Novel Inhibitors of androgen receptor's DNA binding domain identified using an ultra-large virtual screening.

Androgen receptor (AR) inhibition remains the primary strategy to combat the progression of prostate cancer (PC). However, all clinically used AR inhibitors target the ligand-binding domain (LBD), which is highly susceptible to truncations through splicing or mutations that confer drug resistance. Thus, there exists an urgent need for AR inhibitors with novel modes of action. We thus launched a virtual screening of an ultra-large chemical library to find novel inhibitors of the AR DNA-binding domain (DBD) at two sites: protein-DNA interface (P-box) and dimerization site (D-box). The compounds selected through vigorous computational filtering were then experimentally validated. We identified several novel chemotypes that effectively suppress transcriptional activity of AR and its splice variant V7. The identified compounds represent previously unexplored chemical scaffolds with a mechanism of action that evades the conventional drug resistance manifested through LBD mutations. Additionally, we describe the binding features required to inhibit AR DBD at both P-box and D-box target sites.

Dynamic phase separation of the androgen receptor and its coactivators key to regulate gene expression.

Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.

Structure-Based Study to Overcome Cross-Reactivity of Novel Androgen Receptor Inhibitors.

The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.

Development of VPC-70619, a Small-Molecule N-Myc Inhibitor as a Potential Therapy for Neuroendocrine Prostate Cancer.

The Myc family of transcription factors are involved in the development and progression of numerous cancers, including prostate cancer (PCa). Under the pressure of androgen receptor (AR)-directed therapies resistance can occur, leading to the lethal form of PCa known as neuroendocrine prostate cancer (NEPC), characterized among other features by N-Myc overexpression. There are no clinically approved treatments for NEPC, translating into poor patient prognosis and survival. Therefore, there is a pressing need to develop novel therapeutic avenues to treat NEPC patients. In this study, we investigate the N-Myc-Max DNA binding domain (DBD) as a potential target for small molecule inhibitors and utilize computer-aided drug design (CADD) approaches to discover prospective hits. Through further exploration and optimization, a compound, VPC-70619, was identified with notable anti-N-Myc potency and strong antiproliferative activity against numerous N-Myc expressing cell lines, including those representing NEPC.

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

Evaluation of Darolutamide (ODM201) Efficiency on Androgen Receptor Mutants Reported to Date in Prostate Cancer Patients.

Resistance to drug treatments is common in prostate cancer (PCa), and the gain-of-function mutations in human androgen receptor (AR) represent one of the most dominant drivers of progression to resistance to AR pathway inhibitors (ARPI). Previously, we evaluated the in vitro response of 24 AR mutations, identified in men with castration-resistant PCa, to five AR antagonists. In the current work, we evaluated 44 additional PCa-associated AR mutants, reported in the literature, and thus expanded the study of the effect of darolutamide to a total of 68 AR mutants. Unlike other AR antagonists, we demonstrate that darolutamide exhibits consistent efficiency against all characterized gain-of-function mutations in a full-length AR. Additionally, the response of the AR mutants to clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesterone and hydrocortisone), was also investigated. As genomic profiling of PCa patients becomes increasingly feasible, the developed "AR functional encyclopedia" could provide decision-makers with a tool to guide the treatment choice for PCa patients based on their AR mutation status.

Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer.

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

Evaluation of the impact of HMS Plus on postoperative blood loss compared with ACT Plus in cardiac surgery.

BACKGROUND: The standardized management of anticoagulation during the cardiopulmonary bypass seems inaccurate because of patients and surgeries variability. This study evaluates if an individualized management of heparin and protamine guided by the HMS Plus system during cardiopulmonary bypass could reduce postoperative blood loss. METHODS: We conducted a prospective, controlled, unblinded, single-center study. One-hundred and eighthy-eight patients operated for cardiac surgery were included. Patients were divided in ACT Plus group (standardized approach) and HMS Plus group (individualized approach). The primary outcome was blood-loss volume during the first 24 postoperative hours. The main secondary outcomes were the need for allogeneic blood transfusions and the final protamine/heparin ratio. RESULTS: There was no difference between the two groups for baseline characteristics. Medium blood-loss volume in the ACT Plus group was 522±260 mL vs. 527±255 mL in the HMS Plus group (P=0.58). The final protamine/heparin ratio in the ACT Plus group was 0.94±0.1 vs. 0.58±0.1 in the HMS Plus group (P<0.0001). The transfusion rate during surgery in the ACT Plus group was 25% vs. 14% in the HMS Plus group (P=0.09). CONCLUSIONS: HMS Plus did not reduce the mean blood-loss volume during the first 24 postoperative hours compared with ACT Plus. Its utility for potential transfusion rate reduction remains to be proven.

Carotid versus femoral access for transcatheter aortic valve replacement: comparable results in the current era.

OBJECTIVES: The carotid approach for transcatheter aortic valve replacement (TAVR) has been shown to be feasible and safe. The goal of this study was to compare the 30-day outcomes of trans-carotid (TC) and transfemoral (TF) TAVR. METHODS: This retrospective study enrolled 500 consecutive patients treated by TC-TAVR (n = 100) or TF-TAVR (n = 400) with percutaneous closure between January 2018 and January 2020 at the Nantes University Hospital. The primary end-point was the occurrence of cardiovascular death and cerebrovascular events at 30 days. RESULTS: The mean age was 79.9 ± 8.1 in the TC group and 81.3 ± 6.9 (P = 0.069) in the TF group. The TC group had more men (69% vs 50.5%; P = 0.001) and more patients with peripheral vascular disease (86% vs 14.8%; P < 0.0001). Cardiac characteristics were similar between the groups, and the EuroSCORE II was 3.8 ± 2.6% vs 4.6 ± 6.0%, respectively (P = 0.443). The 30-day mortality was 2% in the TC group versus 1% in the TF group (P = 0.345). TC-TAVR was not associated with an increased risk of stroke (2% vs 2.5%; P = 0.999) or major vascular complications (2% vs 4%; P = 0.548). More permanent pacemakers were implanted in the TF group (14.9% vs 5.6%; P = 0.015), and no moderate or severe aortic regurgitation was observed in the TC group (0 vs 3.3%; P = 0.08). TC-TAVR was not associated with an increased risk of mortality or stroke at 30 days (odds ratio 1.32; 95% confidence interval 0.42-4.21; P = 0.63) in the multivariable analysis. CONCLUSIONS: No statistically significant differences between TC-TAVR and TF-TAVR were observed; therefore, TC-TAVR should be the first alternative in patients with anatomical contraindications to the femoral route.

Dual-Inhibitors of N-Myc and AURKA as Potential Therapy for Neuroendocrine Prostate Cancer.

Resistance to androgen-receptor (AR) directed therapies is, among other factors, associated with Myc transcription factors that are involved in development and progression of many cancers. Overexpression of N-Myc protein in prostate cancer (PCa) leads to its transformation to advanced neuroendocrine prostate cancer (NEPC) that currently has no approved treatments. N-Myc has a short half-life but acts as an NEPC stimulator when it is stabilized by forming a protective complex with Aurora A kinase (AURKA). Therefore, dual-inhibition of N-Myc and AURKA would be an attractive therapeutic avenue for NEPC. Following our computer-aided drug discovery approach, compounds exhibiting potent N-Myc specific inhibition and strong anti-proliferative activity against several N-Myc driven cell lines, were identified. Thereafter, we have developed dual inhibitors of N-Myc and AURKA through structure-based drug design approach by merging our novel N-Myc specific chemical scaffolds with fragments of known AURKA inhibitors. Favorable binding modes of the designed compounds to both N-Myc and AURKA target sites have been predicted by docking. A promising lead compound, 70812, demonstrated low-micromolar potency against both N-Myc and AURKA in vitro assays and effectively suppressed NEPC cell growth.

Association between lipodystrophy and length of exposure to ARTs in adult HIV-1 infected patients in Montreal.

BACKGROUND: The aim of this study was to establish the prevalence of lipodystrophy and its association to cumulative exposure to antiretroviral drugs. METHOD: We conducted a cross sectional study in all HIV- infected patients attending the HIV clinic in the Centre hospitalier universitaire de Montréal (CHUM) with DEXA scan. Lipodystrophy was defined as a trunk/limb fat ratio ≥ 1.5. Association between cumulative exposure to antiretroviral (measured in years of use) with trunk/limb fat ratio (coded as a continuous variable) was assessed using univariate and multivariate linear regression for each antiretroviral drug with at least 40 exposed patients. RESULTS: One hundred sixty-six patients were included. Seventy-five percent were male, median age was 56 years, 67% were Caucasian. Overall, prevalence of lipodystrophy was 47%, with a mean trunk/limb fat ratio of 1.87, SD = 1.03, min = 0.6 and max = 5.87. Each 10-year increase in age and HIV infection duration was associated with an average increase of 0.24 and 0.34 for the trunk/limb fat ratio respectively. (p = 0.003, p = 0.002, respectively) Patients classified as lipodystrophic were more likely to be diabetic (50 vs. 28%, p = 0.07) and to have dyslipidemia (47 vs. 19%, p = 0.01). According to viral load at DEXA test, each one log increase was associated with less probability (0.7) of lipodystrophy. (p = 0.01) Among ARV drugs tested, there was an association between years of use of d4T, ritonavir and raltegravir and higher trunk/limb fat ratio (indicating more lipodystrophy) (p < 0.05). CONCLUSION: Lipodystrophy is very common in HIV infected patients and is correlated with duration of some new antiretroviral drugs.

Computer-Aided Discovery of Small Molecules Targeting the RNA Splicing Activity of hnRNP A1 in Castration-Resistant Prostate Cancer.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a versatile RNA-binding protein playing a critical role in alternative pre-mRNA splicing regulation in cancer. Emerging data have implicated hnRNP A1 as a central player in a splicing regulatory circuit involving its direct transcriptional control by c-Myc oncoprotein and the production of the constitutively active ligand-independent alternative splice variant of androgen receptor, AR-V7, which promotes castration-resistant prostate cancer (CRPC). As there is an urgent need for effective CRPC drugs, targeting hnRNP A1 could, therefore, serve a dual purpose of preventing AR-V7 generation as well as reducing c-Myc transcriptional output. Herein, we report compound VPC-80051 as the first small molecule inhibitor of hnRNP A1 splicing activity discovered to date by using a computer-aided drug discovery approach. The inhibitor was developed to target the RNA-binding domain (RBD) of hnRNP A1. Further experimental evaluation demonstrated that VPC-80051 interacts directly with hnRNP A1 RBD and reduces AR-V7 messenger levels in 22Rv1 CRPC cell line. This study lays the groundwork for future structure-based development of more potent and selective small molecule inhibitors of hnRNP A1⁻RNA interactions aimed at altering the production of cancer-specific alternative splice isoforms.

Computer-aided drug discovery of Myc-Max inhibitors as potential therapeutics for prostate cancer.

While Myc is an essential regulator of growth in normal cells, it is also frequently associated with cancer progression, therapy-resistance and lethal outcomes in most human cancers. In prostate cancer (PCa), Myc transcription factors are implicated in the pathogenesis and progression of the full spectrum of PCa, from adenocarcinoma to advanced castration-resistant and neuroendocrine phenotypes. Though a high-value therapeutic target, clinically approved anti-Myc drugs have yet to be discovered. To elicit its oncogenic effects, Myc must form a heterodimer with its partner Max, which together bind DNA and activate transcription of a spectrum of target genes that promote cell growth, proliferation, metabolism, and apoptosis while blocking differentiation. In this study, we identified a binding site on the DNA-binding domain of the structurally ordered Myc-Max complex and employed a computer-aided rational drug discovery approach to identify small molecules that effectively inhibit Myc-Max functionality. A large-scale virtual screening protocol implementing structure-based methodologies was utilized to select a set of top-ranked compounds that were subsequently evaluated experimentally and characterized mechanistically for their ability to inhibit Myc-Max transcriptional activity and subsequent downstream functions, to reduce viability in PCa cell lines, disrupt protein-DNA interactions and to induce apoptosis as their mechanism of action. Among compounds identified that effectively inhibit Myc-Max activity with low to mid-micromolar range potency and no or minimal generic cytotoxicity, VPC-70067, a close analog of the previously identified Myc inhibitor 10058-F4, served as proof-of-concept that our in silico drug discovery strategy performed as expected. Compound VPC-70063, of a chemically different scaffold, was the best performer in a panel of in vitro assays, and the forerunner for future hit-to-lead optimization efforts. These findings lay a foundation for developing more potent, specific and clinically optimized Myc-Max inhibitors that may serve as promising therapeutics, alone or in combination with current anti-cancer treatments, for treatment of specific phenotypes or heterogeneous tumors.

Small molecule-induced degradation of the full length and V7 truncated variant forms of human androgen receptor.

The androgen receptor (AR) is a hormone-activated transcription factor that regulates the development and progression of prostate cancer (PCa) and represents one of the most well-established drug targets. Currently clinically approved small molecule inhibitors of AR, such as enzalutamide, are built upon a common chemical scaffold that interacts with the AR by the same mechanism of action. These inhibitors eventually fail due to the emergence of drug-resistance in the form of AR mutations and expression of truncated AR splice variants (e.g. AR-V7) that are constitutively active, signalling the progression of the castration-resistant state of the disease. The urgent need therefore continues for novel classes of AR inhibitors that can overcome drug resistance, especially since AR signalling remains important even in late-stage advanced PCa. Previously, we identified a collection of 10-benzylidene-10H-anthracen-9-ones that effectively inhibit AR transcriptional activity, induce AR degradation and display some ability to block recruitment of hormones to the receptor. In the current work, we extended the analysis of the lead compounds, and used methods of both ligand- and structure-based drug design to develop a panel of novel 10-benzylidene-10H-anthracen-9-one derivatives capable of suppressing transcriptional activity and protein expression levels of both full length- and AR-V7 truncated forms of human androgen receptor. Importantly, the developed compounds efficiently inhibited the growth of AR-V7 dependent prostate cancer cell-lines which are completely resistant to all current anti-androgens.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.

Cheminformatics Driven Development of Novel Therapies for Drug Resistant Prostate Cancer.

Androgen receptor (AR) is a master regulator of prostate cancer (PCa), and therefore is a pivotal drug target for the treatment of PCa including its castration-resistance form (CRPC). The development of acquired resistance is a major challenge in the use of the current antiandrogens. The recent advancements in inhibiting AR activity with small molecules specifically designed to target areas distinct from the receptor's androgen binding site are carefully discussed. Our new classes of AR inhibitors of AF2 and BF3 functional sites and DBD domains designed using cheminformatics techniques are promising to circumvent various AR-dependent resistance mechanisms.

Role of IL-12 in overcoming the low responsiveness of NK cells to missing self after traumatic brain injury.

Blood samples from 32 patients with severe Traumatic brain injury (TBI) were studied and compared with 11 cardiac surgery patients, and 29 healthy controls. A dramatic decreased expression of HLA class I molecules on monocytes was associated with increased KIR+ NK cell frequency in TBI patients. Overall, the phenotype of TBI NK cells marked by KIR and CD57 expression and lower level of NKp46 and DNAM-1 reflected a differentiated state. The NK-cell response to missing self was marked by lower degranulation and lower IFN-γ production after stimulation with HLA class I deficient cell line. In contrast, the NK-cell ADCC was not altered. IL-12 was able to restore both IFN-γ production and the cytotoxicity capacities of NK cells. This study provides the first extensive description of the phenotype and functions of NK cells in TBI patients. Further evaluation of IL-12 treatment to overcome immunosuppression-induced nosocomial infections is warranted.

Targeting Binding Function-3 of the Androgen Receptor Blocks Its Co-Chaperone Interactions, Nuclear Translocation, and Activation.

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3-directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936-45. ©2016 AACR.

Drug-Discovery Pipeline for Novel Inhibitors of the Androgen Receptor.

The androgen receptor (AR) is an important regulator of genes responsible for the development and recurrence of prostate cancer. Current therapies for this disease rely on small-molecule inhibitors that block the transcriptional activity of the AR. Recently, major advances in the development of novel AR inhibitors resulted from X-ray crystallographic information on the receptor and utilization of in silico drug design synergized with rigorous experimental testing.Herein, we describe a drug-discovery pipeline for in silico screening for small molecules that target an allosteric region on the AR termed the binding-function 3 (BF3) site. Following the identification of potential candidates, the compounds are tested in cell culture and biochemical assays for their ability to interact with and inhibit the AR. The described pipeline is readily accessible and could be applied in drug design efforts toward any surface-exposed region on the AR or other related steroid nuclear receptor.

Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells.

The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.