Small molecule agonist of very late antigen-4 (VLA-4) integrin induces progenitor cell adhesion.

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4ß1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4ß1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4ß1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/ß subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4ß7 as well as α5ß1 and αLß2. When cross-linked to αLß2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/ß subunit interface and not at the ligand-binding site in the inserted ("I") domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level.

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [(18)F]-labeled STI-571 was prepared with high specific activity (75 GBq/µmol) by nucleophilic displacement and an average radiochemical yield of 12%. [(131)I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [(18)F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [(18)F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.

Degrasyn-like symmetrical compounds: possible therapeutic agents for multiple myeloma (MM-I).

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.

Improved synthesis of 17ß-hydroxy-16α-iodo-wortmannin, 17ß-hydroxy-16α-iodoPX866, and the [(131)I] analogue as useful PET tracers for PI3-kinase.

An improved method for the synthesis of 17ß-hydroxy-16α-iodo-wortmannin along with the first synthesis of 17ß-hydroxy-16α-iodoPX866 and [(131)I] radiolabeled 17ß-hydroxy-16α-[(131)I]iodo-wortmannin, as potential PET tracers for PI3K was also described. The differences between wortmannin and its iodo analogue were compared by covalently docking each structure to L833 in PI3K.

Binding partners for curcumin in human schwannoma cells: biologic implications.

Curcumin (diferuloylmethane) is a potent anti-inflammatory and anti-tumorigenic agent that has shown preclinical activity in diverse cancers. Curcumin up-regulates heat shock protein 70 (hsp70) mRNA in several different cancer cell lines. Hsp70 contributes to an escape from the apoptotic effects of curcumin by several different mechanisms including prevention of the release of apoptosis inducing factor from the mitochondria and inhibition of caspases 3 and 9. Previously we showed that the combination of curcumin plus a heat shock protein inhibitor was synergistic in its down-regulation of the proliferation of a human schwannoma cell line (HEI-193) harboring an NF2 mutation, possibly because curcumin up-regulated hsp70, which also binds merlin, the NF2 gene product. In order to determine if curcumin also interacts directly with hsp70 and to discover other binding partners of curcumin, we synthesized biotinylated curcumin (bio-curcumin) and treated HEI-193 schwannoma cells. Cell lysates were prepared and incubated with avidin-coated beads. Peptides pulled down from this reaction were sequenced and it was determined that biotinylated curcumin bound hsp70, hsp90, 3-phosphoglycerate dehydrogenase, and a ß-actin variant. These binding partners may serve to further elucidate the underlying mechanisms of curcumin's actions.

Synthesis of a Macrocycle Based on Linked Amino Acid Mimetics (LAAM).

We report the synthesis of a macrocycle utilizing a novel framework of standard amino acids in combination with subunits that we have named as Linked Amino Acid Mimetics (LAAM's). Macrocycles based on the LAAM concept provide both a peptide targeting region and two independently variable functional regions. In the prototype structure, the commonly known Arg-Gly-Asp (RGD) sequence was used for the targeting region. The functional regions contain a phenyl group, and the linkage was formed via a Ring-Closing Metathesis (RCM) reaction.

Molecular-genetic PET imaging using an HSV1-tk mutant reporter gene with enhanced specificity to acycloguanosine nucleoside analogs.

UNLABELLED: Imaging 2 different molecular-genetic events in a single subject by PET is essential in a variety of in vivo applications. Using herpes simplex virus-1 thymidine kinase (HSV1-tk) mutants with narrower substrate specificities in combination with wild-type HSV1-tk (wtHSV1-tk) would enable differential imaging with corresponding radiotracers, namely 2'-deoxy-2'-(18)F-fluoro-5-ethyl-1-beta-d-arabinofuranosyl-uracil ((18)F-FEAU) and the acycloguanosine derivative 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG). In this study, we evaluated wtHSV1-tk and the A168H mutant, which has been reported to exhibit enhanced acycloguanosine substrate catalytic activity and diminished pyrimidine phosphorylating activity, as PET reporter genes. METHODS: Computational analysis was performed to assess the binding mode of FHBG and FEAU to wtHSV1-tk and the A168H variant. U87 cells were stably transduced with wtHSV1-tk or HSV1-tk(A168H) fused with green fluorescent protein and sorted to obtain equivalent transgene expression. In vitro uptake studies were performed to determine rates of substrate accumulation and retention. Nude mice bearing tumors expressing HSV1-tk variants were subsequently imaged using (18)F-FHBG and (18)F-FEAU. RESULTS: Docking results indicate that binding of FHBG to the A168H variant is unaffected whereas the binding of FEAU is hindered because of a steric clash with the bulkier mutant residues. U87 cells expressing HSV1-tk(A168H) accumulated (18)F-FHBG in in vitro uptake studies at a 3-fold higher rate than did cells expressing wtHSV1-tk without any detectable accumulation of (3)H-FEAU. Furthermore, HSV1-tk(A168H) demonstrated no thymidine phosphorylation activity. In contrast, U87 cells expressing wtHSV1-tk preferentially accumulated (3)H-FEAU at an 18-fold higher rate than they did (18)F-FHBG. Tumors expressing wtHSV1-tk or HSV1-tk(A168H) were distinctly imaged with (18)F-FEAU or (18)F-FHBG, respectively. Hence, tumors expressing HSV1-tk(A168H) accumulated 8.4-fold more (18)F-FHBG than did tumors expressing wtHSV1-tk. In addition, wtHSV1-tk tumors, compared with HSV1-tk(A168H)-expressing tumors (which retained baseline levels of the radiotracer), preferentially accumulated (18)F-FEAU. CONCLUSION: The FEAU and FHBG substrate discrimination capacity of the wtHSV1-tk and HSV1-tk(A168H) reporter enzymes was validated in vivo by PET of mice with tumor xenografts established from U87 cells expressing these different reporters. Thus, HSV1-tk(A168H) may potentially be used as a second reporter gene in combination with wtHSV1-tk to achieve differential PET.

Magnetic Resonance Imaging of Atherosclerotic Plaque at Clinically Relevant Field Strengths (1T) by Targeting the Integrin α4ß1.

Inflammation drives the degradation of atherosclerotic plaque, yet there are no non-invasive techniques available for imaging overall inflammation in atherosclerotic plaques, especially in the coronary arteries. To address this, we have developed a clinically relevant system to image overall inflammatory cell burden in plaque. Here, we describe a targeted contrast agent (THI0567-targeted liposomal-Gd) that is suitable for magnetic resonance (MR) imaging and binds with high affinity and selectivity to the integrin α4ß1(very late antigen-4, VLA-4), a key integrin involved in recruiting inflammatory cells to atherosclerotic plaques. This liposomal contrast agent has a high T1 relaxivity (~2 × 105 mM-1s-1 on a particle basis) resulting in the ability to image liposomes at a clinically relevant MR field strength. We were able to visualize atherosclerotic plaques in various regions of the aorta in atherosclerosis-prone ApoE-/- mice on a 1 Tesla small animal MRI scanner. These enhanced signals corresponded to the accumulation of monocyte/macrophages in the subendothelial layer of atherosclerotic plaques in vivo, whereas non-targeted liposomal nanoparticles did not demonstrate comparable signal enhancement. An inflammatory cell-targeted method that has the specificity and sensitivity to measure the inflammatory burden of a plaque could be used to noninvasively identify patients at risk of an acute ischemic event.

Detection of histone deacetylase inhibition by noninvasive magnetic resonance spectroscopy.

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS)-based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a (19)F MRS molecular marker of HDAC activity together with (31)P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluoro-suberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 micromol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 micromol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by (31)P MRS, increased from 7 to 16 fmol/cell following treatment with FSAHA and also negatively correlated with HDAC activity. Increased phosphocholine is probably due to heat shock protein 90 inhibition as indicated by depletion of client proteins. In summary, (19)F MRS of BLT, combined with (31)P MRS, can be used to monitor HDAC activity in cells. In principle, this could be applied in vivo to noninvasively monitor HDAC activity.

Designing and developing S100P inhibitor 5-methyl cromolyn for pancreatic cancer therapy.

We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 µmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC.

Protein kinase Calpha and epsilon small-molecule targeted therapeutics: a new roadmap to two Holy Grails in drug discovery?

Protein kinase (PK)Calpha and epsilon are rational targets for cancer therapy. However, targeted experimental therapeutics that inhibit PKCalpha or epsilon are unavailable. The authors established recently that covalent modification of an active-site cysteine in human PKCepsilon, Cys452, by small molecules, for example 2-mercaptoethanolamine, is necessary and sufficient to render PKCepsilon kinase-dead. Cys452 is conserved in only eleven human protein kinase genes, including PKCalpha. Therefore, the design of small molecules that bind PKC active sites with an electrophile substituent positioned proximal to the Cys452 side chain may lead to targeted therapeutics that selectively inhibit PKCepsilon, PKCalpha or other PKC isozymes.

A 3D structure model of integrin alpha 4 beta 1 complex: I. Construction of a homology model of beta 1 and ligand binding analysis.

It is well established that integrin alpha 4 beta 1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to alpha 4 beta 1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with alpha 4 beta 1, we began constructing a structure model for the integrin alpha 4 beta 1 complex. As the first step, we have built a homology model of the beta 1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg(2+) ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to alpha 4 beta 1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for beta 1/beta 7 ligand binding selectivity.