A PSMA-targeted doxorubicin small-molecule drug conjugate.

We developed a model small-molecule drug conjugate (SMDC) that employed doxorubicin as a representative chemotherapeutic targeted to the cell membrane biomarker PSMA (prostate-specific membrane antigen) expressed on prostate cancer cells. The strategy capitalized on the clatherin-mediated internalization of PSMA to facilitate the selective uptake and release of doxorubicin in the target cells. The SMDC was prepared and assessed for binding kinetics, plasma stability, cell toxicity, and specificity towards PSMA expressing prostate cancer cell lines. We observed high affinity of the SMDC for PSMA (IC50 5 nM) with irreversible binding, as well as specific effectiveness against PSMA(+) cells. These findings validated the strategy for a small molecule-based approach in targeted cancer therapy.

PSMA-targeted SMART molecules outfitted with SN38.

Herein, we report the modular synthesis and evaluation of a prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugate (SMDC) carrying the chemotherapeutic agent, SN38. Due to the fluorogenic properties of SN38, payload release kinetics from the platform was observed in buffers representing the pH conditions of systemic circulation and cellular internalization. It was found that this platform is stable with minimal payload release at physiological pH with most rapid payload release observed at pH values representing the endosome complex. We confirmed selective payload release and chemotherapeutic efficacy for PSMA(+) prostate cancer cells over PSMA(-) cells. These results demonstrate that chemotherapeutic agents with limited solubility can be conjugated to a water-soluble targeting and linker platform without attenuating efficacy.

PSMA-targeted small-molecule drug-conjugates with valine-citrulline and phosphoramidate cleavable linkers.

In this study, we present a modular synthesis and evaluation of two prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugates (SMDCs) incorporating the potent chemotherapeutic agent monomethyl auristatin E (MMAE). These SMDCs are distinguished by their cleavable linker modules: one utilizing the widely known valine-citrulline linker, susceptible to cleavage by cathepsin B, and the other featuring a novel acid-labile phosphoramidate-based (PhosAm) linker. Both SMDCs maintained nanomolar affinity to PSMA. Furthermore, we confirmed the selective release of the payload and observed chemotherapeutic efficacy specifically within PSMA-positive prostate cancer cells, while maintaining cell viability in PSMA-negative cells. These findings not only validate the efficacy of our approach but also highlight the potential of the innovative pH-responsive PhosAm linker. This study contributes significantly to the field and also paves the way for future advancements in targeted cancer therapy.

Liposomes in Cancer Therapy: How Did We Start and Where Are We Now.

Since their first discovery in the 1960s by Alec Bangham, liposomes have been shown to be effective drug delivery systems for treating various cancers. Several liposome-based formulations received approval by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA), with many others in clinical trials. Liposomes have several advantages, including improved pharmacokinetic properties of the encapsulated drug, reduced systemic toxicity, extended circulation time, and targeted disposition in tumor sites due to the enhanced permeability and retention (EPR) mechanism. However, it is worth noting that despite their efficacy in treating various cancers, liposomes still have some potential toxicity and lack specific targeting and disposition. This explains, in part, why their translation into the clinic has progressed only incrementally, which poses the need for more research to focus on addressing such translational limitations. This review summarizes the main properties of liposomes, their current status in cancer therapy, and their limitations and challenges to achieving maximal therapeutic efficacy.

PRMT1 promotes neuroblastoma cell survival through ATF5.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

Mechanisms and Inhibitors of Histone Arginine Methylation.

Histone methylation plays an important regulatory role in chromatin restructuring and RNA transcription. Arginine methylation that is enzymatically catalyzed by the family of protein arginine methyltransferases (PRMTs) can either activate or repress gene expression depending on cellular contexts. Given the strong correlation of PRMTs with pathophysiology, great interest is seen in understanding molecular mechanisms of PRMTs in diseases and in developing potent PRMT inhibitors. Herein, we reviewed key research advances in the study of biochemical mechanisms of PRMT catalysis and their relevance to cell biology. We highlighted how a random binary, ordered ternary kinetic model for PRMT1 catalysis reconciles the literature reports and endorses a distributive mechanism that the enzyme active site utilizes for multiple turnovers of arginine methylation. We discussed the impacts of histone arginine methylation and its biochemical interplays with other key epigenetic marks. Challenges in developing small-molecule PRMT inhibitors were also discussed.

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR.

Targeting kinase signaling pathways with constrained peptide scaffolds.

Kinases are amongst the largest families in the human proteome and serve as critical mediators of a myriad of cell signaling pathways. Since altered kinase activity is implicated in a variety of pathological diseases, kinases have become a prominent class of proteins for targeted inhibition. Although numerous small molecule and antibody-based inhibitors have already received clinical approval, several challenges may still exist with these strategies including resistance, target selection, inhibitor potency and in vivo activity profiles. Constrained peptide inhibitors have emerged as an alternative strategy for kinase inhibition. Distinct from small molecule inhibitors, peptides can provide a large binding surface area that allows them to bind shallow protein surfaces rather than defined pockets within the target protein structure. By including chemical constraints within the peptide sequence, additional benefits can be bestowed onto the peptide scaffold such as improved target affinity and target selectivity, cell permeability and proteolytic resistance. In this review, we highlight examples of diverse chemistries that are being employed to constrain kinase-targeting peptide scaffolds and highlight their application to modulate kinase signaling as well as their potential clinical implications.

Development of inhibitor-directed enzyme prodrug therapy (IDEPT) for prostate cancer.

Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies.

Rationally designed sulfamides as glutamate carboxypeptidase II inhibitors.

Glutamate carboxypeptidase II (GCPII) is a membrane-bound cell surface peptidase. There is significant interest in the inhibition of GCPII as a means of neuroprotection, while GCPII inhibition as a method to treat prostate cancer remains a topic of further investigation. The key zinc-binding functional group of the well-characterized classes of GCPII inhibitors (phosphonates and phosphoramidates) is tetrahedral and negatively charged at neutral pH, while glutamyl urea class of inhibitors possesses a planar and neutral zinc-binding group. This study explores a new class of GCPII inhibitors, glutamyl sulfamides, which possess a putative net neutral tetrahedral zinc-binding motif. A small library containing six sulfamides was prepared and evaluated for inhibitory potency against purified GCPII in an enzymatic assay. While most inhibitors have potencies in the micromolar range, one showed promising sub-micromolar potency, with the optimal inhibitor in this series being aspartyl-glutamyl sulfamide (2d). Lastly, computational docking was used to develop a tentative binding model on how the most potent inhibitors interact with the ligand-binding site of GCPII.

PSMA-targeted SPECT agents: mode of binding effect on in vitro performance.

BACKGROUND: The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an active target for imaging and therapeutic applications for prostate cancer. The internalization of PSMA has been shown to vary with inhibitors' mode of binding: irreversible, slowly reversible, and reversible. METHODS: In the present study, PSMA-targeted clickable derivatives of an irreversible phosphoramidate inhibitor DBCO-PEG(4) -CTT-54 (IC(50) = 1.0 nM) and a slowly reversible phosphate inhibitor, DBCO-PEG(4) -CTT-54.2 (IC(50) = 6.6 nM) were clicked to (99m) Tc(CO)(3) -DPA-azide to assemble a PSMA-targeted SPECT agent. The selectivity, percent uptake, and internalization of these PSMA-targeted SPECT agents were evaluated in PSMA-positive and PSMA-negative cells. RESULTS: In vitro studies demonstrated that PSMA-targeted SPECT agents exhibited selective cellular uptake in the PSMA-positive LNCaP cells compared to PSMA-negative PC3 cells. More importantly, it was found that (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54 based on an irreversible PSMA inhibitor core, exhibited greater uptake and internalization than (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54.2 constructed from a slowly reversible PSMA inhibitor core. CONCLUSIONS: We have demonstrated that a PSMA-targeted SPECT agent can be assembled efficiently using copper-less click chemistry. In addition, we demonstrated that mode of binding has an effect on internalization and percent uptake of PSMA-targeted SPECT agents; with the irreversible targeting agent demonstrating superior uptake and internalization in PSMA+ cells. The approach demonstrated in this work now supports a modular approach for the assembly of PSMA-targeted imaging and therapeutic agents.

Prolonged androgen deprivation leads to downregulation of androgen receptor and prostate-specific membrane antigen in prostate cancer cells.

Emergence of androgen-independent cancer cells during androgen deprivation therapy presents a significant challenge to successful treatment outcomes in prostate cancer. Elucidating the role of androgen deprivation in the transition from an androgen-dependent to an androgen-independent state may enable the development of more effective therapeutic strategies against prostate cancer. Herein, we describe an in vitro model for assessing the effects of continuous androgen-deprivation on prostate cancer cells (LNCaP) with respect to the expression of two prostate-specific markers: the androgen receptor (AR) and prostate-specific membrane antigen (PSMA). Compared with androgen-containing normal growth medium, androgen-deprived medium apparently induced the concomitant downregulation of AR and PSMA over time. Decreased protein levels were confirmed by fluorescence imaging, western blotting and enzymatic activity studies. In contrast to the current understanding of AR and PSMA in prostate cancer progression, our data demonstrated that androgen-deprivation induced a decrease in AR and PSMA levels in androgen-sensitive LNCaP cells, which may be associated with the development of more aggressive disease-state following androgen deprivation therapy.