Copper, an essential micronutrient, controls multiple fundamental processes throughout all forms of life, such as respiration, cell proliferation and neurotransmitter biosynthesis. High redox activity makes copper a doubleedged sword for cell survival, and abnormal copper metabolism is commonly associated with cancer and other diseases. Recently, advances in copper biology have forged new links between researchers from different disciplines to dissect the use of copper targets in cancer treatment. Copper is crucial for maintaining the rapid growth and proliferation of cancer cells, a process known as 'cuproplasia'. However, excess mitochondrial copper accumulation also triggers 'cuproptosis', a new form of programmed cell death, involving the destabilization of Fe-S cluster proteins and the aggregation of DLAT. Accordingly, both activating cuproptosis by copper ionophores and suppressing cuproplasia through copper depletion are noteworthy anticancer options, but this field currently lacks systematic reviews from both biological and chemical perspectives. This review provides a comprehensive overview of the leading known copper biology and copper-targeted cancer therapy, focusing on copper homeostasis, signal transduction, and copper overload/depletion, which should be beneficial for the development of the next-generation of inorganic anticancer drugs. We expect that our review will provide a balanced perspective to the development and comprehension of copper-based cancer therapy.

Accumulating evidence supports that exosomal RNAs are crucial in tumor microenvironment and may be used as diagnostic biomarkers for cancers. This study aimed to determine the role of exosomal circular RNA_protein tyrosine phosphatase receptor type A (circ_PTPRA) in colorectal cancer (CRC). The morphology of exosomes was identified by transmission electron microscopy (TEM), and several exosome-specific proteins were quantified by western blot. The expression of circ_PTPRA, miR-671-5p and SMAD family member 4 (SMAD4) was detected using quantitative polymerase chain reaction (qPCR). Cell cycle was assessed using flow cytometry assay. Cell proliferation was assessed by MTT assay. Radiosensitivity was observed according to colony growth and cell apoptosis rate by colony formation assay and flow cytometry assay. The protein levels of proliferation- and apoptosis-related markers and SMAD4 were measured by western blot. The predicted relationship between miR-671-5p and circ_PTPRA or SMAD4 was verified by dual-luciferase reporter assay. Animal study was performed to investigate the role of exosomal circ_PTPRA in vivo. Circ_PTPRA expression was declined in serumal exosomes from CRC patients and CRC cell lines. Exosomal circ_PTPRA induced CRC cell cycle arrest and inhibited cell proliferation. Besides, exosomal circ_PTPRA promoted radiosensitivity of CRC cells, leading to inhibitory colony formation and increased apoptotic rate. In mechanism, circ_PTPRA functioned as a competing endogenous RNA (ceRNA) to increasing SMAD4 level by binding to miR-671-5p. Rescue experiments concluded that circ_PTPRA inhibited CRC growth and radioresistance by decreasing miR-671-5p expression, and miR-671-5p inhibition also inhibited CRC growth and radioresistance by enriching SMAD4 expression. Moreover, exosomal circ_PTPRA blocked tumor growth in vivo. Exosomal circ_PTPRA enhanced CRC cell radiosensitivity and inhibited CRC malignant development partially by regulating the miR-671-5p/SMAD4 pathway, hinting that exosomal circ_PTPRA might be used as a potential predicted and therapeutic target for CRC.

As an essential micronutrient element in organisms, copper controls a host of fundamental cellular functions. Recently, copper-dependent cell growth and proliferation have been defined as "cuproplasia". Conversely, "cuproptosis" represents copper-dependent cell death, in a nonapoptotic manner. So far, a series of copper ionophores have been developed to kill cancer cells. However, the biological response mechanism of copper uptake has not been systematically analyzed. Based on quantitative proteomics, we revealed the crosstalk be-tween copper stress and cuproptosis in cancer cells, and also explored the feasibility of curcumin as anticancer copper ionophore. Copper stress not only couples with cuproptosis, but also leads to reactive oxygen species (ROS) stress, oxidative damage and cell cycle arrest. In cancer cells, a feedback cytoprotection mechanism involving cuproptosis mediators was discovered. During copper treatment, the activation of glutamine trans-porters and the loss of Fe-S cluster proteins are the facilitators and results of cuproptosis, respectively. Through copper depletion, glutathione (GSH) blocks the cuproptosis process, rescues the activation of glutamine trans-porters, and prevents the loss of Fe-S cluster proteins, except for protecting cancer cells from apoptosis, protein degradation and oxidative damage. In addition, the copper ionophore curcumin can control the metabolisms of lipids, RNA, NADH and NADPH in colorectal cancer cells, and also up-regulates positive cuproptosis mediators. This work not only established the crosstalk between copper stress and cuproptosis, but also discolored the suppression and acceleration of cuproptosis by GSH and curcumin, respectively. Our results are significant for understanding cuproptosis process and developing novel anticancer reagents based on cuproptosis.

Abstract Chemotherapeutic drugs have been a mainstay of cancer therapy for decades; however, their effectiveness is often hampered by inefficient drug exposures and undesirable toxicity to normal tissues. Here we report on a novel drug delivery system, termed heat shock protein 90 (HSP90) inhibitor-drug conjugates (HDC), based on the property that small molecule inhibitors of HSP90 are preferentially retained in tumors cells in contrast to their rapid clearance from the circulation and normal tissues. By attaching chemotherapeutic drugs to HSP90 inhibitor backbones, HDC technology exploits this inherent retention property to efficiently deliver cytotoxic payloads directly into tumor tissues and provide extended drug exposure. STA-12-8666 is an HDC that comprises an HSP90 inhibitor fused to the topoisomerase inhibitor SN-38 (active metabolite of irinotecan). In vivo modeling showed that the HSP90 inhibitor moiety was required for tumor-selective retention of STA-12-8666. Prolonged exposure of STA-12-8666 provided extended release of active SN-38 within the tumor compartment, generating up to two weeks of biomarker engagement (γ-H2AX) in contrast to 3-4 days with irinotecan. The broad therapeutic window exhibited by STA-12-8666 conferred superior efficacy and durability over irinotecan treatment alone - resulting in complete or near complete responses (CR) across a broad spectrum of solid tumor models, including an irinotecan-insensitive bladder cancer model and an aggressive lung cancer model where biweekly treatment of STA-12-8666 was initiated at a starting tumor volume 5-times greater that of typical studies. CRs were also observed in a human pancreatic PDX model following 3 doses of STA-12-8666, which were maintained for more than a month. Of note, recurrent PDX tumors remained sensitive to subsequent therapeutic challenge with STA-12-8666 suggesting HDC delivery may circumvent common mechanisms of resistance to irinotecan. Preliminary findings from an ongoing Phase 1 dose escalation study in dogs with spontaneous tumors suggest a well-managed safety profile and encouraging tumor responses. Overall, STA-12-8666 is a promising investigational agent prototypical of a platform technology that can be applied to other cytotoxic payloads to improve therapeutic indices as well as generating new pharmaceutical entities for evaluation as novel anticancer drugs. Citation Format: David A. Proia, Donald L. Smith, Junyi Zhang, Dan Zhou, John-Paul Jimenez, Jim Sang, Sarah Rippy, Cheryl London, Luisa S. Ogawa, Jun Jiang, Teresa Przewloka, Manuel Sequeira, Jaime Acquaviva, Suqin He, John Chu, Chaohua Zhang, Yuan Liu, Josephine Ye, Vladimir Khazak, Igor Astsaturov, Takayo Inoue, Noriaki Tatsuta, Richard C. Bates, Andrew Sonderfan, Dinesh Chimmanamada, Weiwen Ying. STA-12-8666: a first-in-class HSP90 inhibitor drug conjugate (HDC) designed to selectively deliver chemotherapy to tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2015-4409

A facile and convenient synthetic method for the anticancer agent elesclomol and its analogues, N-1,N-3-dialkyl-N-1,N-3-bis(arylcarbonothioyl)malonohydrazides, by the direct coupling of N-alkyl-N-(substituted)benzothiohydrazides and substituted malonic acids using propylphosphonic anhydride (T3P((R))) under very mild conditions has been developed.

Abstract Background: Most cytotoxic agents are often broadly active but non-selective, and have the disadvantage of high toxicity due to collateral damage to normal tissues. Drugs that target specific protein drivers of cancer cell growth are more tumor selective, yet often lead to tumor resistance via point mutations in their target or activation of alternative signaling pathways. Targeted delivery strategies, such as Antibody-Drug Conjugates (ADCs), offer a solution to these limitations by delivering potent anti-cancer payloads more directly to tumors. Hsp90 is a chaperone protein required by many cancer cells to maintain the stability and function of numerous proteins that drive cancer cell growth, survival, and metastasis. Small molecule inhibitors of Hsp90 such as ganetespib, PU-H71 and 17-AAG are found to be retained in tumors with half- time up to 65 hours in mouse xenografts. These properties are believed to be due to overexpression of an active form of Hsp90 in cancer cells as compared to normal tissues, and have recently been applied for tumor imaging in patients. Results: We have developed a small molecule drug conjugate platform technology using the unique properties of Hsp90 proteins and Hsp90 inhibitors. We have synthesized HDCs with various Hsp90 inhibitor scaffolds including resorcinol, purine, geldanamycin, etc. A cell-based client protein degradation assay is carried out to confirm the intracellular uptake and the Hsp90 binding of the conjugates. While different Hsp90 inhibitor scaffolds offer different DMPK and toxicology profiles, cellular uptake and Hsp90 binding are not greatly affected by the different chemical classes of Hsp90 inhibitors. Over 30 payloads have been conjugated with Hsp90 inhibitors so far. We have selected the payloads based on the hypothesis that HDC can improve the safety profile of a cytotoxic drug, expand the application of chemotherapeutic agents to different tumor types, combat drug resistance, and enable novel anticancer approach. Examples of payloads include topoisomerase inhibitors (camptothecin), microtubule modulators (taxanes), proteasome inhibitors (carfilzomib), CDK inhibitors (flavopiridol) and others. Unlike most other conjugate technologies, HDC does not require lengthy spacing between the anchor Hsp90 inhibitor and the payload. Linker cleavage mechanism is considered a key feature in the HDC design. We have been able to incorporate several linkers such as disulfide, hydrazone, peptide, carbamate, carboxylate, etc. into our HDC designs. Conclusion: In this HDC platform, we provide a method which can be applied to many well-studied mechanisms for modulating cancer pathways and stopping tumor cell growth. We have created a promising platform technology which can result in many anticancer agents in the near future. Citation Format: Weiwen Ying, Dinesh Chimmanamada, Junyi Zhang, Teresa Przewloka, Jun Jiang, Genliang Lu, Sami Osman, James Loch, Dharma Vutukuri, Shoujun Chen, Robert Stein, John Chu, David Proia, Pat Rao, Takayo Inoue, Luisa Shin Ogawa, Ritu Singh, Noriaki Tatsuta. Hsp90 inhibitor drug conjugates (HDCs): Construct design and preliminary evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2014-1619

Abstract Background: Various strategies have been used to make chemotherapeutic compounds more tumor selective in order to increase their safety and efficacy, but they were met with only limited success. Antibody Drug Conjugates (ADCs) and folate receptor mediated drug delivery (FRDC) are the emerging concepts in the drug delivery field, but they are limited to high potent toxins and narrow sets of indications. We established a novel tumor-directed drug delivery platform technology based on the unique property of Hsp90 inhibitors. One prominent feature of Hsp90 inhibitors is their ability to be retained selectively in tumor cells for a prolonged period of time. The difference in residency time in tumor against plasma and normal tissues is 10-25 fold. Moreover, the chaperone protein Hsp90 is overexpressed 2-3 fold in tumor compared to normal tissues. Combining the overexpression Hsp90 and longer residency of its inhibitors in tumor, a strategy to link anticancer drugs to an Hsp90 inhibitor, where the attached “payload” is released in the tumor selectively for prolonged periods, was conceived. Results: Though the HDC concept is applicable to most small molecule anticancer drugs, we chose a common topoisomerase-1 inhibitor (SN-38) for proof-of-concept studies. More than 100 Hsp90 inhibitor/SN38 conjugates were synthesized; we chose STA-12-8666 as our lead molecule based on its in vitro and in vivo pharmacokinetic properties. The conjugate binds strongly to Hsp90 as determined by client protein degradation assays and has good stability in plasma of several species (human>rat>mouse). In xenograft tumor tissue distribution studies, we demonstrate that mice dosed once weekly with 50 mg/kg STA-12-8666 clear the parental HDC and its cleavage products, SN-38 and the Hsp90 inhibitor, from plasma and normal tissue within 24 hr. However, we detected 0.6 µM and 0.3 µM SN-38 in tumor at 24h and 48h, respectively. SN-38 was not detected in tumor at 24h or 48h with an equivalent dose of the SN-38 prodrug, irinotecan. Xenograft studies using multiple cell lines demonstrated superior efficacy for STA-12-8666 compared to irinotecan. For example, in MCF-7 breast cancer xenografts, STA-12-8666 produced 70% regression in tumor volume compared to moderate tumor growth inhibition by irinotecan with no regression. Similar results were observed in in colon, SCLC, ovarian, bladder, NSCLC, melanoma models. Preliminary toxicological evaluation of STA-12-8666 resulted in better or comparable toxicities to that of irinotecan. Conclusion: We designed a novel tumor-directed drug delivery platform by conjugating previously validated small molecule chemotherapeutics to HSP90 inhibitors. Our lead candidate STA-12-8666, has great tissue distribution profile, superior in vivo antitumor activity and better safety profile than the active control irinotecan, warranting its clinical evaluation. Citation Format: Dinesh Chimmanamada, David Proia, Takayo Inoue, Luisa Shin Ogawa, Suqin He, Manuel Sequeira, Donald Smith, John-Paul Jimenez, Chaohua Zhang, Jim Sang, Jaime Acquaviva, Masazumi Nagai, Yuan Liu, Josephine Ye, Dan Zhou, Andrew Sonderfan, Ritu Singh, Noriaki Tatsuta, Teresa Przewloka, Jun Jiang, Junyi Zhang, Weiwen Ying. Hsp90-inhibitor drug conjugates (HDC): Novel tumor-selective drug delivery platform with unprecedented anticancer activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2509. doi:10.1158/1538-7445.AM2014-2509

A series of N′1,N′3-dialkyl-N′1,N′3-di(alkylcarbonothioyl) malonohydrazides have been designed and synthesized as anticancer agents by targeting oxidative stress and Hsp70 induction. Structure–activity relationship (SAR) studies lead to the discovery of STA-4783 (elesclomol), a novel small molecule that has been evaluated in a number of clinical trials as an anticancer agent in combination with Taxol.

Vascular disrupting agents (VDAs) are an emerging class of therapeutics targeting the existing vascular network of solid tumors. However, their clinical progression has been hampered because of limited single-agent efficacy, primarily caused by the persistence of surviving cells at the well perfused "viable rim" of tumors, which allows rapid tumor regrowth to occur. In addition, off-target adverse events, including cardiovascular toxicities, underscore a need for compounds with improved safety profiles. Here, we characterize the mechanism of action, antitumor efficacy, and cardiovascular safety profile of (S)-2-amino-N-(2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl)-3-phenylpropanamide hydrochloride (STA-9584), a novel tubulin-binding VDA. In vitro, 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)aniline (STA-9122) (active metabolite of STA-9584) displayed increased potency relative to other tubulin-binding agents and was highly cytotoxic to tumor cells. STA-9584 induced significant tumor regressions in prostate and breast xenograft models in vivo and, in an aggressive syngeneic model, demonstrated superior tumor growth inhibition and a positive therapeutic index relative to combretastatin A-4 phosphate (CA4P). It is noteworthy that histological analysis revealed that STA-9584 disrupted microvasculature at both the center and periphery of tumors. Compared with CA4P, STA-9584 induced a 73% increase in central necrotic area, 77% decrease in microvasculature, and 7-fold increase in tumor cell apoptosis in the remaining viable rim 24 h post-treatment. Ultrasound imaging confirmed that STA-9584 rapidly and efficiently blocked blood flow in highly perfused tumor regions. Moreover, cardiovascular effects were evaluated in the Langendorff assay and telemetered dogs, and cardiovascular toxicity was not predicted to be dose-limiting. This bioactivity profile distinguishes STA-9584 from the combretastatin class and identifies the compound as a promising new therapeutic VDA candidate.

A series of novel indolizine 2-oxoacetamides were designed and synthesized as PDE4 inhibitors. Preliminary SAR of this new class of compounds revealed key structural features required for high potency. Compounds 1ab and 2a are among the most potent inhibitors of PDE4 with low single nM IC50. Cellular activity was demonstrated by the inhibition of TNFα production from human PBMC with IC50 ranging from 14 to 72 nM. Docking analyses suggest the OH group in 1ab enhance the binding via an H-bond interaction with the PDE4 enzyme.