Integrated transcriptomics- and structure-based drug repositioning identifies drugs with proteasome inhibitor properties.

Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the ß5 subunit, our in vitro study revealed that these compounds inhibited the ß1, ß2, and ß5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.

Association of KRAS Mutation and Gene Pathways in Colorectal Carcinoma: A Transcriptome- and Methylome-Wide Study and Potential Implications for Therapy.

Kirsten Rat Sarcoma (KRAS) is the most commonly mutated oncogene in colorectal carcinoma (CRC). We have previously reported the interactions between microsatellite instability (MSI), DNA promoter methylation, and gene expression. In this study, we looked for associations between KRAS mutation, gene expression, and methylation that may help with precision medicine. Genome-wide gene expression and DNA methylation were done in paired CRC tumor and surrounding healthy tissues. The results suggested that (a) the magnitude of dysregulation of many major gene pathways in CRC was significantly greater in patients with the KRAS mutation, (b) the up- and down-regulation of these dysregulated gene pathways could be correlated with the corresponding hypo- and hyper-methylation, and (c) the up-regulation of CDKN2A was more pronounced in tumors with the KRAS mutation. A recent cell line study showed that there were higher CDKN2A levels in 5-FU-resistant CRC cells and that these could be down-regulated by Villosol. Our findings suggest the possibility of a better response to anti-CDKN2A therapy with Villosol in KRAS-mutant CRC. Also, the more marked up-regulation of genes in the proteasome pathway in CRC tissue, especially with the KRAS mutation and MSI, may suggest a potential role of a proteasome inhibitor (bortezomib, carfilzomib, or ixazomib) in selected CRC patients if necessary.

Multi-omics analysis identifies repurposing bortezomib in the treatment of kidney-, nervous system-, and hematological cancers.

Repurposing of FDA-approved drugs is a quick and cost-effective alternative to de novo drug development. Here, we identify genes involved in bortezomib sensitivity, predict cancer types that may benefit from treatment with bortezomib, and evaluate the mechanism-of-action of bortezomib in breast cancer (BT-474 and ZR-75-30), melanoma (A-375), and glioblastoma (A-172) cells in vitro. Cancer cell lines derived from cancers of the blood, kidney, nervous system, and skin were found to be significantly more sensitive to bortezomib than other organ systems. The in vitro studies confirmed that although bortezomib effectively inhibited the ß5 catalytic site in all four cell lines, cell cycle arrest was only induced in G2/M phase and apoptosis in A-375 and A-172 after 24h. The genomic and transcriptomic analyses identified 33 genes (e.g. ALDH18A1, ATAD2) associated with bortezomib resistance. Taken together, we identified biomarkers predictive of bortezomib sensitivity and cancer types that might benefit from treatment with bortezomib.

The SAR analysis of dietary polyphenols and their antagonistic effects on bortezomib at physiological concentrations.

Background: Bortezomib (BTZ), a primary treatment for MM, but its effectiveness can be reduced by interactions with vicinal diol moieties (VDMs) in polyphenols. Despite this, it's debated whether BTZ therapy necessitates avoiding polyphenol-rich products, given the low bioavailability of polyphenols. Additionally, it remains unclear whether the structure of polyphenols contributes to their BTZ antagonism. Therefore, our study aims to unravel the structure-activity relationship of dietary polyphenols and their BTZ antagonism at daily diet-achievable physiological concentrations. Methods: We assessed the antagonistic effects of 25 polyphenols against BTZ using cell viability assays in RPMI 8226 cells. ChemGPS-NP helped analyze the structural similarity. Additionally, long-term cytotoxicity assays evaluated these effects at physiologically relevant concentrations. Results: By cell viability assays, we found a positive correlation between the number of VDMs in gallotannins and their BTZ antagonism. Moreover, the origin and configuration of VDMs, rather than the total VDM concentration, play a pivotal role in the combined antagonistic effects against BTZ in gallotannins. Additionally, ChemGPS-NP analysis indicated that the aromaticity and C-3 hydroxyl group in flavonoids' C-rings enhance their BTZ antagonism. Finally, long-term cytotoxicity assays reveal that gallic acid (GA), epigallocatechin (EGC), and epigallocatechin gallate (EGCG), at their physiological concentrations-attainable through tea consumption-significantly and synergistically antagonize BTZ. Conclusion: Due to the potential for these polyphenols to reduce the effectiveness of BTZ, it is advisable for MM patients undergoing BTZ treatment to reduce their consumption of foods high in VDM-containing polyphenols.

Safety of proteasome inhibitor drugs for the treatment of multiple myeloma post-marketing: a pharmacovigilance investigation based on the FDA adverse event reporting system.

BACKGROUND: The use of proteasome inhibitors (PIs), namely Bortezomib and Carfilzomib, revolutionized multiple myeloma (MM) treatment. Understanding their distinct adverse event (AE) profiles aids in tailored treatment plans. RESEARCH DESIGN AND METHODS: We analyzed FDA Adverse Event Reporting System (FAERS) data (Q1 2012-Q4 2023) for Bortezomib and Carfilzomib, utilizing reporting odds ratio (ROR), proportional reporting ratio (PRR), and Bayesian confidence propagation neural network (BCPNN). RESULTS: FAERS yielded 19,720 Bortezomib and 12,252 Carfilzomib AE reports. Males aged 45-65 exhibited higher AE susceptibility. Common AE systems included Infections, Nervous System Disorders, Blood Disorders, General Disorders, Cardiac Disorders, and Renal Disorders. New Bortezomib signals were sepsis and colitis. Carfilzomib exhibited elevated cardiac and renal toxicity but reduced peripheral neuropathy and thrombocytopenia. CONCLUSIONS: FAERS analysis revealed new AE signals (sepsis, colitis) for Bortezomib and highlighted Carfilzomib's heightened cardiac and renal risks compared to Bortezomib. Balancing PIs' benefits and risks is crucial for clinical decision-making.

Corrigendum: Proteasome inhibitor YSY01A abrogates constitutive STAT3 signaling via down-regulation of Gp130 and JAK2 in human A549 lung cancer cells.

[This corrects the article DOI: 10.3389/fphar.2017.00476.].

Carfilzomib shows therapeutic potential for reduction of liver fibrosis by targeting hepatic stellate cell activation.

Because hepatic stellate cells (HSCs) play a major role in fibrosis, we focused on HSCs as a potential target for the treatment of liver fibrosis. In this study, we attempted to identify drug candidates to inactivate HSCs and found that several proteasome inhibitors (PIs) reduced HSC viability. Our data showed that a second-generation PI, carfilzomib (CZM), suppressed the expression of fibrotic markers in primary murine HSCs at low concentrations of 5 or 10 nM. Since CZM was not toxic to HSCs up to a concentration of 12.5 nM, we examined its antifibrotic effects further. CZM achieved a clear reduction in liver fibrosis in the carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis without worsening of liver injury. Mechanistically, RNA sequence analysis of primary HSCs revealed that CZM inhibits mitosis in HSCs. In the CCl4-injured liver, amphiregulin, which is known to activate mitogenic signaling pathways and fibrogenic activity and is upregulated in murine and human metabolic dysfunction-associated steatohepatitis (MASH), was downregulated by CZM administration, leading to inhibition of mitosis in HSCs. Thus, CZM and next-generation PIs in development could be potential therapeutic agents for the treatment of liver fibrosis via inactivation of HSCs without liver injury.

Anti-tumor effect of proteasome inhibitor on canine urothelial carcinoma.

Canine urothelial carcinoma (cUC) is one of the most malignant tumors affecting dogs; however, its proliferative mechanism is yet to be fully elucidated. The ubiquitin-proteasome system (UPS) is an important metabolic pathway regulating protein degradation, and its dysfunction leads to apoptosis. We investigated the antitumor effect of the proteasome inhibitor bortezomib, which blocks the UPS. Bortezomib inhibited cell growth in cUC cell lines by inducing apoptosis in vitro. These findings suggest the potential of bortezomib as a novel therapeutic drug for dogs with cUC.

Ixazomib in POEMS syndrome: 'Ixa'ctly what we need?

The role of the proteasome inhibitor ixazomib in the treatment of POEMS syndrome continues to evolve. He and colleagues present the results of a study investigating ixazomib in combination with cyclophosphamide and dexamethasone in newly diagnosed POEMS patients. The triplet showed excellent efficacy and tolerability, and constitutes an effective treatment option for patients with POEMS. Commentary on: He et al. An open-label, prospective trial to evaluate the efficacy and safety of ixazomib in combination with cyclophosphamide and dexamethasone in patients with newly-diagnosed POEMS syndrome. Br J Haematol 2024;205:478-482.

Establishment of a histiocytic sarcoma cell line and anti-tumor effect of bortezomib in the African pygmy hedgehog (Atelerix albiventris).

The African pygmy hedgehog (Atelerix albiventris) is known to have a high incidence of tumor. However, investigating the tumors of this species has been constrained by the limited availability of research materials such as cell lines and genome information. In this study, we successfully established a novel cell line from a histiocytic sarcoma (HS) of an African pygmy hedgehog, allowing us to conduct a drug screening. We investigated using FDA-approved drug library screening to determine which anticancer drug this tumor cell line is sensitive to, and as a result of apoptosis experiments, bortezomib among the three proteasome inhibitors was found to induce cell death of cancer cells by significantly increasing caspase-3 cleavage (P<0.01). Thus, we elucidated that the proteasome inhibitors, particularly bortezomib, exhibit anti-tumor effects on a cell line derived from an HS in an African pygmy hedgehog through a mechanism comparable to that described in human tumors. This study reports the first characterized cell line from the African pygmy hedgehog and also highlights the potential utility of bortezomib as an anti-tumor treatment for HS in this species.

Multiple Endocrine Neoplasia Type 1 Regulates TGFß-Mediated Suppression of Tumor Formation and Metastasis in Melanoma.

Over the past few decades, the worldwide incidence of cutaneous melanoma, a malignant neoplasm arising from melanocytes, has been increasing markedly, leading to the highest rate of skin cancer-related deaths. While localized tumors are easily removed by excision surgery, late-stage metastatic melanomas are refractory to treatment and exhibit a poor prognosis. Consequently, unraveling the molecular mechanisms underlying melanoma tumorigenesis and metastasis is crucial for developing novel targeted therapies. We found that the multiple endocrine neoplasia type 1 (MEN1) gene product Menin is required for the transforming growth factor beta (TGFß) signaling pathway to induce cell growth arrest and apoptosis in vitro and prevent tumorigenesis in vivo in preclinical xenograft models of melanoma. We further identified point mutations in two MEN1 family members affected by melanoma that led to proteasomal degradation of the MEN1 gene product and to a loss of TGFß signaling. Interestingly, blocking the proteasome degradation pathway using an FDA-approved drug and RNAi targeting could efficiently restore MEN1 expression and TGFß transcriptional responses. Together, these results provide new potential therapeutic strategies and patient stratification for the treatment of cutaneous melanoma.

Targeting the ubiquitin pathway in lymphoid malignancies.

Ubiquitination and related cellular processes control a variety of aspects in human cell biology, and defects in these processes contribute to multiple illnesses. In recent decades, our knowledge about the pathological role of ubiquitination in lymphoid cancers and therapeutic strategies to target the modified ubiquitination system has evolved tremendously. Here we review the altered signalling mechanisms mediated by the aberrant expression of cancer-associated E2s/E3s and deubiquitinating enzymes (DUBs), which result in the hyperactivation of oncoproteins or the frequently allied downregulation of tumour suppressors. We discuss recent highlights pertaining to the several different therapeutic interventions which are currently being evaluated to effectively block abnormal ubiquitin-proteasome pathway and the use of heterobifunctional molecules which recruit the ubiquitination system to degrade or stabilize non-cognate substrates. This review aids in comprehension of ubiquitination aberrance in lymphoid cancers and current targeting strategies and elicits further investigations to deeply understand the link between cellular ubiquitination and lymphoid pathogenesis as well as to ameliorate corresponding treatment interventions.

The Synergistic Effect of Reduced Graphene Oxide and Proteasome Inhibitor in the Induction of Apoptosis through Oxidative Stress in Breast Cancer Cell Lines.

Reduced graphene oxide (rGO) and a proteasome inhibitor (MG-132) are some of the most commonly used compounds in various biomedical applications. However, the mechanisms of rGO- and MG-132-induced cytotoxicity remain unclear. The aim of this study was to investigate the anticancer effect of rGO and MG-132 against ZR-75-1 and MDA-MB-231 breast cancer cell lines. The results demonstrated that rGO, MG-132 or a mix (rGO + MG-132) induced time- and dose-dependent cytotoxicity in ZR-75-1 and MDA-MB-231 cells. Apart from that, we found that treatment with rGO and MG-132 or the mix increased apoptosis, necrosis and induction of caspase-8 and caspase-9 activity in both breast cancer cell lines. Apoptosis and caspase activation were accompanied by changes in the ultrastructure of mitochondria in ZR-75-1 and MDA-MB-231 cells incubated with rGO. Additionally, in the analyzed cells, we observed the induction of oxidative stress, accompanied by increased apoptosis and cell necrosis. In conclusion, oxidative stress induces apoptosis in the tested cells. At the same time, both mitochondrial and receptor apoptosis pathways are activated. These studies provided new information on the molecular mechanisms of apoptosis in the ZR-75-1 and MDA-MB-231 breast cancer cell lines.

Impact of prior treatment on selinexor, bortezomib, dexamethasone outcomes in patients with relapsed/refractory multiple myeloma: Extended follow-up subgroup analysis of the BOSTON trial.

OBJECTIVES: To analyze the impact of prior therapies on outcomes with selinexor, bortezomib, and dexamethasone (SVd) versus bortezomib and dexamethasone (Vd) in 402 patients with relapsed/refractory multiple myeloma (RRMM) in the phase 3 BOSTON trial. METHODS: Post hoc analysis of progression-free survival (PFS), overall survival (OS), and safety for lenalidomide-refractory, proteasome inhibitor (PI)-naïve, bortezomib-naïve, and one prior line of therapy (1LOT) patient subgroups. RESULTS: At a median follow-up of over 28 months, clinically meaningful improvements in PFS were noted across all groups with SVd. The median SVd PFS was longer in all subgroups (lenalidomide-refractory: 10.2 vs. 7.1 months, PI-naïve: 29.5 vs. 9.7; bortezomib-naïve: 29.5 vs. 9.7; 1LOT: 21.0 vs. 10.7; p < .05). The lenalidomide-refractory subgroup had longer OS with SVd (26.7 vs. 18.6 months; HR 0.53; p = .015). In all subgroups, overall response and ≥very good partial response rates were higher with SVd. The manageable safety profile of SVd was similar to the overall patient population. CONCLUSIONS: With over 2 years of follow-up, these clinically meaningful outcomes further support the use of SVd in patients who are lenalidomide-refractory, PI-naïve, bortezomib-naïve, or who received 1LOT (including a monoclonal antibody) and underscore the observed synergy between selinexor and bortezomib.

Proteasome inhibition paradoxically degrades gain-of-function mutant p53 R273H in NSCLC and could have therapeutic implications.

Lung cancer is the leading cause of cancer mortality. Despite therapeutic advances in recent years, new treatment strategies are needed to improve outcomes of lung cancer patients. Mutant p53 is prevalent in lung cancers and drives several hallmarks of cancer through a gain-of-function oncogenic program, and often predicts a poorer prognosis. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. In this study, we report that proteasome inhibition results in degradation of mutant p53 in non-small cell lung cancer (NSCLC) cell lines bearing the R273H mutant protein and show evidence that this was mediated by hsp70. NSCLC cell lines with the mutant R273H allele demonstrated increased susceptibility and apoptosis to proteasome inhibitors. These data suggest that proteasome inhibitors could have therapeutic implications in some subsets of TP53 mutated NSCLC.

Assessment of prolonged proteasome inhibition through ixazomib-based oral regimen on newly diagnosed and first-relapsed multiple myeloma: A real-world Chinese cohort study.

OBJECTIVE: To evaluate the effectiveness, safety, and convenience of in-class transition (iCT) from intravenous bortezomib-based induction to ixazomib-based oral regimens. METHODS: This retrospective real-world study was conducted in 16 Chinese hospitals between October 2017 and April 2023 and analyzed newly diagnosed (NDMM) and first-line relapsed multiple myeloma (FRMM) patients who attained at least a partial response from bortezomib-based induction therapy, followed by an ixazomib-based oral regimen for 2 year or until disease progression or intolerable toxicity. RESULTS: The study enrolled 199 patients, median age: 63 years old, male 55.4%, 53% as high risk (HR), and 47% as standard risk. Cytogenetic risk stratification by metaphase fluorescence in situ hybridization (M-FISH), based on the Mayo Clinic risk stratification system. The median duration of total PI therapy was 11 months, with ixazomib-based treatment spanning 6 months. At the 20-month median follow-up, 53% of patients remained on therapy. The 24-month PFS rate was 84.3% from the initiation of bortezomib-based induction and 83.4% from the start of ixazomib-based treatment. Overall response rate (ORR) was 100% post-bortezomib induction and 90% following 6 cycles of the ixazomib-based regimen. Based on the Sankey diagrams, 89.51% of patients maintained or improved their disease response after 2 cycles of iCT, 6 cycles (90.14%), and 12 cycles (80%). The HR level of Mayo was found to be a significant independent factor in a worse remission (hazard ratio (HR) 2.55; p = 0.033). Ixazomib's safety profile aligned with previous clinical trial data, with 49% of patients experiencing at least one AE of any grade. The most common AEs included peripheral neuropathy, nausea and vomiting, diarrhea, thrombocytopenia, and granulocytopenia. CONCLUSION: In the real-world Chinese MM population, NDMM and FRMM patients responded favorably to PI-based continuous therapy, demonstrating substantial response rates. The ixazomib-based iCT allows for sustained PI-based treatment, offering promising efficacy and tolerable AEs.

Carfilzomib activates ER stress and JNK/p38 MAPK signaling to promote apoptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers in the world, which is frequently diagnosed at a late stage. HCC patients have a poor prognosis due to the lack of an efficacious therapeutic strategy. Approved drug repurposing is a way for accelerating drug discovery and can significantly reduce the cost of drug development. Carfilzomib (CFZ) is a second-generation proteasome inhibitor, which is highly efficacious against multiple myeloma and has been reported to possess potential antitumor activities against multiple cancers. However, the underlying mechanism of CFZ on HCC is still unclear. Here, we show that CFZ inhibits the proliferation of HCC cells through cell cycle arrest at the G2/M phase and suppresses the migration and invasion of HCC cells by inhibiting epithelial-mesenchymal transition. We also find that CFZ promotes reactive oxygen species production to induce endoplasmic reticulum (ER) stress and activate JNK/p38 MAPK signaling in HCC cells, thus inducing cell death in HCC cells. Moreover, CFZ significantly inhibits HCC cell growth in a xenograft mouse model. Collectively, our study elucidates that CFZ impairs mitochondrial function and activates ER stress and JNK/p38 MAPK signaling, thus inhibiting HCC cell and tumor growth. This indicates that CFZ has the potential as a therapeutic drug for HCC.

Impact of prior lenalidomide or proteasome inhibitor exposure on the effectiveness of ixazomib-lenalidomide-dexamethasone for relapsed/refractory multiple myeloma: A pooled analysis from the INSURE study.

OBJECTIVES: To characterize the impact of prior exposure and refractoriness to lenalidomide or proteasome inhibitors (PIs) on the effectiveness and safety of ixazomib-lenalidomide-dexamethasone (IRd) in relapsed/refractory multiple myeloma (RRMM). METHODS: INSURE is a pooled analysis of adult RRMM patients who had received IRd in ≥2 line of therapy from three studies: INSIGHT MM, UVEA-IXA, and REMIX. RESULTS: Overall, 391/100/68 were lenalidomide-naïve/-exposed/-refractory and 37/411/110 were PI-naïve/-exposed/-refractory. Median duration of therapy (DOT) was 15.3/15.6/4.7 months and median progression-free survival (PFS) was 21.6/25.8/5.6 months in lenalidomide-naïve/exposed/refractory patients. Median DOT and PFS in PI-naïve/exposed/refractory patients were 20.4/15.2/6.9 months and not reached/19.8/11.4 months, respectively. The proportion of lenalidomide-naïve/exposed/refractory patients in INSIGHT and UVEA-IXA who discontinued a study drug due to adverse events (AEs) was ixazomib, 31.6/28.2/28.0% and 18.6/6.7/10.5%; lenalidomide, 21.9/28.2/16.0% and 16.1/6.7/10.5%; dexamethasone, 18.4/20.5/16.0% and 10.6/0/10.5%, respectively. The proportion of PI-naïve/exposed/refractory patients in INSIGHT and UVEA-IXA who discontinued a study drug due to AEs was: ixazomib, 44.4/28.8/27.8% and 22.2/16.7/15.7%; lenalidomide, 33.3/22.0/19.4% and 16.7/15.9/11.8%; dexamethasone, 33.3/17.4/16.7% and 16.7/9.5/7.8%, respectively. REMIX AE discontinuation rates were unavailable. CONCLUSION: IRd appeared to be effective in RRMM patients in routine clinical practice regardless of prior lenalidomide or PI exposure, with better outcomes seen in lenalidomide- and/or PI-nonrefractory versus refractory patients.

Proteasome inhibitor-associated histiocytoid Sweet's syndrome: Clinical and histological similarities to Nakajo-Nishimura syndrome suggest a potential mechanism.

Histiocytoid Sweet's syndrome (HSS) is a variant of Sweet's syndrome (SS) that clinically resembles SS but differs histologically by infiltrates, predominantly composed of immature cells of the myeloid lineage. Medications such as proteasome inhibitors have been reported to cause HSS but there has been little discussion on the underlying mechanism. Here we report two cases of HSS associated with a proteasome inhibitor. Both patients were on ixazomib for the treatment of multiple myeloma and presented with acute erythematous plaques on the upper half of the body. Pathological findings were consistent with HSS. Similarities between proteasome inhibitor-induced HSS and Nakajo-Nishimura syndrome, an inherited inflammatory disease, can be identified both clinically and histologically, suggesting a potential explanation of the mechanism behind proteasome inhibitor-associated HSS.

The role of proteasomes in tumorigenesis.

Protein homeostasis is the basis of normal life activities, and the proteasome family plays an extremely important function in this process. The proteasome 20S is a concentric circle structure with two α rings and two ß rings overlapped. The proteasome 20S can perform both ATP-dependent and non-ATP-dependent ubiquitination proteasome degradation by binding to various subunits (such as 19S, 11S, and 200 PA), which is performed by its active subunit ß1, ß2, and ß5. The proteasome can degrade misfolded, excess proteins to maintain homeostasis. At the same time, it can be utilized by tumors to degrade over-proliferate and unwanted proteins to support their growth. Proteasomes can affect the development of tumors from several aspects including tumor signaling pathways such as NF-κB and p53, cell cycle, immune regulation, and drug resistance. Proteasome-encoding genes have been found to be overexpressed in a variety of tumors, providing a potential novel target for cancer therapy. In addition, proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib have been put into clinical application as the first-line treatment of multiple myeloma. More and more studies have shown that it also has different therapeutic effects in other tumors such as hepatocellular carcinoma, non-small cell lung cancer, glioblastoma, and neuroblastoma. However, proteasome inhibitors are not much effective due to their tolerance and singleness in other tumors. Therefore, further studies on their mechanisms of action and drug interactions are needed to investigate their therapeutic potential.