Drug-Class Specific Impact of Antivirals on the Reproductive Capacity of HIV

Predictive markers linking drug efficacy to clinical outcome are a key component in the drug discovery and development process. In HIV infection, two different measures, viral load decay and phenotypic assays, are used to assess drug efficacy in vivo and in vitro. For the newly introduced class of integrase inhibitors, a huge discrepancy between these two measures of efficacy was observed. Hence, a thorough understanding of the relation between these two measures of drug efficacy is imperative for guiding future drug discovery and development activities in HIV. In this article, we developed a novel viral dynamics model, which allows for a mechanistic integration of the mode of action of all approved drugs and drugs in late clinical trials. Subsequently, we established a link between in vivo and in vitro measures of drug efficacy, and extract important determinants of drug efficacy in vivo. The analysis is based on a new quantity—the reproductive capacity—that represents in mathematical terms the in vivo analog of the read-out of a phenotypic assay. Our results suggest a drug-class specific impact of antivirals on the total amount of viral replication. Moreover, we showed that the (drug-)target half life, dominated by immune-system related clearance processes, is a key characteristic that affects both the emergence of resistance as well as the in vitro–in vivo correlation of efficacy measures in HIV treatment. We found that protease- and maturation inhibitors, due to their target half-life, decrease the total amount of viral replication and the emergence of resistance most efficiently

Contributions to the Mathematical Systems Medicine of Antimicrobial Therapy and Genotype-Phenotype Inference.

The following summary of my publications describes the main ideas in the corresponding research articles and clarfifies my contribution in multi-author publications. I decided to apply for habilitation according to x2.I.1.(c) of the Habilitationsordnung (this path is usually referred as Kumulative Habilitation"). I selected 13 first- or last author publications for this habilitation that concern contributions to the mathematical systems medicine of antiviral therapy [tMH10, tMS+11, FtK+11, tMMS12, DSt12, DWSt15, Dt16, DSt16, DDKt18, DSD+19, DDKt19], as well as inference of genotype-phenotype associations [SDH+15, SSJ+18]. The selected publications represent my major contributions in this research eld since submitting my doctoral thesis in September 2009

Pandemics, public health emergencies and antimicrobial resistance - putting the threat in an epidemiologic and risk analysis context

abstract: Public health messaging about antimicrobial resistance (AMR) sometimes conveys the problem as an epidemic. We outline why AMR is a serious endemic problem manifested in hospital and community-acquired infections. AMR is not an epidemic condition, but may complicate epidemics, which are characterised by sudden societal impact due to rapid rise in cases over a short timescale. Influenza, which causes direct viral effects, or secondary bacterial complications is the most likely cause of an epidemic or pandemic where AMR may be a problem. We discuss other possible causes of a pandemic with AMR, and present a risk assessment formula to estimate the impact of AMR during a pandemic. Finally, we flag the potential impact of genetic engineering of pathogens on global risk and how this could radically change the epidemiology of AMR as we know it. Understanding the epidemiology of AMR is key to successfully addressing the problem. AMR is an endemic condition but can play a role in epidemics or pandemics, and we present a risk analysis method for assessing the impact of AMR in a pandemic.The electronic version of this article is the complete one and can be found online at: https://archpublichealth.biomedcentral.com/articles/10.1186/s13690-017-0223-

The impact of sex, gender and pregnancy on 2009 H1N1 disease

Children and young adults of reproductive age have emerged as groups that are highly vulnerable to the current 2009 H1N1 pandemic. The sex of an individual is a fundamental factor that can influence exposure, susceptibility and immune responses to influenza. Worldwide, the incidence, disease burden, morbidity and mortality rates following exposure to the 2009 H1N1 influenza virus differ between males and females and are often age-dependent. Pregnancy and differences in the presentation of various risk factors contribute to the worse outcome of infection in women. Vaccination and antiviral treatment efficacy also vary in a sex-dependent manner. Finally, sex-specific genetic and hormonal differences may contribute to the severity of influenza and the clearance of viral infection. The contribution of sex and gender to influenza can only be determined by a greater consideration of these factors in clinical and epidemiological studies and increased research into the biological basis underlying these differences

2019 meeting of the global virus network

The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9–11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals.info:eu-repo/semantics/submittedVersio

The utility of efavirenz-based prophylaxis against HIV infection. A systems pharmacological analysis

Pre-exposure prophylaxis (PrEP) is considered one of the five “pillars” by UNAIDS to reduce HIV transmission. Moreover, it is a tool for female self-protection against HIV, making it highly relevant to sub-Saharan regions, where women have the highest infection burden. To date, Truvada is the only medication for PrEP. However, the cost of Truvada limits its uptake in resource-constrained countries. Similarly, several currently investigated, patent-protected compounds may be unaffordable in these regions. We set out to explore the potential of the patent-expired antiviral efavirenz (EFV) as a cost-efficient PrEP alternative. A population pharmacokinetic model utilizing data from the ENCORE1 study was developed. The model was refined for metabolic autoinduction. We then explored EFV cellular uptake mechanisms, finding that it is largely determined by plasma protein binding. Next, we predicted the prophylactic efficacy of various EFV dosing schemes after exposure to HIV using a stochastic simulation framework. We predicted that plasma concentrations of 11, 36, 1287 and 1486ng/mL prevent 90% sexual transmissions with wild type and Y181C, K103N and G190S mutants, respectively. Trough concentrations achieved after 600 mg once daily dosing (median: 2017 ng/mL, 95% CI:445–9830) and after reduced dose (400 mg) efavirenz (median: 1349ng/mL, 95% CI: 297–6553) provided complete protection against wild-type virus and the Y181C mutant, and median trough concentrations provided about 90% protection against the K103N and G190S mutants. As reduced dose EFV has a lower toxicity profile, we predicted the reduction in HIV infection when 400 mg EFV-PrEP was poorly adhered to, when it was taken “on demand” and as post-exposure prophylaxis (PEP). Once daily EFV-PrEP provided 99% protection against wild-type virus, if ≥50% of doses were taken. PrEP “on demand” provided complete protection against wild-type virus and prevented ≥81% infections in the mutants. PEP could prevent >98% infection with susceptible virus when initiated within 24 h after virus exposure and continued for at least 9 days. We predict that 400 mg oral EFV may provide superior protection against wild-type HIV. However, further studies are warranted to evaluate EFV as a cost-efficient alternative to Truvada. Predicted prophylactic concentrations may guide release kinetics of EFV long-acting formulations for clinical trial design

Cyclodextrins as medicine and therapeutic adjuvants in drug delivery systems

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.Ao longo de várias décadas desde a sua descoberta, as ciclodextrinas foram sendo usadas como excipientes e veículos de muitas substâncias ativas para aumentar a solubilidade aquosa, estabilidade físico-química e fisiológica e a veiculação destes fármacos. Atualmente, as ciclodextrinas estão presentes nas mais diversas formulações de fármacos, como comprimidos, pomadas, gotas oftálmicas, entre outras. Adicionalmente a esta função de excipientes, recentemente as ciclodextrinas também são encontradas como substância ativa no tratamento de algumas patologias. Esta monografia foca-se em avanços recentes e encorajantes sobre a aplicação de ciclodextrinas como fármacos e adjuvantes terapêuticos em sistemas de veiculação de fármacos. As primeiras três secções fornecem uma perspetiva histórica e descrição das ciclodextrinas e ciclodextrinas modificadas, enquanto que a quarta secção fornece uma revisão dos avanços atuais no uso terapêutico das ciclodextrinas. O potencial terapêutico das ciclodextrinas é maioritariamente derivado da sua habilidade em formar complexos com colesterol e outras moléculas. Deste modo, estas moléculas estão a ser estudadas como novo agente terapêutico em muitas patologias conhecidas onde outras classes terapêuticas não conseguem reduzir a severidade da doença e/ou controlar a mesma, e até como entidades químicas para algumas patologias sem terapia conhecida. Na maioria das situações, as ciclodextrinas modificadas são frequentemente usadas devido ao facto de terem uma maior afinidade para o alvo terapêutico, colesterol, quando comparadas às ciclodextrinas “convencionais”, tendo também uma menor taxa de efeitos adversos e toxicidade. Em algumas patologias, o mecanismo exato da atividade das ciclodextrinas é desconhecido. Assim é necessário continuar a investigação, incluindo a iniciação e conclusão de muitos estudos clínicos antes que estas moléculas cheguem a farmácias, hospitais e doentes como fármaco. A produção destes dados, incluindo considerações toxicológicas, são relevantes para a sua aprovação e uso posterior ou comparticipação dos diversos sistemas nacionais de saúde. Assim, num futuro próximo, poderemos ter novas estratégias disponíveis com diferentes ações benéficas no organismo humano, com a máxima segurança e benefício para o doente.Over several decades since its discovery, cyclodextrins have been used as excipients and vehicles of many active pharmaceutical ingredients to enhance aqueous solubility, physicochemical and physiological stability, and the deliverability of these drugs. Currently, cyclodextrins are present in diverse drug formulations, such as pills, ointments, ophthalmic drops, among other formulations. In addition to the function of pharmaceutical excipients, cyclodextrins have also found recent use as active substances in the treatment of some pathologies. This monograph focuses on some recent and encouraging advances in the application of cyclodextrin as medicine and therapeutic adjuvants in drug delivery systems. The first three sections provides an historical overview and description of cyclodextrins and modified cyclodextrins while the fourth section reviews the current advances in the therapeutic use of cyclodextrin molecules. Cyclodextrin’s therapeutic potential is mainly derived from their ability to form complexes with cholesterol and other molecules. As such they are being studied as new therapeutic agents in many known pathologies where other therapeutic classes are not able to reduce the severity of the disease and/or even control it, and as new chemical entities for some pathologies with no known therapy. In most situations, modified cyclodextrins are more frequently used as they will have greater affinity for the therapeutic target, cholesterol, when compared to “conventional” cyclodextrins, also having a lower rate of adverse effects and toxicity. In some pathologies, the exact mechanism of cyclodextrins activity is unknown. Further research activities, including the initiation and completion of many clinical trials are required before these molecules reach pharmacies, hospitals and patients as active substance. The production of these data, including toxicity consideration, will be relevant for its approval and later use or reimbursement by multiple national health systems. Thus, in a near future, we could have new strategies available with different beneficial actions in the human body, with maximum safety and benefit for the patient

Ebola Virus Entry: From Molecular Characterization to Drug Discovery

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013\u207b2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD

Advancing antiviral strategies against emerging RNA viruses by phenotypic drug discovery

Pathogenic RNA viruses can emerge from unexpected sources at unexpected times and cause severe disease in humans, as exemplified by the ongoing coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV) outbreaks from the past decade. Despite the increasing impact of emerging viruses to health and economy worldwide, our preparedness to stand against these diseases is hampered by the lack of approved and effective antiviral therapies. Thus, the development of novel antivirals is of urgent need. To date, antiviral drug discovery has primarily focused on targeting specific viral proteins, but these treatments often suffer from viral resistance and are limited to only one or few viruses. Instead, phenotypic drug discovery enables the identification of drug candidates that are active in the disease-relevant model and not restricted to previously characterized biological processes. As RNA viruses are highly dependent on the host cell pathways due to their relatively small genome, targeting virus vulnerabilities within the host cell has been a promising antiviral strategy for broad spectrum antivirals, but is relatively unexplored so far. In fact, phenotypic approaches can additionally identify host-directed antivirals due to the unbiased nature. The focus of this doctoral thesis was to identify novel antiviral compounds with broad spectrum activity and investigate the compound mechanism of action and target pathways from the host cell and virus perspective. To achieve these goals, multiple cutting-edge phenotype-based methodologies were implemented that additionally advanced the antiviral drug discovery landscape. In Paper I, we developed an image-based phenotypic antiviral assay and screened our in-house chemical library targeting cellular oxidative stress and nucleotide metabolism pathways in Hazara virus (HAZV)-infected cells. Screening hit compounds TH3289 and TH6744 activity was validated by their therapeutic window and both compounds were also active beyond HAZV, especially TH3289 that was tested and displayed activity against EBOV, CCHFV, SARS-CoV-2 and a common cold coronavirus 229E (CoV-229E). We also excluded the intended target 8-oxoguanine DNA glycosylase (OGG1) protein to be responsible for TH6744 antiviral activity and characterized host cell chaperone and co-chaperone network as target pathways of TH6744 by implementing thermal proteome profiling methodology. In Paper II, we transferred our image-based phenotypic assay to ZIKV-infected brain cells in order to screen structural analogs of TH3289 and TH6744 against a pathogenic RNA virus. TH3289 and TH6744 again appeared among the screening hits and presented a promising therapeutic window in various cellular models, further confirming their broad activity. Moreover, TH6744 reduced ZIKV infection and progeny release in cerebral organoid model and impressively rescued ZIKV-induced cytotoxicity in organoids. Additionally, treatment with TH6744 rapidly diminished ZIKV progeny release during late replication cycle stages, elucidating the antiviral mechanism of action. In Paper III, we established an untargeted morphological profiling method to provide in-depth host cell responses during antiviral screening. We combined the Cell Painting protocol with antibody-based virus detection in a single assay followed by automated image analysis pipeline providing segmentation and classification of infected cells and extraction of cell morphological features. We demonstrated how our assay reliably distinguished CoV-229E infected human lung fibroblasts from non-infected controls based on cellular morphological features. Furthermore, our method can be applied in phenotypic drug screening as validated by nine host- and virus-targeting antivirals. Effective antivirals Remdesivir and E-64d treatment reversed the infection-specific signatures in host cells. Thereby, the developed method can be implemented for antiviral phenotypic drug discovery by morphological profiling of drug candidates