A targeted low molecular weight near-infrared fluorescent probe for prostate cancer

Prostate-specific membrane antigen (PSMA) remains an active target for imaging and therapeutic applications for prostate cancer. Although radionuclide-based imaging is generally more sensitive and also has been deeply explored, near-infrared fluorescence imaging agents are simple to prepare and compatible with long-term storage conditions. In the present study, a near-infrared fluorescent imaging probe (Cy5.5-CTT-54.2) has been developed by chemical conjugation of Cy5.5N-hydroxysuccinimide ester (Cy5.5-NHS) with a potent PSMA inhibitor CTT-54.2 (IC(50)=144 nM). The probe displays a highly potency (IC(50)=0.55 nM) against PSMA and has demonstrated successful application for specifically labeling PSMA-positive prostate cancer cells in both two and three-dimensional cell culture conditions. These results suggest that the potent, near-infrared Cy5.5-PSMA inhibitor conjugate may be useful for the detection of prostate tumor cells by optical in vivo imaging.     

68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging

Urea-based inhibitors of the prostate-specific membrane antigen (PSMA) represent low-molecular-weight pepidomimetics showing the ability to image PSMA-expressing prostate tumors. The highly efficient, acyclic Ga(III) chelator N,N'-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N'- diacetic acid (HBED-CC) was introduced as a lipophilic side chain into the hydrophilic pharmacophore Glu-NH-CO-NH-Lys which was found favorable to interact with the PSMA "active binding site". This report describes the syntheses, in vitro binding analyses, and biodistribution data of the radiogallium labeled PSMA inhibitor Glu-NH-CO-NH-Lys(Ahx)-HBED-CC in comparison to the corresponding DOTA conjugate. The binding properties were analyzed using competitive cell binding and enzyme-based assays followed by internalization experiments. Compared to the DOTA-conjugate, the HBED-CC derivative showed reduced unspecific binding and considerable higher specific internalization in LNCaP cells. The (68)Ga complex of the HBED-CC ligand exhibited higher specificity for PSMA expressing tumor cells resulting in improved in vivo properties. (68)Ga labeled Glu-NH-CO-NH-Lys(Ahx)-HBED-CC showed fast blood and organ clearances, low liver accumulation, and high specific uptake in PSMA expressing organs and tumor. It could be demonstrated that the PET-imaging property of a urea-based PSMA inhibitor could significantly be improved with HBED-CC.     

Targeting prostate cancer cells with a multivalent PSMA inhibitor-guided streptavidin conjugate

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin-biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG(4)-CTT54, and biotin-PEG(12)-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC(50) values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG(12)-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG(12)-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC(50)=1.86 nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA's biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.     

Selective gene therapy for prostate cancer cells using liposomes conjugated with IgM type monoclonal antibody against prostate-specific membrane antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by beta-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast, no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine kinase gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.     

Synthesis and biological evaluation of PSMA-targeting paclitaxel conjugates

Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.     

Structure-Activity Relationships of 2′,5′-Oligoadenylate Analogue Modifications of Prostate-Specific Membrane Antigen (PSMA) Antagonists

Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A previously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly reduced its biological activity. The results will provide a path to agents for targeted imaging and treatment of prostate cancer.     

Synthesis and evaluation of a novel near-infrared fluorescent probe based on succinimidyl-Cys-C(O)-Glu that targets prostate-specific membrane antigen for optical imaging

Prostate-specific membrane antigen (PSMA), which is highly expressed in both localized and metastatic prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported radiolabeled asymmetric urea derivatives as a PSMA-targeting radiotracer for single-photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging. Here, based on these radiopharmaceutical probes, we designed a novel near-infrared (NIR) fluorescent imaging probe (800CW-SCE) by chemical conjugation between IRDye 800CW-Maleimide and an asymmetric urea compound, known as PSMA inhibitor, for optical imaging. In the in vitro cellular uptake study, 800CW-SCE was internalized into PSMA-positive PCa cells (LNCaP cells) but not into PSMA-negative PCa cells (PC-3 cells). Moreover, in the in vivo imaging study, the probe was highly accumulated in LNCaP tumors but not in PC-3 tumors, and remained in LNCaP tumors until 24 h after intravenous administration. These results suggest that the potent NIR conjugate may contribute to clinical intraoperative optical imaging.     

Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody-indocyanine green (ICG) conjugate

In patients with prostate cancer, a positive surgical margin is associated with an increased risk of cancer recurrence and poorer outcome, yet margin status cannot be determined during the surgery. An in vivo optical imaging probe that could identify the tumor margins during surgery could result in improved outcomes. The design of such a probe focuses on a highly specific targeting moiety and a near-infrared (NIR) fluorophore that is activated only when bound to the tumor. In this study, we successfully synthesized an activatable monoclonal antibody-fluorophore conjugate consisting of a humanized anti-Prostate-Specific Membrane Antigen (PSMA) antibody (J591) linked to an indocyanine green (ICG) derivative. Prior to binding to PSMA and cellular internalization, the conjugate yielded little light; however, after binding an 18-fold activation was observed permitting the specific detection of PSMA+ tumors up to 10 days after injection of a low dose (0.25 mg/kg) of the reagent. This agent demonstrates promise as a method to image the extent of prostate cancer in vivo and could assist with real-time resection of extracapsular extension of tumor and positive lymph nodes.     

Evidence for the involvement of iron siderophore in the transport of molybdenum in cowpeaRhizobium

The production of a catechol type of siderophore by an iron-depleted culture of cowpeaRhizobium decreased with the increase in the concentration of molybdenum in the growth medium above 1 mM. In vitro addition of molybdenum at pH 5 and 7 changed the absorbance maxima of siderophore, indicating the interaction of molybdenum with siderophore. Tungsten, which is a competitive inhibitor of molybdenum, was unable to dissociate the molybdenum-siderophore conjugate. In the presence of iron, siderophore increased the uptake of molybdenum. Under these conditions, the addition of 2,3-dihydroxybenzoic acid did not show an increase in the uptake. This suggests that an entire siderophore molecule is involved in the transport of molybdenum.     

Synthesis and biological evaluation of a carbocyclic azanoraristeromycin siderophore conjugate

Synthesis and biological evaluation of a carbocyclic azanoraristeromycin siderophore conjugate 22 is reported. Coupling of previously prepared L-alanyl-4'-azanoraristeromycin 19 with protected tripeptide trihydroxamate 20, followed by hydrogenolytic removal of all protecting groups, provided the first carbocylic azanoraristeromycin siderophore conjugate (22, 8 with iron). Compounds 19 and 22 showed inhibitory activity against tumor cells, and conjugate 22, in particular, displayed significant activity against those viruses (i.e. reo, parainfluenza, vaccinia, cytomegalo) that are known to be inhibited by S-adenosylhomocysteine hydrolase inhibitors.     

Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12–14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. K_i values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.     

Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography

Prostate-specific membrane antigen (PSMA) is a type II membrane protein that has attracted significant attention as a target for immunioscintigraphic and radioimmunotherapeutic applications for prostate cancer. However, definitive studies on its substrate and inhibitor specificity as well as protein-protein interactions have been somewhat limited by difficulties in the purification of native PSMA. In this study, we optimized the purification of native PSMA from LNCaP cells using conformational epitope-specific antibody-affinity chromatography. Western blot analysis and an HPLC-based enzymatic activity assay were used to compare the yield and activity of PSMA purified by different methods. The ratio of purified PSMA in a native and active conformation was determined by quantifying the amount of non-native PSMA not retained in a second antibody-affinity isolation. The addition of both a neutralization step and the inclusion of Zn(2+) to the equilibration buffer in desalting step provides considerable enhancement in the yield of active PSMA from LNCaP cells.     

The molecular pruning of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen

To identify the pharmacophore of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen (PSMA), a small analog library was designed and screened for inhibitory potency against PSMA. The design of the lead inhibitor was based upon N-acyl derivatives of endogenous substrate folyl-gamma-Glu and incorporates a phosphoramidate group to interact with the PSMA catalytic zinc atoms. The scope of the analog library was designed to test the importance of various functional groups to the inhibitory potency of the lead phosphoramidate. The IC(50) for the lead phosphoramidate inhibitor was 35 nM while the IC(50) values for the analog library presented a range from 0.86 nM to 4.1 microM. Computational docking, utilizing a recently solved X-ray crystal structure of the recombinant protein, along with enzyme inhibition data, was used to propose a pharmacophore model for the PSMA active site.     

Synthesis and biological evaluation of Doxorubicin-containing conjugate targeting PSMA

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), has recently emerged as a prominent biomarker of prostate cancer (PC) and as an attractive protein trap for drug targeting. At the present time, several drugs and molecular diagnostic tools conjugated with selective PSMA ligands are actively evaluated in different preclinical and clinical trials. In the current work, we discuss design, synthesis and a preliminary biological evaluation of PSMA-specific small-molecule carrier equipped by Doxorubicin (Dox). We have introduced an unstable azo-linker between Dox and the carrier hence the designed compound does release the active substance inside cancer cells thereby providing a relatively high Dox concentration in nuclei and a relevant cytotoxic effect. In contrast, we have also synthesized a similar conjugate with a stable amide linker and it did not release the drug at all. This compound was predominantly accumulated in cytoplasm and did not cause cell death. Preliminary in vivo evaluation has showed good efficiency for the degradable conjugate against PC3-PIP(PSMA⁺)-containing xenograft mine. Thus, we have demonstrated that the conjugate can be used as a template to design novel analogues with improved targeting, anticancer activity and lower rate of potential side effects. 3D molecular docking study has also been performed to elucidate the underlying mechanism of binding and to further optimization of the linker area for improving the target affinity.     

Expression of Prostate-Specific Membrane Antigen in Lung Cancer Cells and Tumor Neovasculature Endothelial Cells and Its Clinical Significance

BackgroundProstate-specific membrane antigen (PSMA) has been found in tumor neovasculature endothelial cells (NECs) of non-prostate cancers and may become the most promising target for anti-tumor therapy. To study the value of PSMA as a potential new target for lung cancer treatment, PSMA expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) tissues and its relationship with clinicopathology were investigated in the current study.MethodsImmunohistochemistry was used to detect PSMA expression in a total of 150 lung specimens of patients with lung cancer. The data were analyzed using univariate and multivariate statistical analyses.ResultsThe percentages of NSCLC patients who had PSMA (+) tumor cells and PSMA (+) NECs were 54.02% and 85.06%, respectively. The percentage of patients younger than 60 years old who had PSMA (+) tumor cells was 69.05%, which was significantly greater than the percentage of patients aged 60 years or older (40.00%, p<0.05). A significant difference was observed in the percentage of NSCLC patients with PMSA (+) NECs and stage I or II cancer (92.98%) and those patients with stage III or IV cancer (76.77%). In the SCLC tissues, NEC PSMA expression (70.00%) did not differ significantly from NSCLC. SCLC tumor cells and normal lung tissues cells were all negative. There was no significant correlation between the presence of PSMA (+) NECs in SCLC patients and the observed clinicopathological parameters.ConclusionsPSMA is expressed not only in NECs of NSCLC and SCLC but also in tumor cells of most NSCLC patients. The presence of PSMA (+) tumor cells and PSMA (+) NECs in NSCLC was negatively correlated with age and the clinicopathological stage of the patients, respectively.     

The clinical application of 68Ga-PSMA PET/CT and regulating mechanism of PSMA expression in patients with brain metastases of lung cancer

Brain metastases (BMs) of lung cancer are common malignant intracranial tumours associated with severe neurological symptoms and an abysmal prognosis. Prostate-specific membrane antigen (PSMA) has been reported to express significantly in a variety of solid tumours. However, the clinical applications of ⁶⁸Ga-PSMA PET/CT and the mechanism of PSMA expression in patients with BMs of lung cancer have rarely been reported. Experiments with ⁶⁸Ga-PSMA PET/CT and immunohistochemical staining were conducted to evaluate the expression of PSMA from seven patients with BMs of lung cancer who accepted surgical treatment in Fudan University Shanghai Cancer Center between October 2020 and October 2021. The mechanism of PSMA expression in BMs of lung cancer was explored by using single-cell RNA sequencing. The median maximum standardized uptake value (SUVmax) in BMs was higher than that in primary lung cancer (8.6 ± 2.8 vs. 3.6 ± 1.3, P < 0.01). The mean SUVmax in BMs was 1.76-fold higher than that in the liver, which indicated the potential of PSMA radioligand therapy (PSMA-RLT) for BMs. BMs showed intense PSMA staining, while normal lung tissue had no PSMA staining and there was only faint primary lung cancer staining by immunohistochemistry (IHC). Single-cell RNA sequencing (scRNA-seq) analysis found that PSMA was mainly expressed in oligodendrocytes of BMs, whereas it was expressed at lower levels in solid cells of lung cancer. PSMA expression in oligodendrocytes might be regulated by the factors ATF3 and NR4A1, which were associated with ER stress.     

Conformational and SAR analysis of NAALADase and PSMA inhibitors

Prostate specific membrane antigen (PSMA) is a 110 kDa type II transmembrane protein that is expressed exclusively by prostate tumor cells and as such is a clear cellular target in the development of a new method for fast and reliable diagnosis of prostate cancer. PSMA is highly homologous to the neuropeptidase NAALADase, and it has been shown that inhibitors of NAALADase also strongly bind to PSMA. In an effort to better understand the structural basis of the inhibitory activity of more than 60NAALADase inhibitors synthesized and tested by our group, we used Monte Carlo calculations employing the Merck Molecular Force Field to explore the conformational space available to a set of PSMA inhibitors. Conformational analysis indicated that the lower the number of unique conformations accessible by an inhibitor, the greater the biological activity displayed by the compound against LnCAP cells. This suggests that the difference in activity is largely entropy based. The key conformations associated with high activity are used to develop a simple pharmacophore model that led to the design of new, conformationally restricted analogues with potentially high activity in rational drug design.     

Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia

The prostatic membrane antigen (PSMA) is a protein that is expressed in the prostatic epithelium. We studied the expression of PSMA in a series of 55 patients with different stages of prostate cancer and we compared the PSMA staining in prostate cancer cells, in high-grade prostatic intraepithelial neoplasia (PIN) and in histologically benign prostatic epithelium for the same specimen. For this purpose archival paraffin-embedded specimens were studied by immunohistochemistry with a monoclonal antibody 7E11-C5.3 against PSMA using the streptavidin-biotin method. The mean percentage of PSMA immunoreactivity was 56.67% in prostate cancer (CaP) cells, and 48.6% in PIN cells, which was significantly higher than benign-appearing prostatic epithelium (5.72%) (for each pair, p<0.001). PSMA expression was greater in CaP with a higher Gleason score (p=0.01), but no relationship was found with serum PSA value. We conclude that PSMA overexpression is detected in high-grade PIN and is associated with a higher Gleason score of prostate cancer. It is a potential marker for studying carcinogenesis and progression of prostate cancer.     

Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics

The mode of inhibition for phosphoramidate peptidomimetic inhibitors of prostate-specific membrane antigen was determined by inhibition reversibility experiments. The results revealed that these inhibitors can be classified into three types: pseudoirreversible (compounds 1-3), moderately reversible (compounds 4-9), and rapidly reversible inhibitors (compounds 10 and 11). Representative compounds from each class were further evaluated for their ability to induce cellular internalization of PSMA. Results from these experiments revealed that the pseudoirreversible inhibitor 1 induced the greatest PSMA internalization. The discovery of pseudoirreversible PSMA inhibitors is expected to provide a new avenue of investigation and therapeutic applications for prostate cancer and neurological disorders.     

Synthesis and Biological Evaluation of a Carbocyclic Azanoraristeromycin Siderophore Conjugate

Abstract

Synthesis and biological evaluation of a carbocyclic azanoraristeromycin siderophore conjugate 22 is reported. Coupling of previously prepared L-alanyl-4′-azanoraristeromycin 19 with protected tripeptide trihydroxamate 20, followed by hydrogenolytic removal of all protecting groups, provided the first carbocylic azanoraristeromycin siderophore conjugate (22, 8 with iron). Compounds 19 and 22 showed inhibitory activity against tumor cells, and conjugate 22, in particular, displayed significant activity against those viruses (i.e. reo, parainfluenza, vaccinia, cytomegalo) that are known to be inhibited by S-adenosylhomocysteine hydrolase inhibitors.