Cláudia Bessa

New insights into cancer‐related proteins provided by the yeast model

Cancer is a devastating disease with a profound impact on society. In recent years, yeast has provided a valuable contribution with respect to uncovering the molecular mechanisms underlying this disease, allowing the identification of new targets and novel therapeutic opportunities. Indeed, several attributes make yeast an ideal model system for the study of human diseases. It combines a high level of conservation between its cellular processes and those of mammalian cells, with advantages such as a short generation time, ease of genetic manipulation and a wealth of experimental tools for genome‐ and proteome‐wide analyses. Additionally, the heterologous expression of disease‐causing proteins in yeast has been successfully used to gain an understanding of the functions of these proteins and also to provide clues about the mechanisms of disease progression. Yeast research performed in recent years has demonstrated the tremendous potential of this model system, especially with the validation of findings obtained with yeast in more physiologically relevant models. The present review covers the major aspects of the most recent developments in the yeast research area with respect to cancer. It summarizes our current knowledge on yeast as a cellular model for investigating the molecular mechanisms of action of the major cancer‐related proteins that, even without yeast orthologues, still recapitulate in yeast some of the key aspects of this cellular pathology. Moreover, the most recent contributions of yeast genetics and high‐throughput screening technologies that aim to identify some of the potential causes underpinning this disorder, as well as discover new therapeutic agents, are discussed.

Contribution of Yeast Models to Neurodegeneration Research

As a model organism Saccharomyces cerevisiae has greatly contributed to our understanding of many fundamental aspects of cellular biology in higher eukaryotes. More recently, engineered yeast models developed to study endogenous or heterologous proteins that lay at the root of a given disease have become powerful tools for unraveling the molecular basis of complex human diseases like neurodegeneration. Additionally, with the possibility of performing target-directed large-scale screenings, yeast models have emerged as promising first-line approaches in the discovery process of novel therapeutic opportunities against these pathologies. In this paper, several yeast models that have contributed to the uncovering of the etiology and pathogenesis of several neurodegenerative diseases are described, including the most common forms of neurodegeneration worldwide, Alzheimers, Parkinsons, and Huntingtons diseases. Moreover, the potential input of these cell systems in the development of more effective therapies in neurodegeneration, through the identification of genetic and chemical suppressors, is also addressed.

Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction

One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53-MDM2 interaction inhibitors.

New Therapeutic Strategies for Cancer and Neurodegeneration Emerging from Yeast Cell-based Systems

Despite great advances in understanding the molecular etiology of cancer and neurodegeneration, therapeutic strategies against these diseases are still largely lacking. Hence, acceleration of the discovery of new therapeutic agents against these pathologies is of enormous interest. This review is focused on the role of multi-faceted and expanding yeast cell-based systems in the search for new drugs and therapeutic targets in cancer and neurodegeneration. Though the obvious limitations of using a microorganism to address human diseases, when used in the early phase and with complementary mammalian systems, it can have a tremendous impact in the discovery of new therapeutic opportunities. In this review, many evidence are provided demonstrating the valuable contribution of yeast in this area. Additionally, several yeast target-based drug screening approaches based on a readily screenable phenotype on genomic technologies increasingly oriented towards genetic and chemical high-throughput analysis are addressed. Altogether, with this review, we intend not only to recognize previous successes and ongoing work in this area, but also to point out new opportunities that may be of interest for yeast as a model organism and as a powerful system in the discovery of new lead compounds that have the potential to become novel drugs in cancer and neurodegeneration.

Novel simplified yeast‐based assays of regulators of p53–MDMX interaction and p53 transcriptional activity

Yeast has proven to be an efficient model system for functional and pharmacological studies of the p53 tumour suppressor protein. In this work, the human p53–MDMX regulatory pathway was reconstituted in yeast. Additionally, by using the known inhibitor of p53–MDMX interaction, SJ‐172550, the efficacy of a simplified yeast‐based screening assay to search for inhibitors of p53–MDMX interaction is demonstrated for the first time. Moreover, further insights on p53 transcriptional activity in yeast are provided. In particular, it is shown that the reported wild‐type (wt) p53‐induced yeast growth inhibition and cell cycle arrest is associated with actin depolarization and with an increase of actin mRNA and protein expression levels. The increase of actin protein levels was not observed with the p53 R273H mutant (a loss of function p53 mutation hotspot) and was further intensified with the toxic p53 V122A mutant (reported to exhibit higher transcriptional activity than wt p53 for selected p53 target sequences). Moreover, it is shown that the wt p53‐induced actin protein levels are modulated by natural (MDM2 and MDMX) and chemical (pifithrin‐α, nutlin‐3a and SJ‐172550) regulators of p53 activity. Furthermore, wt p53 could stimulate transcription from a minimal promoter containing a fragment of the ACT1 upstream sequence. Thus, ACT1 is proposed as a putative endogenous p53 target gene. This finding may open the way for the development of simpler yeast p53 transactivation assays, not based on artificial reporter constructs, for the analysis of the impact of mutants, cofactors and small molecules on p53 transcriptional activity.

Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1, a novel anticancer small-molecule

Restoration of the p53 pathway, namely by reactivation of mutant (mut) p53, represents a valuable anticancer strategy. Herein, we report the identification of the enantiopure tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a novel reactivator of wild-type (wt) and mut p53, using a yeast-based screening strategy. SLMP53-1 has a p53-dependent anti-proliferative activity in human wt and mut p53R280K-expressing tumor cells. Additionally, SLMP53-1 enhances p53 transcriptional activity and restores wt-like DNA binding ability to mut p53R280K. In wt/mut p53-expressing tumor cells, SLMP53-1 triggers p53 transcription-dependent and mitochondrial apoptotic pathways involving BAX, and wt/mut p53 mitochondrial translocation. SLMP53-1 inhibits the migration of wt/mut p53-expressing tumor cells, and it shows promising p53-dependent synergistic effects with conventional chemotherapeutics. In xenograft mice models, SLMP53-1 inhibits the growth of wt/mut p53-expressing tumors, but not of p53-null tumors, without apparent toxicity. Collectively, besides the potential use of SLMP53-1 as anticancer drug, the tryptophanol-derived oxazoloisoindolinone scaffold represents a promissing starting point for the development of effective p53-reactivating drugs.

Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions.

Endocytosis inhibition during H2O2-induced apoptosis in yeast

Yeast revealed to be a versatile organism for studying endocytosis. Here, inhibition of endocytosis by H(2)O(2) and its correlation with apoptotic cell death were ascertained in Saccharomyces cerevisiae. We found that H(2)O(2) causes alterations in vacuolar morphology and a concentration-dependent inhibition of endocytosis. We also found that H(2)O(2)-induced endocytosis inhibition is a reversible process that occurs in the early phase of the apoptotic cascade, preceding chromatin condensation and DNA fragmentation. Additionally, mutants affecting early steps of the endocytic pathway display sensitivity to H(2)O(2). As endocytosis inhibition was also observed with acetic acid, it may be a broader cellular dysfunction of oxidative stress-induced toxicity in yeast.

Chronological aging in conidia of pathogenic Aspergillus: Comparison between species.

Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger are common airborne fungi, and the most frequent causative agents of human fungal infections. However, the resistance and lifetime persistence of these fungi in the atmosphere, and the mechanism of aging of Aspergillus conidia are unknown.With this work, we intended to study the processes underlying conidial aging of these four relevant and pathogenic Aspergillus species. Chronological aging was therefore evaluated in A. fumigatus, A. flavus, A. terreus and A. niger conidia exposed to environmental and human body temperatures. The results showed that the aging process in Aspergillus conidia involves apoptosis,with metacaspase activation, DNA fragmentation, and reactive oxygen species production, associated with secondary necrosis. Distinct results were observed for the selected pathogenic species. At environmental conditions, A. niger was the species with the highest resistance to aging, indicating a higher adaption to environmental conditions, whereas A. flavus followed by A. terreus were the most sensitive species. At higher temperatures (37 °C), A. fumigatus presented the longest lifespan, in accordance with its good adaptation to the human body temperature. Altogether,with this work new insights regarding conidia aging are provided, which may be useful when designing treatments for aspergillosis.

Discovery of a small-molecule protein kinase Cδ-selective activator with promising application in colon cancer therapy

Protein kinase C (PKC) isozymes play major roles in human diseases, including cancer. Yet, the poor understanding of isozymes-specific functions and the limited availability of selective pharmacological modulators of PKC isozymes have limited the clinical translation of PKC-targeting agents. Here, we report the first small-molecule PKCδ-selective activator, the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), which binds to the PKCδ-C1-domain. Roy-Bz potently inhibited the proliferation of colon cancer cells by inducing a PKCδ-dependent mitochondrial apoptotic pathway involving caspase-3 activation. In HCT116 colon cancer cells, Roy-Bz specifically triggered the translocation of PKCδ but not other phorbol ester responsive PKCs. Roy-Bz caused a marked inhibition in migration of HCT116 cells in a PKCδ-dependent manner. Additionally, the impairment of colonosphere growth and formation, associated with depletion of stemness markers, indicate that Roy-Bz also targets drug-resistant cancer stem cells, preventing tumor dissemination and recurrence. Notably, in xenograft mouse models, Roy-Bz showed a PKCδ-dependent antitumor effect, through anti-proliferative, pro-apoptotic, and anti-angiogenic activities. Besides, Roy-Bz was non-genotoxic, and in vivo it had no apparent toxic side effects. Collectively, our findings reveal a novel promising anticancer drug candidate. Most importantly, Roy-Bz opens the way to a new era on PKC biology and pharmacology, contributing to the potential redefinition of the structural requirements of isozyme-selective agents, and to the re-establishment of PKC isozymes as feasible therapeutic targets in human diseases.