Browsing by Subject "Drug Discovery"
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Item Open Access A new trial design to accelerate tuberculosis drug development: the Phase IIC Selection Trial with Extended Post-treatment follow-up (STEP).(BMC Med, 2016-03-23) Phillips, Patrick PJ; Dooley, Kelly E; Gillespie, Stephen H; Heinrich, Norbert; Stout, Jason E; Nahid, Payam; Diacon, Andreas H; Aarnoutse, Rob E; Kibiki, Gibson S; Boeree, Martin J; Hoelscher, MichaelBACKGROUND: The standard 6-month four-drug regimen for the treatment of drug-sensitive tuberculosis has remained unchanged for decades and is inadequate to control the epidemic. Shorter, simpler regimens are urgently needed to defeat what is now the world's greatest infectious disease killer. METHODS: We describe the Phase IIC Selection Trial with Extended Post-treatment follow-up (STEP) as a novel hybrid phase II/III trial design to accelerate regimen development. In the Phase IIC STEP trial, the experimental regimen is given for the duration for which it will be studied in phase III (presently 3 or 4 months) and patients are followed for clinical outcomes of treatment failure and relapse for a total of 12 months from randomisation. Operating characteristics of the trial design are explored assuming a classical frequentist framework as well as a Bayesian framework with flat and sceptical priors. A simulation study is conducted using data from the RIFAQUIN phase III trial to illustrate how such a design could be used in practice. RESULTS: With 80 patients per arm, and two (2.5 %) unfavourable outcomes in the STEP trial, there is a probability of 0.99 that the proportion of unfavourable outcomes in a potential phase III trial would be less than 12 % and a probability of 0.91 that the proportion of unfavourable outcomes would be less than 8 %. With six (7.5 %) unfavourable outcomes, there is a probability of 0.82 that the proportion of unfavourable outcomes in a potential phase III trial would be less than 12 % and a probability of 0.41 that it would be less than 8 %. Simulations using data from the RIFAQUIN trial show that a STEP trial with 80 patients per arm would have correctly shown that the Inferior Regimen should not proceed to phase III and would have had a high chance (0.88) of either showing that the Successful Regimen could proceed to phase III or that it might require further optimisation. CONCLUSIONS: Collection of definitive clinical outcome data in a relatively small number of participants over only 12 months provides valuable information about the likelihood of success in a future phase III trial. We strongly believe that the STEP trial design described herein is an important tool that would allow for more informed decision-making and accelerate regimen development.Item Open Access Advancing drug discovery for glomerulopathies using stem-cell-derived kidney models.(Trends in pharmacological sciences, 2023-04) Barreto, Amanda D; Burt, Morgan A; Musah, SamiraChronic kidney disease (CKD) is an epidemic that affects millions worldwide. The glomerulus, a specialized unit of the nephron, is highly susceptible to injury. Human induced pluripotent stem cells (iPSCs) have emerged as an attractive resource for modeling kidney disease and therapeutic discovery.Item Open Access AI is a viable alternative to high throughput screening: a 318-target study.(Scientific reports, 2024-04) Atomwise AIMS ProgramHigh throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery.Item Open Access Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.(Nature communications, 2019-09) Juvvadi, Praveen R; Fox, David; Bobay, Benjamin G; Hoy, Michael J; Gobeil, Sophie MC; Venters, Ronald A; Chang, Zanetta; Lin, Jackie J; Averette, Anna Floyd; Cole, D Christopher; Barrington, Blake C; Wheaton, Joshua D; Ciofani, Maria; Trzoss, Michael; Li, Xiaoming; Lee, Soo Chan; Chen, Ying-Lien; Mutz, Mitchell; Spicer, Leonard D; Schumacher, Maria A; Heitman, Joseph; Steinbach, William JCalcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.Item Open Access HIV-1 envelope gp41 broadly neutralizing antibodies: hurdles for vaccine development.(PLoS Pathog, 2014-05) Verkoczy, Laurent; Kelsoe, Garnett; Haynes, Barton FItem Open Access Human endotoxin administration as an experimental model in drug development.(Clin Pharmacol Ther, 2014-10) Suffredini, AF; Noveck, RJLinking human physiology to inflammatory mechanisms discovered in vitro or in animal models is essential to determine their importance. Innate immunity underlies many of these inflammatory responses in health and disease. Bacterial endotoxin is the quintessential trigger of innate immune responses. When administered to humans, endotoxin has been an important means of demonstrating key inflammatory mechanisms in vivo. Furthermore, endotoxin challenges have provided opportunities to test the effects of novel inflammation-modifying agents in humans.Item Open Access Impact of economic, regulatory, and patent policies on innovation in cancer chemoprevention.(Cancer Prev Res (Phila), 2008-07) Moe, Jeffrey LChemoprevention agents are an emerging new scientific area that holds out the promise of delaying or avoiding a number of common cancers. These new agents face significant scientific, regulatory, and economic barriers, however, which have limited investment in their research and development (R&D). These barriers include above-average clinical trial scales, lengthy time frames between discovery and Food and Drug Administration approval, liability risks (because they are given to healthy individuals), and a growing funding gap for early-stage candidates. The longer time frames and risks associated with chemoprevention also cause exclusivity time on core patents to be limited or subject to significant uncertainties. We conclude that chemoprevention uniquely challenges the structure of incentives embodied in the economic, regulatory, and patent policies for the biopharmaceutical industry. Many of these policy issues are illustrated by the recently Food and Drug Administration-approved preventive agents Gardasil and raloxifene. Our recommendations to increase R&D investment in chemoprevention agents include (a) increased data exclusivity times on new biological and chemical drugs to compensate for longer gestation periods and increasing R&D costs; chemoprevention is at the far end of the distribution in this regard; (b) policies such as early-stage research grants and clinical development tax credits targeted specifically to chemoprevention agents (these are policies that have been very successful in increasing R&D investment for orphan drugs); and (c) a no-fault liability insurance program like that currently in place for children's vaccines.Item Embargo Investigation of Heat Shock Protein 90 in Plasmodium Parasites(2024) Mansfield, Christopher RockyMalaria is an infectious disease caused by apicomplexan Plasmodium species. These protozoan parasites are transmitted by a mosquito vector to a human host, wherein they undergo an asymptomatic liver stage followed by a symptomatic blood stage of infection. Despite eradication efforts, Plasmodium remain accountable for hundreds of thousands of mortalities per year, mostly caused by P. falciparum. The spreading resistance to the front-line antimalarials that broadly disrupt parasite proteostasis demands further characterization of their adaptive stress responses and novel multi-stage drug targets. This work focuses on the essential P. falciparum molecular chaperone heat shock protein 90 (PfHsp90). Specifically, PfHsp90 is expected to directly interact with a subset of parasite proteins to facilitate their ATP-dependent maturation, stabilization, and regulation. Despite this critical function, the scope of its chaperoning interactions—as well as its consequent contributions to mitigate cellular stress and maintain parasite proteome integrity throughout development—remains largely unresolved. To enable its functional interrogation, we first aimed to establish chemical inhibitors of PfHsp90 with greater affinity to the parasite compared to the conserved human homolog (HsHsp90). In general, our testing supports the particular utility of compounds that bind at the chaperone’s nucleotide-binding domain, as opposed to a putative C-terminal allosteric site, based on their high affinity and resolved mode of ATP-competitive inhibition. From this class of competitive inhibitors, we identified XL888 as exhibiting moderate selectivity to PfHsp90, despite that it was initially developed as a HsHsp90 inhibitor. Subsequent structural evaluation indicated that the PfHsp90 lid subdomain contributes to the parasite chaperone’s higher affinity interaction with XL888’s tropane scaffold. Considering this molecular basis, we were able to develop Tropane 1 as a novel XL888 analog with nanomolar affinity and approximately 10-fold selectivity to PfHsp90, which further demonstrated dual-stage, anti-Plasmodium activity. We next surveyed the PfHsp90-dependent proteome using innovative chemical biology strategies. Based on their thermal stability after chaperone inhibition, we identified 50 candidates as putative PfHsp90 interactors. A significant enrichment of proteasome regulatory particle components was represented in this analysis, from which we subsequently validated that PfHsp90 chaperones the 26S proteasome to support the controlled recycling of cellular proteins. Additionally, we adopted bio-orthogonal labeling with unnatural amino acids to track proteome dynamics in Plasmodium parasites. To date, we have implemented this approach to support that compromised PfHsp90 activity coordinates translation attenuation as a stress response. However, this work sets the foundation to employ such labeling to quantify PfHsp90-coordinated proteome dynamics across multiple parasite life stages. Collectively, these findings broaden our understanding of PfHsp90’s regulation of Plasmodium parasite proteostasis and further establish the potential of this molecular chaperone as a novel, multi-stage antimalarial drug target.
Item Open Access Priorities for the Priority Review Voucher.(Am J Trop Med Hyg, 2017-01-11) Ridley, David BThe U.S. Congress created the priority review voucher program in 2007 to encourage development of drugs for neglected diseases. Under the voucher program, the developer of a drug for a neglected or rare pediatric disease that is approved by the U.S. Food and Drug Administration receives a bonus priority review voucher for another drug. As of 2016, four vouchers have sold for an average price of $200 million. Recent experience with the voucher program indicates strengths and weaknesses of the program, as well as a need for legislative changes.Item Open Access Targeting the SUMO pathway for neuroprotection in brain ischaemia.(Stroke and vascular neurology, 2016-09) Yang, Wei; Sheng, Huaxin; Wang, HaichenSmall ubiquitin-like modifier (SUMO) conjugation (SUMOylation) is a post-translational protein modification that modulates almost all major cellular processes, and has been implicated in many human diseases. A growing body of evidence from in vitro and in vivo studies demonstrates that increasing global levels of SUMO conjugated proteins (global SUMOylation) protects cells against ischaemia-induced damage, while suppressing global SUMOylation promotes cell injury after ischaemia. Indeed, SUMOylation has emerged as a potential therapeutic target for neuroprotection in brain ischaemia, including global brain ischaemia and focal brain ischaemia (ischaemic stroke). Here, we summarise findings on the role of SUMOylation in human diseases, brain ischaemia in particular, and review recent developments in drug discovery targeting SUMOylation with a major focus on its neuroprotective applications.Item Open Access The cost of drug development.(N Engl J Med, 2015-05-14) DiMasi, Joseph A; Grabowski, Henry G; Hansen, Ronald WItem Open Access Therapeutic Development of Apolipoprotein E Mimetics for Acute Brain Injury: Augmenting Endogenous Responses to Reduce Secondary Injury.(Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics, 2020-04) James, Michael L; Komisarow, Jordan M; Wang, Haichen; Laskowitz, Daniel TOver the last few decades, increasing evidence demonstrates that the neuroinflammatory response is a double-edged sword. Although overly robust inflammatory responses may exacerbate secondary tissue injury, inflammatory processes are ultimately necessary for recovery. Traditional drug discovery often relies on reductionist approaches to isolate and modulate specific intracellular pathways believed to be involved in disease pathology. However, endogenous brain proteins are often pleiotropic in order to regulate neuroinflammation and recovery mechanisms. Thus, a process of "backward translation" aims to harness the adaptive properties of endogenous proteins to promote earlier and greater recovery after acute brain injury. One such endogenous protein is apolipoprotein E (apoE), the primary apolipoprotein produced in the brain. Robust preclinical and clinical evidence demonstrates that endogenous apoE produced within the brain modulates the neuroinflammatory response of the acutely injured brain. Thus, one innovative approach to improve outcomes following acute brain injury is administration of exogenous apoE-mimetic drugs optimized to cross the blood-brain barrier. In particular, one promising apoE mimetic peptide, CN-105, has demonstrated efficacy across a wide variety of preclinical models of brain injury and safety and feasibility in early-phase clinical trials. Preclinical and clinical evidence for apoE's neuroprotective effects and downregulation of neuroinflammatory and the resulting translational therapeutic development strategy for an apoE-based therapeutic are reviewed.Item Open Access Understanding and Targeting Fatty Acid-CoA Ligase ACSL4 in Advanced Human Prostate Cancer(2024) Wu, JinjinProstate cancer (PCa) ranks as the most prevalent non-cutaneous malignancy among men. While localized prostate cancer can often be cured, the recurrence of metastatic castration-resistant prostate cancer (mCRPC) remains a formidable challenge and is the leading cause of mortality in PCa patients. Recent findings from our lab and others highlights the pivotal role of RB1 loss in driving lineage plasticity, metastasis, and lethality of prostate tumors. One downstream target of RB1 loss/E2F activation is acyl-CoA synthetase long-chain family member 4 (ACSL4), a fatty acid ligase crucial for the utilization of long-chain fatty acids. Dysregulated fatty acid metabolism fuels oncogenic processes and tumor progression through excessive energy production and lipogenesis, and reprogramming fatty acid metabolism shows promise in advanced cancer therapy. ACSL4 has been reported to be upregulated in cancers of various histological origins, including prostate cancer, and its elevated expression is correlated with cancer aggressiveness. However, the metabolic regulation and clinical significance of ACSL4 in prostate tumorigenesis and progression remains elusive. Through both gain-of-function and loss-of-function approach aiming to examine the outcome of high ACSL4 level in PCa patients, we revealed ACSL4 as an oncogenic driver that promoted prostate cancer progression via enhanced cell proliferation and ATP production. Further characterization via untargeted lipidomic profiling and RNA sequencing has unveiled a distinct metabolic profile and transcriptomic landscape resulting from ACSL4 overexpression, characterized by altered organelle membrane composition, extracellular matrix component and increased neuroendocrine markers that support cancer progression. These findings underscore the potential of ACSL4 as a novel target for advanced cancer therapy. By employing a structure-based virtual screening approach targeting ATP binding domains of ACSL4, followed by in vitro drug testing, we successfully identified four candidate ACSL4 inhibitors. These inhibitors demonstrated significant efficacy in suppressing prostate cancer (PCa) cell proliferation in a dose-response manner. Notably, we showed that these candidate inhibitors exhibit enhanced effectiveness in inhibiting cell proliferation in isogenic ACSL4-high PCa cells. Moreover, we assessed inhibition selectivity through an isotope-labeled ACSL4 activity assay for these inhibitors. Moving forward, our focus will shift towards analyzing pharmacodynamics/pharmacokinetics (PD/PK) for these inhibitors and modifying drug structures to facilitate in vivo application.The first three chapters of this dissertation are introductory information that reviews the role of lipid metabolism in cancer progression and therapy resistance through the regulation of energy production, phospholipid composition and lipid storage, and the clinical significance of targeting lipid metabolism and fatty acid activation as a cancer therapy. Chapter 1 provides a comprehensive review of the current knowledge of how altered lipid metabolism contributes to aberrant cellular activities and oncogenic events in cancer cells, particularly in prostate cancer. Fatty acid activation is key to lipid utilization, and upregulation of multiple Acyl-CoA synthetase, including ACSL4, is frequently observed in cancer patients. The dissertation aims to bridge the existing gap in knowledge by unraveling the biological mechanisms underlying the positive correlation between prostate tumorigenesis and ACSL4 as a long-chain fatty acid activator. Furthermore, it seeks to explore the therapeutic potential of targeting ACSL4 as a viable option for managing advanced prostate cancer. Chapter 2 provided the background of lipid metabolism, and the impact of dysregulated lipid metabolism on cancer cell activities as a result of oncogenic signaling. In this chapter, we highlight the current understanding of the intricate interplay between cancer lipid metabolism and well-known oncogenic signaling. By elucidating these connections, we illuminate how altered lipid metabolic pathways contribute to the reshaping of cellular components, modulation of energy production levels, and perturbation of lipid storage mechanisms, all of which drive cancer progression. Chapter 3 discusses the knowledge and considerations in the current field on the potential of targeting various pathways in lipid metabolism as effective cancer therapies. Given the pivotal role of dysregulated lipid metabolism during tumorigenesis, there has been significant clinical interest in developing pharmacological inhibitors to target altered lipid metabolic activities in cancer. Recent studies have discovered multiple targets as key fatty acid metabolic regulators during oncogenesis with several inhibitors demonstrating effectiveness in both preclinical studies and clinical trials. Most of these drugs exert their effects on tumorigenesis and tumor expansion by targeting enzymes in different lipid metabolic pathways, including de novo fatty acid synthesis, metabolic substrates, fatty acid degradation and lipid storage. The major obstacle in this field has been related to target specificity, as many enzymes within the same family share high structural similarities, leading to off-target issues for many lipogenesis inhibitors. In addition, inhibition of some crucial steps in fatty acid metabolism can cause severe side effects due to accumulation of unexpected side products and toxicity to normal tissues. In many cases, single treatment with lipid synthesis inhibitors shows only moderate efficacy. Therefore, it is imperative for future studies to discover novel targets more specific to oncogenesis and explore the potential of combinatorial treatment with other therapeutic options such as chemotherapy and androgen deprivation therapy to achieve best efficacy with minimized toxicity. Chapter 4 provides the basis for this thesis project based on our previous findings and preliminary data on the key modulators of advanced prostate cancer progression and metastasis. The Retinoblastoma Transcriptional Corepressor 1 (RB) is a key cell cycle regulator and tumor suppressor through regulating essential transcriptional activities via the RB1/E2F signaling axis. Loss of RB1 activity is known to drive cancer cell proliferation and correlates with cancer aggressiveness. Consistently, our preliminary data also showed that co-deletion of RB1 and PTEN in a mouse model led to a highly aggressive and lethal metastatic prostate cancer compared with PTEN deletion only. In addition to its well-established role in mitotic cell cycle regulation, RB1 has been reported to execute tumor-suppressing function through controlling important oncogenic transcriptional activities. Importantly, our previous study identified an RB1 downstream effector called ACSL4, also known as Acyl-CoA Synthetase Long Chain Family Member 4. We showed that RB1/E2F negatively regulates ACSL4 transcription through multiple binding sites on its promoter region, and ACSL4 levels are found to be significantly upregulated in RB-loss PCa cells and patient samples. ACSL4 catalyzes the activation and membrane incorporation of fatty acids by promoting fatty acid utilization and subsequently regulates cell lipid metabolism. Moreover, ACSL4 has been reported to be overexpressed in advanced cancers, including metastatic castration-resistant prostate cancer (mCRPC) and neuroendocrine prostate cancer (NEPC), and its upregulation is often associated with therapy resistance and poor prognosis. In addition to RB1, multiple genes have been reported to regulate ACSL4 level in cancer cells, including the well-known oncogenic signaling AR. However, the role of ACSL4 in reshaping cancer fatty acid metabolism thus promoting tumorigenesis remains elusive. Understanding the biological function of ACSL4 in the context of cancer metabolic landscape will provide great insight into novel targeted therapy by inhibiting ACSL4 activity. Chapter 5 discusses the findings from the first part of this thesis project, which focuses on characterizing the impact of ACSL4 on prostate tumorigenesis through its regulation on cancer cell activities. To explore the role of ACSL4 in cancer cell proliferation and to understand the underlying mechanism, we examined ACSL4 levels in common human PCa cell lines, and generated ACSL4 overexpression cell lines to represent the clinical observation of ACSL4 upregulation. Examination of cell proliferation through crystal violet assay showed that ACSL4 overexpression led to significantly increased cell growth in human PCa cell lines. In addition, ACSL4-overexpressing xenograft tumors had increased growth rate in vivo. Further characterization through the seahorse assay revealed that these cells showed significantly higher oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), indicating boosted ATP-dependent cell viability that accounts for the increased cell proliferation. ACSL4 activates intracellular fatty acids, preferably long-chain fatty acids by esterification of CoA to free fatty acids and generate Acyl-CoA. One metabolic fate of Acyl-CoA is to be diverted into mitochondria, where they are further catalyzed into Acetyl-CoA and participate in tricarboxylic acid (TCA) cycle. Acceleration of TCA cycle due to increased Acetyl-CoA production can cause excessive ATP production supporting cancer cell growth. This result is also consistent with the previous finding that cancer cells tend to shift fatty acid utilization towards de novo synthesis. To confirm these results through loss-of-function studies, we sought to generate ACSL4 knockout and knockdown cell lines. Interestingly, shRNA knockdown of ACSL4 could not be stabilized due to impaired cell viability in parental DU145 and PC-3 cell lines. Alternatively, we found that cells with double knockdown of RB1 and ACSL4 can be stably passed with the knockdown effects well maintained. Our previous study found that RB1 loss granted PCa cells growth advantage and supported cancer progression, and that RB1 deficient PCa is highly lethal and lacks effective therapy options. Examination of the double knockdown PCa cells revealed that the proliferation of these cells was significantly impaired compared. Consistently, we found that ACSL4 knockout PCa cells had a significantly decreased cell proliferation rate and ATP production level. This result provides strong rationale for targeting ACSL4 activity as an efficient way to treat advanced prostate cancers with RB1 deficiency. To further investigate the role of ACSL4 in reshaping cancer cell fatty acid metabolic landscape, in chapter 6 we aim to dissect the mechanism behind the effect of ACSL4 on PCa tumor growth. To this end, we performed untargeted lipidomic profiling analysis and RNA sequencing analysis on ACSL4 overexpression cells and control cells. Here we emphasize the role of ACSL4 in reshaping cancer cell lipid metabolic landscape to support cancer cell growth and drug resistance. Our lipidomic profiling analysis revealed upregulated lipid species in ACSL4 overexpressing PCa cells including triglyceride (TG), bismethyl phosphatidic acid (BisMePA), cholesteryl ester (ChE) and phosphatidylethanolamine (PE). Notably, upregulation of TG and PE has been associated with increased risk of tumor metastasis and progression. ACSL4 preferably consumes long-chain fatty acids including arachidonic acid (AA), eicosatetraenoic acid (EA), stearic acid (SA) and palmitic acid (PA) as substrates. Particularly, BisMePA and ChE with AA, EA and SA acyl tails are also enriched in ACSL4 overexpressing PCa cells. Interestingly, upregulation of BisMePA was observed for both ACSL4-specific acyl chains and in total amount. BisMePA is a major component of lysosomal and endosomal membranes, regulating endo-lysosomal capacity and cell response to stress, and elevated BisMePA could promote cancer cell survival and drug resistance. Interestingly, RNA-seq analysis on ACSL4-overexpressing cells revealed significant change of a set of genes that regulates lysosome stability and activity, consistent with our lipidomic data. Moreover, we found that ACSL4 OE may play a role in remodeling cancer cell extracellular matrix (ECM) to favor cancer cell migration and drug resistance as indicated by significant changes in the ADAMTS family and A2M. Intriguingly, we also noticed that ACSL4 OE cells had a distinct pattern of neuroendocrine prostate carcinomas (NEPC) signature genes expression independent of AR signaling compared with the wildtype cells, which warrants further study of how ACSL4 is involved in lineage plasticity during the progression of NEPC. Given these results, it is imperative to explore the feasibility of pharmacological inhibition of ACSL4 for clinical application. Chapter 7 focuses on the discovery of novel ACSL4 inhibitors as potential treatment for advanced prostate cancer. Structure-based virtual screening (SBVS) utilizes computational docking approach based on the protein 3D structures, which has been widely applied in the discovery of novel small molecule ligands in early-stage drug discovery. To identify ACSL4 inhibitors, we performed SBVS by rendering and docking ACSL4 structure against 100,000 ligands obtained from three Maybridge compound libraries. These ligands were docked against ATP binding pocket and K572 acyl group accommodating channel on ACSL4 structure, respectively to optimize target selectivity. Standard docking followed by precision docking of the top hits generated each round was performed to maximize targeting accuracy. As a result, we identified 158 hits in total on ACSL4 structure, from which we selected top 15 hit compounds for subsequent in vitro screening. SBVS complements high-throughput screening (HTS) through computational approach and provides valuable insights into novel compound discovery, however, downstream in vitro validation is required to examine the pharmacological effects of these candidate molecules. Therefore, we established an in vitro drug testing system using human PCa cell lines to test the potency of the 15 top candidates. We found that 4 out of 15 compounds effectively inhibited PCa cell proliferation in a dose-response manner and their inhibitory effects positively correlated with cellular levels of ACSL4. Notably, we highlight the potency of our candidate inhibitors on suppressing PCa cell growth compared with the only previously reported ACSL4 inhibitor PRGL493, particularly in aggressive ACSL4-high PCa cells such as RB1-deficient PC-3 and neuroendocrine prostate cancer (NEPC) cell line H660. Achieving inhibitor selectivity for desired targets in the development of new drugs is often challenging and can lead to severe side effects due to off targeting. Here we performed an isotope-labeled enzyme activity assay to assess the inhibitor selectivity of our candidate inhibitors on ACSL4 activity. Through measuring the consumption and conversion of arachidonic acid (AA) to AA-CoA by ACSL4 activity, we confirmed that 2 of our candidate inhibitors showed high selectivity towards ACSL4 inhibition. Chapter 8 highlights the potential of ACSL4 inhibitors in the context of clinical application. ACSL4 is known to regulate cancer cell sensitivity to ferroptosis through increased lipid peroxidation. Chemotherapeutic agents inducing cancer cell apoptosis such as docetaxel have been widely used to treat patients with metastatic prostate cancer. It has been debated that the effect of chemotherapy in certain contexts also involves other types of cell death including ferroptosis, arguing that ACSL4 inhibitor could weaken the potency of chemotherapy. Therefore, we investigated the mechanism of action of docetaxel and ACSL4 inhibitors in the context of prostate cancer. We showed that docetaxel treatment mainly induced apoptosis in PCa cell lines PC-3 and DU145, given that the inhibition of cell growth was only rescued by apoptosis inhibitors Z-VAD, not ferroptosis inhibitors ferrostatin and deferoxamine (DFO). This result indicates that inhibition of ACSL4 will not attenuate the effect of chemotherapy. Consistently, ACSL4 overexpressing PCa cells are more resistant to docetaxel treatment. Therefore, we further sought to maximize the therapeutic efficacy for ACSL4 inhibitors, and we found that combinational treatment of ACSL4 inhibitors and docetaxel displayed a synergistic effect on PCa cells, which was partially rescued by apoptosis inhibitor Z-VAD. Our data provides evidence for a novel precision therapeutic option for advanced PCa patients with high ACSL4 expression. Chapter 9 concludes the findings of this study and offers directions for future research. It underscores the significance of targeting fatty acid metabolism and ACSL4 as a pivotal metabolic regulator and oncogenic driver in tumorigenesis and progression, providing valuable insights into treatment options for targeted therapy in advanced prostate cancer.
Item Open Access Vaccines in 2017: Closing in on a Zika virus vaccine.(Nature reviews. Immunology, 2018-02) Diamond, Michael S; Coyne, Carolyn B