Kuiying Xu

A Radiotracer Strategy to Quantify PARP-1 Expression In Vivo Provides a Biomarker That Can Enable Patient Selection for PARP Inhibitor Therapy.

Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [(125)I] KX1: as a PARP-1-selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo The pharmacologic properties of the PARP radiotracer [(125)I] KX1: was characterized in multiple cell lines where single-agent sensitivity was correlated with [(125)I] KX1: binding to PARP-1. In vivo evaluation of [(125)I] KX1: verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [(125)I] KX1: correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516-24. ©2016 AACR.

Development of a Positron Emission Tomography Radiotracer for Imaging Elevated Levels of Superoxide in Neuroinflammation

Reactive oxygen species (ROS) are believed to play a major role in the proinflammatory, M1-polarized form of neuroinflammation. However, it has been difficult to assess the role of ROS and their role in neuroinflammation in animal models of disease because of the absence of probes capable of measuring their presence with the functional imaging technique positron emission tomography (PET). This study describes the synthesis and in vivo evaluation of [18F]ROStrace, a radiotracer for imaging superoxide in vivo with PET, in an LPS model of neuroinflammation. [18F]ROStrace was found to rapidly cross the blood-brain barrier (BBB) and was trapped in the brain of LPS-treated animals but not the control group. [18F] ox-ROStrace, the oxidized form of [18F]ROStrace, did not cross the BBB. These data suggest that [18F]ROStrace is a suitable radiotracer for imaging superoxide levels in the central nervous system with PET.

Iodinated benzimidazole PARP radiotracer for evaluating PARP1/2 expression in vitro and in vivo

BACKGROUND PARP inhibitors (PARPi) have the potential to impact cancer therapy in a selective patient population; however, despite current patient selection methods clinical trials have shown mixed response rates. It is therefore clinically useful to determine which patients will respond prior to receiving PARPi therapy. One essential biomarker is to measure the level of PARP enzyme expression in tumors. Small molecule radiotracers have been developed to accurately quantify PARP-1 expression in vitro and in vivo. [125I]KX-02-019 is the first report of a radioiodinated analogue of the benzimidazole class of PARPi. Herein, we studied the pharmacological properties of [125I]KX-02-019 as well as the in vivo biodistribution. METHODS [125I]KX-02-019 was evaluated in both cancer and non-cancer cell lines. We evaluated the pharmacologic properties of [125I]KX-02-019 in live cells by measuring enzyme association and dissociation kinetics, saturation, and specificity. In addition, competitive inhibition experiments were carried out with commercially available PARPi. Protein expression was analyzed by Western blot to compare PARP-1 and PARP-2 expression across cell lines studied. The biodistribution was studied in a mouse EMT6 tumor model at time points of 0.5, 1, 2, 4 and 6h. RESULTS [125I]KX-02-019 showed subtle differences in pharmacological properties in the absence of PARP-2. In addition, [125I]KX-02-019 was competitively displaced by clinical PARPi. In vivo biodistribution studies showed an increasing tumor to muscle ratio over 6h as well as fast clearance from healthy tissues. CONCLUSION [125I]KX-02-019 has binding sites in both PARP1 KO cells as well as PARP2 KO cells showing higher affinity for PARP-2. This observation is supported by a decrease in binding affinity in PARP2 KO cells compared to PARP1 KO cells. The pharmacologic and biological properties of [125I]KX-02-019 studied in vitro and in vivo showed that this analogue may be useful in determining pharmacokinetic and pharmacodynamic properties of clinical PARPi.

A PET imaging agent for evaluating PARP-1 expression in ovarian cancer

BACKGROUND. Poly(ADP-ribose) polymerase (PARP) inhibitors are effective in a broad population of patients with ovarian cancer; however, resistance caused by low enzyme expression of the drug target PARP-1 remains to be clinically evaluated in this context. We hypothesize that PARP-1 expression is variable in ovarian cancer and can be quantified in primary and metastatic disease using a novel PET imaging agent. METHODS. We used a translational approach to describe the significance of PET imaging of PARP-1 in ovarian cancer. First, we produced PARP1-KO ovarian cancer cell lines using CRISPR/Cas9 gene editing to test the loss of PARP-1 as a resistance mechanism to all clinically used PARP inhibitors. Next, we performed preclinical microPET imaging studies using ovarian cancer patient–derived xenografts in mouse models. Finally, in a phase I PET imaging clinical trial we explored PET imaging as a regional marker of PARP-1 expression in primary and metastatic disease through correlative tissue histology. RESULTS. We found that deletion of PARP1 causes resistance to all PARP inhibitors in vitro, and microPET imaging provides proof of concept as an approach to quantify PARP-1 in vivo. Clinically, we observed a spectrum of standard uptake values (SUVs) ranging from 2–12 for PARP-1 in tumors. In addition, we found a positive correlation between PET SUVs and fluorescent immunohistochemistry for PARP-1 (r2 = 0.60). CONCLUSION. This work confirms the translational potential of a PARP-1 PET imaging agent and supports future clinical trials to test PARP-1 expression as a method to stratify patients for PARP inhibitor therapy. TRIAL REGISTRATION. Clinicaltrials.gov NCT02637934. FUNDING. Research reported in this publication was supported by the Department of Defense OC160269, a Basser Center team science grant, NIH National Cancer Institute R01CA174904, a Department of Energy training grant DE-SC0012476, Abramson Cancer Center Radiation Oncology pilot grants, the Marsha Rivkin Foundation, Kaleidoscope of Hope Foundation, and Paul Calabresi K12 Career Development Award 5K12CA076931.

Rapid Cu-Catalyzed [211At]Astatination and [125I]Iodination of Boronic Esters at Room Temperature

Access to 211At- and 125I-radiolabeled compounds in excellent RCCs and RCYs was achieved in just 10 min at room temperature using a Cu catalyst. The reaction conditions are applicable to a broad class of aryl and heteroaryl boronic reagents with varying steric and electronic properties as well as late-stage astatination and iodination of anticancer PARP inhibitors. This protocol eliminates the traditional need for toxic organotin reagents, elevated temperatures, and extended reaction times, providing a more practical and environmentally friendly approach to developing α-emitting radiotherapeutics.

Highly Selective Dopamine D3 Receptor Antagonists with Arylated Diazaspiro Alkane Cores

A series of potent and selective D3 receptor (D3R) analogues with diazaspiro alkane cores were synthesized. Radioligand binding of compounds 11, 14, 15a, and 15c revealed favorable D3R affinity (Ki = 12-25.6 nM) and were highly selective for D3R vs D3R (ranging from 264- to 905-fold). Variation of these novel ligand architectures can be achieved using our previously reported 10-20 min benchtop C-N cross-coupling methodology, affording a broad range of arylated diazaspiro precursors.

Abstract 5197: Targeting PARP-1 to deliver alpha-particles to cancer chromatin

Introduction: Neuroblastoma (NB) is a radiosensitive pediatric cancer that develops in the sympathetic nervous system and typically affects children under the age of 10. High-risk NB is associated with a 40% 5-year survival rate. Nuclear enzyme poly (ADP-ribose) polymerase 1 (PARP-1) is overexpressed in high-risk NB, making it an attractive target for alpha-particle therapy. Alpha-particles have a short path length and high linear energy transfer, causing dense ionizations across DNA, inducing double stranded breaks that result in cell death. The purpose of this study was to explore a newly developed radiotherapeutic ([ 211 At]MM4) that combines the targeting potential of a small molecule PARP inhibitor (PARPi) with the cytotoxic effects of 211 At in high-risk NB. Methods: In vitro cytotoxicity was performed in a panel of high-risk NB cell lines to evaluate the relative potency of [ 211 At]MM4. Next, DNA damage was assessed by measuring gH2AX foci formation at 1, 4, and 24 hrs after [ 211 At]MM4 treatment. In parallel, PARP-1 expression was measured in response to therapy and cleaved PARP-1 was quantified to assess apoptosis. Cell cycle analysis was performed after treatment to identify therapy related effects. The in-vivo biodistribution of [ 211 At]MM4 was performed alongside ex-vivo autoradiography. Tumor cytology for PARP-1, gH2AX, and Ki-67 was performed in response to therapy. Finally, in-vivo therapy experiments were performed. Results: Cytotoxicity data indicated a significant reduction in cell viability following treatment for all six NB cell lines, in-vitro co-incubation studies confirmed the specificity of [ 211 At]-MM4 to its drug target. Immunofluorescence analysis showed a dose-dependent increase in both gH2AX and PARP-1 expression. The in-vivo biodistribution of [ 211 At]MM4 revealed rapid tumor targeting at 1 hr and clearance from all tissues at 4 hrs. Ex-vivo autoradiography showed a tumor-muscle ratio greater than 6. Tumor cytology revealed DNA damage measured by gH2AX and PARP-1 expression increased following treatment. Small colonies of proliferating tumor cells were detected after treatment using Ki-67 staining. In-vivo therapy efficacy studies revealed low fractionated doses were tolerable and resulted in significant delay in tumor regrowth. Conclusion: [ 211 At]MM4 is a novel alpha-emitting radiotherapeutic that specifically targets nuclear PARP-1 overexpression in neuroblastoma and incites double-stranded breaks in cancer DNA. The cytotoxic effects of [ 211 At]MM4 have been experimentally validated both in-vitro and in-vivo; the results of these experiments confirm the therapeutic potential of [ 211 At]MM4 as a viable treatment option for high-risk neuroblastoma. Citation Format: Laura Puentes, Kuiying Xu, Catherine Hou, Robert H. Mach, John M. Maris, Daniel A. Pryma, Mehran Makvandi. Targeting PARP-1 to deliver alpha-particles to cancer chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5197. doi:10.1158/1538-7445.AM2017-5197

Dopamine D3 receptor partial agonist LS-3-134 attenuates cocaine-motivated behaviors

Aims The dopamine D3 receptor (D3R) is a pharmacotherapeutic target for drug dependence. We have successfully imaged human D3Rs using radiolabeled LS‐3‐134, an arylamide phenylpiperazine with moderate selectivity for the D3R over D2R and low efficacy at the D2 and D3R. In this study, we screened for effects of LS‐3‐134 as a potential anti‐cocaine therapeutic. Methods Male rats were pretreated with LS‐3‐134 (0, 1.0, 3.2, or 5.6 mg/kg, IP) 15 min prior to tests for its effects on spontaneous and cocaine‐induced locomotion. We next investigated the effects of LS‐3‐134 (0, 1.0, 3.2, 5.6, or 10.0 mg/kg, IP) on operant responding on a multiple variable‐interval (VI) 60‐second schedule with alternating cocaine (0.375 mg/kg, IV) and sucrose (45 mg) reinforcer components. Additionally, we tested LS‐3‐134 (5.6 mg/kg, IP) effects on a progressive ratio (PR) schedule of cocaine reinforcement, on extinction of cocaine‐seeking behavior, and on reinstatement of extinguished cocaine‐seeking behavior by cocaine‐associated light/tone cues. Results LS‐3‐134 did not alter spontaneous locomotion, but at 5.6 mg/kg, it reduced cocaine‐induced locomotion, break points on the high‐effort progressive ratio schedule of reinforcement, and responding during extinction and cue reinstatement. In contrast, LS‐3‐134 did not alter cocaine or sucrose reinforcement on the low‐effort multiple VI 60‐second schedule. Conclusions The effects of LS‐3‐134 are similar to other dopamine D3 low efficacy partial agonists and antagonists in attenuating cocaine intake under high effort schedules of reinforcement and in attenuating cocaine‐seeking behavior elicited by cocaine‐associated cues. These findings are consistent with the anti‐craving profile of other dopamine D3 drugs. HighlightsLS‐3‐134 is a moderately selective dopamine D3 receptor partial agonist.LS‐3‐134 reduced cocaine‐induced hyperlocomotion, but not spontaneous locomotion.No effects were seen on low‐effort self‐administration of cocaine and sucrose.LS‐3‐134 reduced high‐effort self‐administration and cocaine‐seeking behavior.LS‐3‐134 effects are consistent with other anti‐craving dopamine D3 drugs.

Chalcones and Five-Membered Heterocyclic Isosteres Bind to Alpha Synuclein Fibrils in Vitro

A series of chalcone and heterocyclic isosteres, in which the enone moiety was replaced with an isoxazole and pyrazole ring system, was synthesized and their affinities for alpha synuclein (Asyn), amyloid beta (Aβ), and tau fibrils were measured in vitro. The compounds were found to have a modest affinity and selectivity for Asyn versus Aβ fibrils and low affinity for tau fibrils. Insertion of a double bond to increase the extendable surface area resulted in an increase in affinity and improvement in selectivity for Asyn versus Aβ and tau fibrils. The results of this study indicate that compound 11 is a secondary lead compound for structure–activity relationship studies aimed at identifying a suitable compound for positron emission tomography-imaging studies of insoluble Asyn aggregates in Parkinson’s disease.

Analogs of Arylamide Phenylpiperazine Ligands to Investigate the Factors Influencing D3 Dopamine Receptor Bitropic Binding and Receptor Subtype Selectivity

We have previously reported on the ability of arylamide phenylpiperazines to bind selectively to the D3 versus the D2 dopamine receptor subtype. For these studies, we used LS-3-134 as the prototypic arylamide phenylpiperazine ligand because it binds with high affinity at D3 dopamine receptor (0.17 nM) and exhibits >150-fold D3 vs D2 receptor binding selectivity. Our goal was to investigate how the composition and size of the nonaromatic ring structure at the piperazine position of substituted phenylpiperazine analogues might influence binding affinity at the human D2 and D3 dopamine receptors. Two factors were identified as being important for determining the binding affinity of bitropic arylamide phenylpiperazines at the dopamine D3 receptor subtype. One factor was the strength of the salt bridge between the highly conserved residue Asp3.32 with the protonated nitrogen of the nonaromatic ring at the piperazine position. The second factor was the configuration of the unbound ligand in an aqueous solution. These two factors were found to be related to the logarithm of the affinities using a simple correlation model, which could be useful when designing high affinity subtype selective bitropic ligands. While this model is based upon the interaction of arylamide phenylpiperazines with the D2 and D3 D2-like dopamine receptor subtypes, it provides insights into the complexity of the factors that define a bitropic mode of the binding at GPCRs.