Crizotinib-based proteolysis targeting chimera suppresses gastric cancer by promoting MET degradation.

As one of the common malignant cancer types, gastric cancer (GC) is known for late-stage diagnosis and poor prognosis. Overexpression of the receptor tyrosine kinase MET is associated with poor prognosis among patients with advanced stage GC. However, no MET inhibitor has been used for GC treatment. Like other tyrosine kinase inhibitors that fit the "occupancy-driven" model, current MET inhibitors are prone to acquired resistance. The emerging proteolysis targeting chimera (PROTAC) strategy could overcome such limitations through direct degradation of the target proteins. In this study, we successfully transformed the MET-targeted inhibitor crizotinib into a series of PROTACs, recruiting cereblon/cullin 4A E3 ubiquitin ligase to degrade the MET proteins. The optimized lead PROTAC (PRO-6 E) effectively eliminated MET proteins in vitro and in vivo, inhibiting proliferation and motility of MET-positive GC cells. In the MKN-45 xenograft model, PRO-6 E showed pronounced antitumor efficacy with a well-tolerated dosage regimen. These results validated PRO-6 E as the first oral PROTAC for MET-dependent GC.

Targeted Protein Degradation Technology and Nanomedicine: Powerful Allies against Cancer.

Targeted protein degradation (TPD) is an emerging therapeutic strategy with the potential of targeting undruggable pathogenic proteins. After the first proof-of-concept proteolysis-targeting chimeric (PROTAC) molecule was reported, the TPD field has entered a new era. In addition to PROTAC, numerous novel TPD strategies have emerged to expand the degradation landscape. However, their physicochemical properties and uncontrolled off-target side effects have limited their therapeutic efficacy, raising concerns regarding TPD delivery system. The combination of TPD and nanotechnology offers great promise in improving safety and therapeutic efficacy. This review provides an overview of novel TPD technologies, discusses their clinical applications, and highlights the trends and perspectives in TPD nanomedicine.

Cytotoxic and antitumor peptides as novel chemotherapeutics.

Covering: up to 2020Treatment resistance and drug-induced refractory malignancies pose significant challenges for current chemotherapy drugs. There have been increasing research efforts aimed at developing novel chemotherapeutics, especially from natural products and related derivatives. Natural cytotoxic peptides, an emerging source of chemotherapeutics, have exhibited the advantage of overcoming drug resistance and displayed broad-spectrum antitumor activities in the clinic. This highlight examines the increasingly popular cytotoxic peptides from isolated natural products. In-depth review of several peptides provides examples for how this novel strategy can lead to the improved anti-tumor effects. The mechanisms and current application of representative natural cytotoxic peptides (NCPs) have also been discussed, with a particular focus on future directions for interdisciplinary research.

Biochemical and Anti-Triple Negative Metastatic Breast Tumor Cell Properties of Psammaplins.

Breast tumors reprogram their cellular metabolism, nutrient uptake, and utilization-associated biochemical processes. These processes become further transformed as genetically predisposed metastatic breast tumor cells colonize specific organs. Breast tumor cells often metastasize to the brain, bone, lung and liver. Massagué and colleagues isolated organotropic subclones and established organ-specific gene signatures associated with lung-, bone-, and brain-specific metastatic triple-negative breast cancer (TNBC) MDA-MB-231 cells. Using these genetically characterized metastatic subclones specific to lung (LM4175), bone (BoM1833), and brain (BrM-2a), we evaluated marine natural products for the ability to differentially suppress metastatic breast cancer cells in a target organ-dependent manner. Psammaplin-based histone deacetylase (HDAC) inhibitors were found to differentially inhibit HDAC activity, induce activation of hypoxia-inducible factor-1 (HIF-1), and disrupt organotropic metastatic TNBC subclone growth. Further, psammaplins distinctly suppressed the outgrowth of BoM1833 tumor spheroids in 3D-culture systems. Similar results were observed with the prototypical HDAC inhibitor trichostatin A (TSA). These organotropic tumor cell-based studies suggest the potential application of HDAC inhibitors that may yield new directions for anti-metastatic breast tumor research and drug discovery.

Hypoxia-inducible factor-1α inhibition modulates airway hyperresponsiveness and nitric oxide levels in a BALB/c mouse model of asthma.

Hypoxia-inducible factor (HIF)-1α is a master regulator of inflammation and is upregulated in alveolar macrophages and lung parenchyma in asthma. HIF-1α regulates select pathways in allergic inflammation, and thus may drive particular asthma phenotypes. This work examines the role of pharmacologic HIF-1α inhibition in allergic inflammatory airway disease (AIAD) pathogenesis in BALB/c mice, which develop an airway hyperresponsiveness (AHR) asthma phenotype. Systemic treatment with HIF-1α antagonist YC-1 suppressed the increase in HIF-1α expression seen in control AIAD mice. Treatment with YC-1 also decreased AHR, blood eosinophilia, and allergic inflammatory gene expression: IL-5, IL-13, myeloperoxidase and iNOS. AIAD mice had elevated BAL levels of NO, and treatment with YC-1 eliminated this response. However, YC-1 did not decrease BAL, lung or bone marrow eosinophilia. We conclude that HIF-1α inhibition in different genetic backgrounds, and thus different AIAD phenotypes, decreases airway resistance and markers of inflammation in a background specific manner. CAPSULE SUMMARY: Asthma is a common disease that can be difficult to control with current therapeutics. We describe how pharmacologic targeting of a specific transcription factor, HIF-1α, suppresses asthmatic airway reactivity and inflammation.

Sampangine (a Copyrine Alkaloid) Exerts Biological Activities through Cellular Redox Cycling of Its Quinone and Semiquinone Intermediates.

The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity.

Kalkitoxin inhibits angiogenesis, disrupts cellular hypoxic signaling, and blocks mitochondrial electron transport in tumor cells.

The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

Oncolytic Peptide-Nanoplatform Drives Oncoimmune Response and Reverses Adenosine-Induced Immunosuppressive Tumor Microenvironment.

The application of oncolytic peptides has become a powerful approach to induce complete and long-lasting remission in multiple types of carcinomas, as affirmed by the appearance of tumor-associated antigens and adenosine triphosphate (ATP) in large quantities, which jumpstarts the cancer-immunity cycle. However, the ATP breakdown product adenosine is a significant contributor to forming the immunosuppressive tumor microenvironment, which substantially weakens peptide-driven oncolytic immunotherapy. In this study, a lipid-coated micelle (CA@TLM) loaded with a stapled oncolytic peptide (PalAno) and an adenosine 2A receptor (A2AR) inhibitor (CPI-444) is devised to enact tumor-targeted oncolytic immunotherapy and to overcome adenosine-mediated immune suppression simultaneously. The CA@TLM micelle accumulates in tumors with high efficiency, and the acidic tumor microenvironment prompts the rapid release of PalAno and CPI-444. Subsequently, PalAno induces swift membrane lysis of tumor cells and the release of antigenic materials. Meanwhile, CPI-444 blocks the activation of the immunosuppressive adenosine-A2AR signaling pathway. This combined approach exhibits pronounced synergy at stalling tumor growth and metastasis in animal models for triple-negative breast cancer and melanoma, providing a novel strategy for enhanced oncolytic immunotherapy.

Semisynthetic studies identify mitochondria poisons from botanical dietary supplements--geranyloxycoumarins from Aegle marmelos.

Bioassay-guided isolation and subsequent structure elucidation of a Bael tree Aegle marmelos lipid extract yielded two unstable acylated geranyloxycoumarin mixtures (1-2), six geranyloxycoumarins (3-8), (+)-9'-isovaleroxylariciresinol (9), and dehydromarmeline (10). In a T47D cell-based reporter assay, 1 and 2 potently inhibited hypoxia-induced HIF-1 activation (IC50 values 0.18 and 1.10 µgmL(-1), respectively). Insufficient material and chemical instability prevented full delineation of the fatty acyl side chain olefin substitution patterns in 1 and 2. Therefore, five fatty acyl geranyloxycoumarin ester derivatives (11-15) were prepared from marmin (3) and commercial fatty acyl chlorides by semisynthesis. The unsaturated C-6' linoleic acid ester derivative 14 that was structurally most similar to 1 and 2, inhibited HIF-1 activation with comparable potency (IC50 0.92 µM). The octanoyl (11) and undecanoyl (12) ester derivatives also suppressed HIF-1 activation (IC50 values 3.1 and 0.87 µM, respectively). Mechanistic studies revealed that these geranyloxycoumarin derivatives disrupt mitochondrial respiration, primarily at complex I. Thus, these compounds may inhibit HIF-1 activation by suppressing mitochondria-mediated hypoxic signaling. One surprising observation was that, while less potent, the purported cancer chemopreventive agent auraptene (8) was found to act as a mitochondrial poison that disrupts HIF-1 signaling in tumors.

Comparative study of chromatographic medium-associated mass and potential antitumor activity loss with bioactive extracts.

Natural product drug discovery programs often rely on the use of silica (Si) gel, reversed-phase media, or size-exclusion resins (e.g., RP-C18, Sephadex LH-20) for compound purification. The synthetic polymer-based sorbent Diaion HP20SS (cross-linked polystyrene matrix) is used as an alternative to prepare purified natural product libraries. To evaluate the impact of chromatographic media on the isolation of biologically active, yet chromatographically unstable natural products, Diaion HP20SS was evaluated side-by-side with normal-phase sorbents for irreversible binding of extract constituents and their effects on bioactivity. An array of chemically diverse natural product-rich extracts was selected as a test panel, and a cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) was employed to monitor potential change(s) in bioactivity. Silica gel caused significant irreversible binding of three out of 10 extracts. Curcuma longa, Saururus cernuus, and Citrus reticulata extracts showed decreased HIF-1 inhibitory activity after elution through Si gel. An additional nonpolar column wash of HP20SS with EtOAc retained considerable bioactivities of active extracts. In general, Si gel produced the greatest loss of bioactivity. However, HP20SS elution reduced significantly HIF-1 inhibitory activity of certain extracts (e.g., Asimina triloba).

Inducers of hypoxic response: marine sesquiterpene quinones activate HIF-1.

The hypoxia-inducible factor-1 (HIF-1) transcription factor regulates cellular oxygen homeostasis. Agents that activate HIF-1 and downstream HIF targets represent potential drug leads for the prevention and/or treatment of ischemic disorders. In a search for small-molecule HIF-1 activators, 1936 marine invertebrate and algal extract samples (U.S. National Cancer Institute's Open Repository) were evaluated for HIF-1 activation activity in a cell-based reporter assay. Bioassay-guided fractionation of two active extracts of the sponge Dactylospongia elegans afforded four new sesquiterpene quinones (2-5), one new sesquiterpene phenol (6), the known Golgi disruptor ilimaquinone (1), and three previously reported ilimaquinone analogues (7-9). While antiproliferative activity was observed at higher concentrations, the sesquiterpene quinones (1-3) possessing a 2-hydroxy-5-methoxy-1,4-benzoquinone moiety activated HIF-1 and increased the expression of HIF-1 target gene vascular endothelial growth factor (VEGF) in T47D cells.

Structures and potential antitumor activity of sesterterpenes from the marine sponge Hyrtios communis.

The extract of marine sponge Hyrtios communis was found to inhibit activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) in T47D human breast tumor cells. Bioassay-guided isolation led to the identification of six new (1-6) and five previously reported (7-11) sesterterpene analogues and two unrelated sesterterpenes. Two new sesterterpenes, thorectidaeolide A (1) and 4-acetoxythorectidaeolide A (2), and luffariellolide (11) were among the most potent inhibitors of hypoxia (1% O2)-induced HIF-1 activation (IC50 values of 3.2, 3.5, and 3.6 µM, respectively). Luffariellolide (11) exhibited a significant level of cytotoxicity that mirrored its HIF-1 inhibitory activity. Neither compound 1, compound 2, nor any of the other less active sesterterpenes suppressed breast tumor T47D or MDA-MB-231 cell viability.

Structures and mechanisms of antitumor agents: xestoquinones uncouple cellular respiration and disrupt HIF signaling in human breast tumor cells.

The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1-7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1-7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC(50) value of 0.2 µM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.

Glycolysis inhibitor screening identifies the bis-geranylacylphloroglucinol protonophore moronone from Moronobea coccinea.

Tumor cells exhibit enhanced glucose consumption and lactate production even when supplied with adequate oxygen (a phenomenon known as the Warburg effect, or aerobic glycolysis). Pharmacological inhibition of aerobic glycolysis represents a potential tumor-selective approach that targets the metabolic differences between normal and malignant tissues. Human breast tumor MDA-MB-231 cells were used to develop an assay system to discover natural product-based glycolysis inhibitors. The assay employed was based on hypersensitivity to glycolytic inhibition in tumor cells treated with the mitochondrial electron transport inhibitor rotenone. Under such conditions, ATP supply, and hence cell viability, depends exclusively on glycolysis. This assay system was used to evaluate 10648 plant and marine organism extracts from the U.S. National Cancer Institute's Open Repository. Bioassay-guided isolation of an active Moronobea coccinea extract yielded the new bis-geranylacylphloroglucinol derivative moronone (1). Compound 1 exhibited enhanced antiproliferative/cytotoxic activity in the presence of rotenone-imposed metabolic stress on tumor cells. Surprisingly, mechanistic studies revealed that 1 did not inhibit glycolysis, but rather functions as a protonophore that dissipates the mitochondrial proton gradient. In the presence of rotenone, tumor cells may be hypersensitive to protonophores due to increased ATP utilization by the ATP synthase.

CD47: Beyond an immune checkpoint in cancer treatment.

The transmembrane protein, CD47, is recognized as an important innate immune checkpoint, and CD47-targeted drugs have been in development with the aim of inhibiting the interaction between CD47 and the regulatory glycoprotein SIRPα, for antitumor immunotherapy. Further, CD47 mediates other essential functions such as cell proliferation, caspase-independent cell death (CICD), angiogenesis and other integrin-activation-dependent cell phenotypic responses when bound to thrombospondin-1 (TSP-1) or other ligands. Mounting strategies that target CD47 have been developed in pre-clinical and clinical trials, including antibodies, small molecules, siRNAs, and peptides, and some of them have shown great promise in cancer treatment. Herein, the authors endeavor to provide a retrospective of ligand-mediated CD47 regulatory mechanisms, their roles in controlling antitumor intercellular and intracellular signal transduction, and an overview of CD47-targetd drug design.

Polymer chimera of stapled oncolytic peptide coupled with anti-PD-L1 peptide boosts immunotherapy of colorectal cancer.

Rationale: Scarce tumor mutation burden and neoantigens create tremendous obstacles for an effective immunotherapy of colorectal cancer (CRC). Oncolytic peptides rise as a promising therapeutic approach that boosts tumor-specific immune responses by inducing antigenic substances. However, the clinical application of oncolytic peptides has been hindered because of structural instability, proteolytic degradation, and undesired toxicity when administered systemically. Methods: Based on wasp venom peptide, an optimized stapled oncolytic peptide MP9 was developed with rigid α-helix, protease-resistance, and CRC cell cytotoxicity. By incorporating four functional motifs that include D-peptidomimetic inhibitor of PD-L1, matrix metalloproteinase-2 (MMP-2) cleavable spacer, and MP9 with 4-arm PEG, a novel peptide-polymer conjugate (PEG-MP9-aPDL1) was obtained and identified as the most promising systemic delivery vehicle with PD-L1 targeting specificity and favorable pharmacokinetic properties. Results: We demonstrated that PEG-MP9-aPDL1-driven oncolysis induces a panel of immunogenic cell death (ICD)-relevant damage-associated molecular patterns (DAMPs) both in vitro and in vivo, which are key elements for immunotherapy with PD-L1 inhibitor. Further, PEG-MP9-aPDL1 exhibited prominent immunotherapeutic efficacy in a CRC mouse model characterized by tumor infiltration of CD8+ T cells and induction of cytotoxic lymphocytes (CTLs) in the spleens. Conclusion: Our findings suggest that PEG-MP9-aPDL1 is an all-in-one platform for oncolytic immunotherapy and immune checkpoint blockade (ICB).

Small-molecule PROTAC mediates targeted protein degradation to treat STAT3-dependent epithelial cancer.

The aberrant activation of STAT3 is associated with the etiology and progression in a variety of malignant epithelial-derived tumors, including head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). Due to the lack of an enzymatic catalytic site or a ligand-binding pocket, there are no small-molecule inhibitors directly targeting STAT3 that have been approved for clinical translation. Emerging proteolysis targeting chimeric (PROTAC) technology-based approach represents a potential strategy to overcome the limitations of conventional inhibitors and inhibit activation of STAT3 and downstream genes. In this study, the heterobifunctional small-molecule-based PROTACs are successfully prepared from toosendanin (TSN), with 1 portion binding to STAT3 and the other portion binding to an E3 ubiquitin ligase. The optimized lead PROTAC (TSM-1) exhibits superior selectivity, potency, and robust antitumor effects in STAT3-dependent HNSCC and CRC - especially in clinically relevant patient-derived xenografts (PDX) and patient-derived organoids (PDO). The following mechanistic investigation identifies the reduced expression of critical downstream STAT3 effectors, through which TSM-1 promotes cell cycle arrest and apoptosis in tumor cells. These findings provide the first demonstration to our knowledge of a successful PROTAC-targeting strategy in STAT3-dependent epithelial cancer.

Natural and semisynthetic mammea-type isoprenylated dihydroxycoumarins uncouple cellular respiration.

In an effort to identify natural product-based molecular-targeted antitumor agents, mammea-type coumarins from the tropical/subtropical plant Mammea americana were found to inhibit the activation of HIF-1 (hypoxia-inducible factor-1) in human breast and prostate tumor cells. In addition to the recently reported mammea E/BB (15), bioassay-guided fractionation of the active extract yielded 14 mammea-type coumarins including three new compounds, mammea F/BB (1), mammea F/BA (2), and mammea C/AA (3). The absolute configuration of C-1' in 1 was determined by the modified Mosher's method on a methylated derivative. These coumarins were evaluated for their effects on mitochondrial respiration, HIF-1 signaling, and tumor cell proliferation/viability. Acetylation of 1 afforded a triacetoxylated product (A-2) that inhibited HIF-1 activation with increased potency in both T47D (IC(50) 0.83 µM for hypoxia-induced) and PC-3 cells (IC(50) 0.94 µM for hypoxia-induced). Coumarins possessing a 6-prenyl-8-(3-methyloxobutyl) substituent pattern exhibited enhanced HIF-1 inhibitory effects. The O-methylated derivatives were less active at inhibiting HIF-1 and suppressing cell proliferation/viability. Mechanistic studies indicate that these compounds act as anionic protonophores that potently uncouple mitochondrial electron transport and disrupt hypoxic signaling.

Mitochondrial respiration inhibitors suppress protein translation and hypoxic signaling via the hyperphosphorylation and inactivation of translation initiation factor eIF2α and elongation factor eEF2.

Over 20,000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods.