prepared from the top-30 list. Most synthesized compounds exhibited good to excellent binding affinity to CXCR4. Compounds Ig and Im demonstrated notable in vivo suppressive activity against xylene-induced mouse ear inflammation (with 56% and 54% inhibition). Western blot analyses revealed that Ig significantly blocked CXCR4/CXCL12-mediated phosphorylation of Akt. Moreover, Ig attenuated the amount of TNF-α secreted by pathogenic E. coli-infected macrophages. More importantly, Ig had no observable cytotoxicity. Our results demonstrated that FRESH virtual high throughput screening program of targeted chemical class could successfully find potent lead compounds, and the amide-sulfamide pharmacophore was a novel and effective framework blocking CXCR4 function.

Combination of an Integrin-Targeting NIR Tracer and an Ultrasensitive Spectroscopic Device for Intraoperative Detection of Head and Neck Tumor Margins and Metastatic Lymph Nodes.

Despite major advances in targeted drug therapy and radiation therapy, surgery remains the most effective treatment for most solid tumors. The single most important predictor of patient survival is a complete surgical resection of the primary tumor, draining lymph nodes, and metastatic lesions. Presently, however, 20%-30% of patients with head and neck cancer who undergo surgery still leave the operating room without complete resection because of missed lesions. Thus, major opportunities exist to develop advanced imaging tracers and intraoperative instrumentation that would allow surgeons to visualize microscopic tumors during surgery. The cell adhesion molecule integrin αvß3 is specifically expressed by tumor neovasculature and invading tumor cells, but not by quiescent vessels or normal cells. Here we report the combined use of an integrin-targeting near-infrared tracer (RGD-IRDye800CW) and a handheld spectroscopic device, an integrated point spectroscopy with wide-field imaging system, for highly sensitive detection of integrin overexpression on infiltrating cancer cells. By using an orthotopic head and neck cancer animal model, we show that this tracer-device combination allows intraoperative detection of not only invasive tumor margins but also metastatic lymph nodes. Correlated histological analysis further reveals that microscopic clusters of 50-100 tumor cells can be detected intraoperatively with high sensitivity and specificity, raising new possibilities in guiding surgical resection of microscopic tumors and metastatic lymph nodes.

Symmetrical bis-tertiary amines as novel CXCR4 inhibitors.

CXCR4 inhibitors are promising agents for the treatment of cancer metastasis and inflammation. A series of novel tertiary amine derivatives targeting CXCR4 were designed, synthesized, and evaluated. The central benzene ring linker and side chains were modified and optimized to study the structure-activity relationship. Seven compounds displayed much more potent activity than the reference drug, AMD3100, in both the binding affinity assay and the blocking of Matrigel invasion functional assay. These compounds exhibited effective concentration ranging from 1 to 100 nM in the binding affinity assay and inhibited invasion from 65.3% to 100% compared to AMD3100 at 100 nM. Compound IIn showed a 50% suppressive effect against carrageenan-induced paw inflammation in a mouse model, which was as effective as the peptidic antagonist, TN14003 (48%). These data demonstrate that symmetrical bis-tertiary amines are unique CXCR4 inhibitors with high potency.

Benzenesulfonamides: a unique class of chemokine receptor type 4 inhibitors.

The interaction of CXCR4 with CXCL12 (SDF-1) plays a critical role in cancer metastasis by facilitating the homing of tumor cells to metastatic sites. Based on our previously published work on CXCR4 antagonists, we have synthesized a series of aryl sulfonamides that inhibit the CXCR4/CXCL12 interaction. Analogue bioactivities were assessed with binding affinity and Matrigel invasion assays. Computer modeling was employed to evaluate a selection of the new analogues docked into the CXCR4 X-ray structure and to rationalize discrepancies between the affinity and Matrigel in vitro assays. A lead compound displays nanomolar potency in the binding affinity assay (IC(50)=8.0 nM) and the Matrigel invasion assay (100 % blockade of invasion at 10 nM). These data demonstrate that benzenesulfonamides are a unique class of CXCR4 inhibitors with high potency.

Monocarbonyl curcumin analogues: heterocyclic pleiotropic kinase inhibitors that mediate anticancer properties.

Curcumin is a biologically active component of curry powder. A structurally related class of mimetics possesses similar anti-inflammatory and anticancer properties. Mechanism has been examined by exploring kinase inhibition trends. In a screen of 50 kinases relevant to many forms of cancer, one member of the series (4, EF31) showed ≥85% inhibition for 10 of the enzymes at 5 µM, while 22 of the proteins were blocked at ≥40%. IC50 values for an expanded set of curcumin analogues established a rank order of potencies, and analyses of IKKß and AKT2 enzyme kinetics for 4 revealed a mixed inhibition model, ATP competition dominating. Our curcumin mimetics are generally selective for Ser/Thr kinases. Both selectivity and potency trends are compatible with protein sequence comparisons, while modeled kinase binding site geometries deliver a reasonable correlation with mixed inhibition. Overall, these analogues are shown to be pleiotropic inhibitors that operate at multiple points along cell signaling pathways.

Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis.

BACKGROUND: A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. METHODOLOGY/PRINCIPAL FINDINGS: The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. CONCLUSIONS/SIGNIFICANCE: CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.