Diverse applications of cell-based assays for drug discovery

19/07/2022

A descoberta de medicamentos é um processo demorado conhecido por altas taxas de atrito associadas a gastos dispendiosos. Assim sendo, o modelo atual enfrenta desafios de produtividade. Apenas 10% dos compostos que entram nos ensaios clínicos da Fase I acabam por se tornar terapias aprovadas. Apesar do desenvolvimento de vacinas e medicamentos antivirais, o surto pandémico causado pelo vírus SARS-CoV-2 apenas exacerbou a necessidade de revisar os modelos atuais de descoberta de medicamentos. Quando comparados a uma abordagem tradicional baseada em alvos específicos, os ensaios tridimensionais (3D) baseados em células provaram alavancar as vantagens de uma descoberta de drogas baseada em fenótipos (PDD). Por sua vez, quando comparado a sistemas bidimensionais (2D), o mencionado anteriormente pode ser aplicado a ensaios de alto rendimento (HTS) em microescala, diminuindo assim o custo dos produtos inerentemente associado a uma escala menor em que esses ensaios são realizados. Além disso, a complexidade fenotípica celular é aumentada devido à replicação mais precisa de condições bioquímicas e biofísicas encontradas no microambiente in vivo. Aqui, as células estaminais com interesse especial em células pluripotentes induzidas humanas (hiPSCs) específicos do paciente, solicitam um PDD que permite a potencial resposta à droga de estratificação. Além disso, estes podem ser usados para obter vários tipos de células, induzindo sua diferenciação para camadas germinativas específicas do desenvolvimento embrionário. Como tal, vários processos biológicos e novas entidades biológicas podem ser investigados através da modulação do destino das células estaminais, sendo também possível de forma HTS utilizando plataformas de microescala baseadas em células 3D. Nesta tese, procuramos fornecer várias aplicações de tais ensaios baseados em células como ferramentas eficazes de triagem de drogas. Primeiro, fornecemos uma visão geral dos desenvolvimentos tecnológicos atuais que foram aplicados para melhorar a produtividade do pipeline de descoberta de medicamentos. Drug discovery is a lengthy process known for high attrition rates associated with costly expenditures. The current model faces productivities challenges. Only 10% of compounds entering Phase I clinical trials will eventually become approved therapeutics. Albeit the sift development of vaccines and antiviral medications, the pandemic outbreak caused by the SARS-CoV-2 virus only exacerbated the need to revise the current drug discovery models. When compared to a traditional target-based approach, threedimensional (3D) cell-based assays have proven to leverage the advantages of a phenotypic-based drug discovery (PDD). In turn, when compared to two-dimensional (2D) systems, the aforementioned can be applied to microscale high-throughput screens (HTS), thus decreasing the cost-of-goods inherently associated to a smaller scale at which these assays are carried. Moreover, cell phenotypic complexity is enhanced due to the more accurate replication of biochemical and biophysical cues found in the in vivo microenvironment. Herein, stem cells with special interest in patient specific human induced pluripotent stem cells (hiPSCs), prompt a PDD that allow the potential stratification drug response. Moreover, these can be used to obtain several cell types by inducing its differentiation towards specific germ layers of the embryonic development. As such, several biological processes and new biological entities can be enquired through modulation of stem cell fate, this being also possible in HTS manner by using 3D cellbased microscale platforms. In this thesis, we sought to provide several applications of such cell-based assays as effective drug screening tools. First, we provide an overview of the current technological developments that have been applied to improve the productivities of the drug discovery pipeline. We highlight several modalities that these assays can take both in 2D and 3D, also proving a comparison in its effective use. We then apply different current technologies to probe (1) stem cell differentiation, (2) differential drug responses in hiPSCs that are phenotypic-dependent, (3) antiviral properties of known compounds and finally (4) the circadian influence over drug metabolism. To that extend, we exploit the use of the well-establish micropillar-microwell system to the use of 3D cell aggregates. We were able to effectively differentiate hiPSC into neural progenitors both in 2D and 3D, further identifying differential drug responses when the same compound challenged both cell phenotypes. We have adapted a protocol to produce SARS-CoV-2 pseudoviral particles that was then used to demonstrate the effective antiviral properties several compounds, including commercially available antibodies, an approved drug with known antiviral property, and polysulfated glycosaminoglycans (GAGs) – heparin, Rahman sulfate (RS), pentosan polysulfate (PPS) and mucopolysaccharide polysulfate (MPS). Additionally, we aimed to provide the initial steps to establish an antiviral HTS platform by applying the previous protocols to the 3D microwell-micropillar platform. Finally, we demonstrated the influence of circadian rhythms in gene and protein expression of P450 enzymes when hepatic cells are cultured in 3D. We compared gene and protein expression of key drug metabolizing enzymes as well as important circadian modulators both in a 2D and 3D cell-based assays. We also provide a statistical quantification of the relation between the several players of the molecular circadian clock and DME involved in phase I and II of drug metabolism. We demonstrated the overall effect of a 3D microenvironment in the oscillatory profile of such genes. Moreover, we provide a transcriptomic analysis of several timepoints collected both in 2D and 3D, further demonstrating changes in gene expression when cells are and are not synchronized. As such, we provided the basis for the consideration of circadian factors as key parameters that affect experimental outcomes in 3D drug screens.

Autores da comunidade :

• 

  A

  André Lopes Rodrigues

  ist426192

Orientadores desta instituição:

• 

  M

  Maria Margarida Fonseca Rodrigues Diogo

  ist24804

101720572

Doutoramento em Bioengenharia

- Biotecnologia Industrial

Palavras-chave

• 3D cell based assays
• cellular microarray
• high-throughput screening
• SARS-CoV-2
• circadian rhythm

- Inglês

Instituto Superior Técnico

Entidade financiadora da bolsa/projeto

Fundação para a Ciência e a Tecnologia