Recent Patents on Anti-Cancer Drug Discovery - Volume 16, Issue 4, 2021

Background: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated9 (Cas9) endonuclease system is a facile, highly efficient and selective site-directed mutagenesis tool for RNA-guided genome-editing. CRISPR/Cas9 genome-editing strategy uses designed guide-RNAs that recognizes a 3 base-pair protospacer adjacent motif (PAM) sequence in the target-DNA. CRISPR/Cas-editing tools have mainly been employed in crop plants in relation to yield and stress tolerance. However, the immense potential of this technology has not yet been fully utilized in medicinal plants in deciphering or modulating secondary metabolic pathways producing therapeutically active phytochemicals against cancer and other diseases. Objective: The present review elucidates the use of CRISPR-Cas9 as a promising genome-editing tool in plants and plant-derived natural products with anticancer and other therapeutic applications. It also includes recent patents on the therapeutic applications of CRISPR-CAS systems implicated to cancer and other human medical conditions. Methods: Popular search engines, such as PubMed, Scopus, Google Scholar, Google Patents, Medline, ScienceDirect, SpringerLink, EMBASE, Mendeley, etc., were searched in order to retrieve literature using relevant keywords viz. CRISPER/Cas, plant natural product research, anticancer, therapeutics, etc., either singly or in various combinations. Results: Retrieved citations and further cross-referencing among the literature have resulted in a total number of 71 publications and 3 patents are being cited in this work. Information presented in this review aims to support further biotechnological and clinical strategies to be carried using CRISPER/ Cas mediated optimization of plant natural products against cancer and an array of other human medical conditions. Conclusion: Off late, knock-in and knock-out, point mutation, controlled tuning of gene-expression and targeted mutagenesis have enabled the versatile CRISPR/Cas-editing device to engineer medicinal plants’ genomes. In addition, by combining CRISPR/Cas-editing tool with next-generation sequencing (NGS) and various tools of system biology, many medicinal plants have been engineered genetically to optimize the production of valuable bioactive compounds of industrial significance.

Background: Santacruzamate A (SCA) is a natural product isolated from a marine cyanobacterium. Activity test results revealed that SCA is a highly potent HDAC2 inhibitor with an IC50 value of 0.112 nM. The IC50 of SCA in inhibiting cancer cell proliferation is 28.3 μM and 1.3μM on HCT116 and HuT-78 cells, respectively. Objective: To develop HDAC inhibitors with improved activity, SCA analogs were synthesized for the Structure-Activity Relationship (SAR) studies. Methods: Various substituted groups were introduced into the zinc binging group, linker, and cap regions of SCA by various chemical synthetic methods. Results: Compared with SCA, the derivatives of SCA did not exhibit improved HDAC2 inhibitory activity. Nevertheless, several molecules such as III-32, III-33, IV-4b, and IV-11 showed improved activity in inhibiting cell proliferation on HCT116 and HuT-78 cells. Conclusion: Collectively, a potent HDAC2 inhibitor SCA was discovered as a lead compound for further development of selective HDAC inhibitors.

Background: The design of anti-cancer therapies with high anti-tumour efficacy and reduced toxicity continues to be challenging. Anti-cancer prodrug and antibody-drug-conjugate (ADC) strategies that can specifically and efficiently deliver cytotoxic compounds to cancer cells have been used to overcome some of the challenges. The key to the success of many of these strategies is a self-immolative linker, which after activation can release the drug payload. Various types of triggerable self-immolative linkers are used in prodrugs and ADCs to improve their efficacy and safety. Objective: Numerous patents have reported the significance of self-immolative linkers in prodrugs and ADCs in cancer treatment. Based on the recent patent literature, we summarise methods for designing the site-specific activation of non-toxic prodrugs and ADCs in order to improve selectivity for killing cancer cells. Methods: In this review, an integrated view of the potential use of prodrugs and ADCs in cancer treatment are provided. This review presents recent patents and related publications over the past ten years uptill 2020. Results: The recent patent literature has been summarised for a wide variety of self-immolative PABC linkers, which are cleaved by factors including responding to the difference between the extracellular and intracellular environments (pH, ROS, glutathione) through over-expressed enzymes (cathepsin, plasmin, β-glucuronidase) or bioorthogonal activation. The mechanism for self-immolation involves the linker undergoing a 1,4- or 1,6-elimination (via electron cascade) or intramolecular cyclisation to release cytotoxic drug at the targeted site. Conclusion: This review provides the commonly used strategies from recent patent literature in the development of prodrugs based on targeted cancer therapy and antibody-drug conjugates, which show promise in therapeutic applications.

Background: Existing cancer treatment methods have many undesirable side effects that greatly reduce the quality of life of cancer patients. Objective: This review will focus on the use of ultrasound-responsive liposomes and polymeric micelles in cancer therapy. Methods: This review presents a survey of the literature regarding ultrasound-triggered micelles and liposomes using articles recently published in various journals, as well as some new patents in this field. Results: Nanoparticles have proven promising as cancer theranostic tools. Nanoparticles are selective in nature, have reduced toxicity, and controllable drug release patterns making them ideal carriers for anticancer drugs. Numerous nanocarriers have been designed to combat malignancies, including liposomes, micelles, dendrimers, solid nanoparticles, quantum dots, gold nanoparticles, and, more recently, metal-organic frameworks. The temporal and spatial release of therapeutic agents from these nanostructures can be controlled using internal and external triggers, including pH, enzymes, redox, temperature, magnetic and electromagnetic waves, and ultrasound. Ultrasound is an attractive modality because it is non-invasive, can be focused on the diseased site, and has a synergistic effect with anticancer drugs. Conclusion: The functionalization of micellar and liposomal surfaces with targeting moieties and the use of ultrasound as a triggering mechanism can help improve the selectivity and enable the spatiotemporal control of drug release from nanocarriers.

Background: Pseudo-ginsenoside-Rh2 (pseudo-G-Rh2), a novel derivative of ginsenoside Rh2, is reported to exert a pro-apoptotic effect on various malignancies. However, whether this anti-cancer action of pseudo-G-Rh2 involves autophagy remains to be determined and explored. Objective: The objective of this study was to investigate the pseudo-G-Rh2-induced apoptosis and autophagy and the underlying mechanism. Methods: In the present study, the MTT assay was used for evaluating cell viability, and the lactate dehydrogenase (LDH) assay was performed to assess cell toxicity. Autophagy evaluation was performed using monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM). The levels of autophagy-associated and apoptosis-associated proteins were determined using Western blotting. The Annexin V-FITC/propidium iodide (PI) assay was used to assess apoptosis. Results: The Annexin V-FITC/PI assay revealed that the percentage of apoptotic cells in HepG2 cells at concentrations 0, 20, 40, and 60 μM was 3.75%±1.37%, 5.70%±1.04%, 12.30%±2.10%, and 34.26%±4.73%, respectively. Pseudo-G-Rh2 was observed to significantly increase the expressions of BAX, cleaved-caspase-3, and cleaved-caspase-9, while it decreased the Bcl-2 expression. MDC and TEM analysis revealed that pseudo-G-Rh2 at concentrations 20, 40, and 60 μM significantly facilitated the accumulation of autophagosomes and autolysosomes within the HepG2 cells. Moreover, pseudo-G-Rh2 significantly increased the expressions of LC3 II/LC3 I and Beclin-1 and decreased the expression of p62. The Annexin V-FITC/PI assay also revealed that in comparison to the pseudo-G-Rh2 group, the concurrent treatment with pseudo-G-Rh2 and an autophagy inhibitor (CQ or 3-MA) significantly induced distinct apoptosis. In addition, pseudo-G-Rh2 activated AMPK and inhibited the PI3K/Akt/mTOR pathway in a concentration-dependent manner. Pseudo- G-Rh2 is similar to the current patents, which enhanced its anti-cancer activity by combining with autophagy inhibitors. Conclusion: Pseudo-G-Rh2 could induce protective autophagy in HepG2 cells, at least in part, via AMPK and the PI3K/Akt/mTOR pathway.

Background: The prognosis of Epithelial Ovarian Cancer (EOC) is poor, but the prognostic biomarkers are neither sensitive nor specific. Therefore, it is very important to search novel prognostic biomarkers for EOC. Objectives: The present study aimed to investigate Myosin Light Chain 9(MYL9) expression in Epithelial Ovarian Cancer (EOC) tissues (including paraffin-embedded and fresh tissue samples) and its relationship with clinicopathological characteristics, as well as its potential prognostic value in patients with EOC. Methods: Between March 2009 and December 2018, all of 184 paraffin-embedded cancer tissues from patients with EOC and 41 paratumor tissues, pathologically confirmed at the Memorial Hospital of Sun Yat-sen University and Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, were collected for the present study and were assessed for MYL9 protein expression patterns using Immunohistochemistry (IHC). Furthermore, from August 2013 to November 2019, 16 fresh EOC tissues and their paired paratumor tissues, pathologically confirmed at the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University were analyzed using Reverse-Transcription Quantitative PCR (RT-qPCR) to detect MYL9 mRNA expression levels. Results: The results showed that MYL9 expression was higher in cancer tissues compared with that in paratumor tissues, and MYL9 overexpression was associated with shorter Recurrence Free Survival (RFS) and Overall Survival (OS) of EOC patients. Furthermore, multivariate Cox model analysis indicated that MYL9 overexpression was an independent poor survival prediction in patients with EOC. Conclusion: MYL9 is upregulated in EOC and may serve as a useful patent of prognostic biomarker in EOC, and it may demonstrate an important value for the clinical treatment and supervision of patients with EOC.

Background: Lung cancer is the most common malignant cancer worldwide. Targeted therapies have emerged as a promising treatment strategy for lung cancers. Objective: To evaluate the current landscape of targets and find promising targets for future new drug discovery for lung cancers, this research identified the science-technology-clinical development pattern and mapped the interaction network of targets. Methods: Targets for cancers were classified into 3 groups based on a paper published in Nature. We searched for scientific pieces of literature, patent documents and clinical trials of targets in Group 1 and Group 2 for lung cancers. Then, a target-target interaction network of Group 1 was constructed, and the science-technology-clinical (S-T-C) development patterns of targets in Group 1 were identified. Finally, based on the cluster distribution and the development pattern of targets in Group 1, interactions between the targets were employed to predict potential targets in Group 2 for drug development. Results: The target-target interaction (TTI) network of group 1 resulted in 3 clusters with different developmental stages. The potential targets in Group 2 are divided into 3 ranks. Level-1 is the first priority and level-3 is the last. Level-1 includes 16 targets, such as STAT3, CRKL, and PTPN11, that are mostly involved in signaling transduction pathways. Level-2 and level-3 contain 8 and 6 targets, respectively, related to various biological functions. Conclusion: This study will provide references for drug development in lung cancers, emphasizing that priorities should be given to targets in Level-1, whose mechanisms are worth further exploration.

Objectives: The aim of this study was to formulate fluorescent-labeled targeted immunoliposome to visualize the delivery and distribution of drugs in real-time. Methods: In this study, fluorescent-labeled liposomes were decorated with anti-HER2 VHH or Herceptin to improve the monitoring of intracellular drug delivery and tumor cell tracking with minimal side effects. The conjugation efficiency of antibodies was analyzed by SDS-PAGE silver staining. In addition, the physicochemical characterization of liposomes was performed using DLS and TEM. Finally, confocal microscopy visualized nanoparticles in the target cells. Results: Quantitative and qualitative methods characterized the intracellular uptake of 110±10 nm particles with near 70% conjugation efficiency. In addition, live-cell trafficking during hours of incubation was monitored by wide-field microscopy imaging. The results show that the fluorescent- labeled nanoparticles can specifically bind to HER2-positive breast cancer with minimal off-target delivery. Conclusion: These nanoparticles can have several applications in personalized medicine, especially drug delivery and real-time visualization of cancer therapy. Moreover, this method also can be applied in the targeted delivery of contrast agents in imaging and thermotherapy.