Rafael Radi | Professor and Chair, Department of Biochemistry, Faculty of Medicine and Director of the Center for Biomedical Research (CEINBIO), Universidad de la República, Uruguay

Introduction

This manuscript describes a journey that connects basic biomedical research with science advise responsibilities in Uruguay in the context of the COVID-19 pandemic. This selected topic fits within the overall theme of the Pontifical Academy of Sciences Plenary Session 2022 on “Basic science for human development, peace, and planetary health” (8-9 Sept, 2022 | Casina Pio IV | Vatican City) as it exemplifies how a solid background on basic and curiosity-driven research generates the potential for the fast provision and construction of scientific evidence-based solutions on emergent community or planetary problems.

Redox Metabolism in Humans: from Respiration and Bioenergetics to Redox Signaling and Oxidative Damage

Humans consume oxygen as an essential process for life. The lungs are the main organs that allow the transport of atmospheric oxygen in the gas phase to dissolved oxygen in the blood; once in the blood, oxygen is transported bound to hemoglobin in the red blood cells to the capillary of the different tissues where it is released and diffuses to the cells. Once inside cells, under normal conditions more than 99% of oxygen is consumed in the process of cellular respiration, where it is utilized on mitochondria (the key energy producing organelle) as the terminal acceptor of electrons arising from the oxidation of biomolecules such as carbohydrates, fatty acids and amino acids. The oxidation process permits to obtain energy for the cells and is coupled to the four-electron reduction of oxygen to water (i.e. at the terminal site of the mitochondrial electron transport chain) (Eq. 1).

O₂ + 4e^- + 4H⁺  2H₂O                                                                                  [1]

Still, biological redox[1] processes that use molecular oxygen also involve reduction by one- or two-electrons to yield “partially” reduced oxygen intermediates such as superoxide radical[2] (O₂^.-, Eq. 2) and hydrogen peroxide (H₂O₂, Eq. 3).

O₂ + e^-  O₂^.-                                                                                                 [2]

O₂ + 2e^- + 2H⁺  H₂O₂                                                                                                             [3]

Both, O₂^.- and H₂O₂ are reactive and short-lived intermediates (collectively grouped with other related biomolecules as “reactive oxygen species”, ROS) (1) and can promote oxidative modifications in biomolecules including proteins, lipids and DNA. Depending on the steady-state levels of ROS, these species can play cell regulatory actions that promote adaptation and proliferation (i.e. cell signaling, low to moderate ROS levels) or promote cellular dysfunction and death (i.e. oxidative damage, high ROS levels). The latter condition also named as “oxidative stress” is associated to a variety of acute and chronic disease conditions and the process of aging, and was fully established as a concept in biomedicine in the mid 80s (reviewed in (2)).

Nitric Oxide and Superoxide Radical interactions: The Birth of the Biological Chemistry of Peroxynitrite

In the mid to late 80s, nitric oxide (NO) was discovered as a free radical that could play physiological functions. In fact, ^.NO initially characterized as a vasodilator, was later demonstrated to also exert actions as neurotransmitter and immunomodulator, among several other physiological functions (reviewed in (3)). The discoveries related to the “^.NO pathway” in the regulation of vascular tone in humans led to the Nobel Prize in Physiology or Medicine in 1998. While the physiologists were at that time becoming increasingly interested in the “good” functions of ^.NO, a group of biochemists and biomedical scientists became interested in revealing some potentially “bad” or toxic effects of ^.NO when generated in excess and in conjunction with increased levels of ROS. In fact, increasing evidence was indicating that high levels of ^.NO could promote neurotoxicity and participate in processes such as mammalian cell death and pathogen killing (reviewed in (4)). Observing the “radical nature” of ^.NO and knowing that different radicals tend to react at extremely fast rates with each other, together with other emerging biological evidence, it was postulated that conditions that favor excess and concomitant formation of ^.NO and O₂^.- in tissues promote the formation of peroxynitrite anion (ONOO^-), a strong oxidant and potentially cytotoxic intermediate (5–7).

^.NO + O₂^.-  ONOO^-                                                                         [4]

Early experiments found that peroxynitrite anion and its conjugated acid, peroxynitrous acid (ONOOH), could efficiently promote oxidations in biomolecules. These early discoveries expanded the view on how ^.NO and ROS could synergistically participate in oxidative and inflammatory pathophysiology (8). This hypothesis was explored by a large number of groups worldwide and, at present, peroxynitrite is considered a pathogenic mediator in neurodegenerative processes, inflammatory processes and cardiovascular diseases, among several other conditions (reviewed in (9)). Thus, basic biochemical ideas on how peroxynitrite could be formed and participate in oxidative damage to biomolecules in vivo was progressively adopted as a new concept by the biomedical community and gradually translated to medicine. One of the early papers on the reaction of peroxynitrite with protein and non-protein thiols, contained what turned to be a valuable scheme to illustrate the general hypothesis (6); after two decades, this work was recognized as a citation classic (10). The original proposal and the updated view on the biological chemistry of peroxynitrite was presented in a more recent review (Fig. 1)(11).

From Redox Biology to COVID-19 Scientific Advice

The work on redox biochemistry and medicine through three decades of research (12) provided me with a unique opportunity to work in interdisciplinary projects, with joined contributions with chemists, physical-chemists, molecular and cell biologists, physiologists, pharmacologists and clinicians. But a major (and surprising) interdisciplinary challenge was presented to me on 2020. On March 13, 2020 the government of Uruguay declared a state of health emergency due to the diagnosis of the first cases of COVID-19 (Fig. 2)(13). Soon after, on April 3, 2020, I was invited together with other scientists to a virtual meeting by a government of Uruguay top official and I was unexpectedly requested to create and lead a scientific advisory group to the Presidency to assist in the the management of the COVID-19 pandemic. The request was based on my background and track record on interdisciplinary research[3] and extensive knowledge of the local research community. Thus, after requesting some time to decide and eventually generate a general working plan, the Scientific Advisory Group (i.e. GACH[4]) was formally created in an ad honorem fashion on April 16, 2020 and announced by the President of the República Oriental del Uruguay, Dr. Luis Lacalle Pou, to the population on a national press conference. The creation of GACH was occurring in parallel to several other government measures and academic sector actions as shown in Fig. 2.

The Group was designed with two main areas, the Health Area lead by Prof. Henry Cohen (MD, clinician) and the Data Science and Modelling Area led by Prof. Fernando Paganini (PhD in mathematics and engineer); I served as the General Coordinator of the group. The three coordinators worked jointly to select an interdisciplinary group of 60 top national scientists and experts. The GACH was divided into subgroups depending on topics and expertise of the different participating scientists and regularly generated weekly reports to the government on COVID-19 issues; the GACH was also in permanent exchange with the rest of the scientific community and the Ministry of Health and other government agencies. The GACH members also provided interviews to the press and the coordinators participated in five national press conferences that delivered scientific evidence to the society on the biological, epidemiological and pathological dimensions of the problem and the key elements to be considered for disease mitigation. Some of the key reports are listed in Table 1. A major effort was made so that all GACH members maintained a cohesive and coherent vision of the problem, so plenty of analysis, discussions and exchanges of information and possible recommendations were internally processed before making public statements. All the reports were presented weekly to the President of the Republic and his direct team, and within 24-48 hrs were posted in the public domain (see Table 1); this methodology provided transparency and credibility to the advisory process. In late 2020 and early 2021, key expert advice was provided in relation to the fundamental issue of vaccine selection and design of the vaccination program in collaboration with the Ministry of Health. A complete report was presented to the Presidency on December 2020 and in a session of the Health Commission of the National Parliament on January 2021. The vaccination program started on March 1, 2021, and was delivered at a fast rate and counted with a large adherence of the population.

The eight fundamentals guiding the activities of the GACH towards the mitigation of the COVID-19 event were:

1. Four conceptual axis to reach a new normality: progressiveness, regulation, monitoring and evidence.
2. Diversity of disciplines in the Areas of Health and Data Science and Modelling.
3. Selection of excellent scientists, willing to work as a team and at a fast pace.
4. Generation of regular reports providing analysis and recommendations, taking into consideration and documenting all the national and international evidence.
5. Independent group, no political interference.
6. Weekly meetings with the government, and daily exchanges as needed, transferring the information generated by GACH.
7. Clear separation of roles: the GACH to provide scientific advice, the government to take the final decisions.
8. Transparent and frequent communication with society.

All the ca. 90 public reports generated by GACH at: https://www.presidencia.gub.uy/gach

Table 1. Key Public Reports by GACH

• Non-pharmacological intervention (NPI) strategies
• School re-opening
• Impact of COVID-19 in children
• Encouragement of the use of public spaces
• Impact of the health emergency on non-COVID health issues
• COVID-19 and nutrition
• COVID-19 and dental health practice
• Impact of COVID-19 on mental health
• COVID-19 and social behavioral changes
• Contingency plans for Intensive Care Medicine
• New treatments for COVID-19 patients
• Molecular tools for diagnosis
• Emphasis and reinforcement of the TETRIS strategy
• Data analysis, modeling and projections
• Epidemiological and integrated analysis: public health recommendations
• Mitigation strategies during the end of the year celebrations and summer tourism
• Vaccines and vaccination program (in collaboration with the Ministry of Health)

All reports are uploaded at https://www.presidencia.gub.uy/gach

The key role of scientific evidence-based decisions during the pandemic

In many periods of the evolution of the pandemic, especially in 2020, Uruguay had an exceedingly good control (commented in (14)), in spite of being geographically located in a region that did not have the pandemic under control. The analysis of the different phases of the pandemic in Uruguay and the evaluation of its overall management is under scrutiny now by a group of former members of GACH and will be object of a technical communication elsewhere in the future. Our preliminary analysis indicates that within the Latin American region, Uruguay was, on one hand, the country with the least impact on mortality and excess deaths (Fig. 3) (normalized by population) while, on the other hand, its “stringency index”[5] over time was for the most part in moderate values. Adherence of the population to non-pharmacological interventions during 2020 and to the vaccination program in 2021 was remarkable and there is a general national and international agreement that the participation of scientific advice by an independent group was very relevant on these individual and community behaviors (15).

The GACH experience was highlighted in the international medical literature as a valid approach in the COVID-19 event control, when comparing the responses to the pandemic of 28 countries (15). In addition, the scientific system in Uruguay largely increased social and public visibility[6] and gained influence with the political sector that shows more willingness to request scientific advice for other public policy decisions and to discuss issues related to science budget.[7] Finally, the GACH did a great effort to minimize partisan divisions during the pandemic, as they constitute an additional risk factor within the pandemic (16, 17); the GACH made it clear to both the political sector and society, each time it had the capacity to communicate this, especially during the most difficult times of the evolution of the COVID-19 event in Uruguay (February to May 2021).

The group resumed activities in July 2021, when the percentage of fully vaccinated population was over 50% and decoupling between mobility and infections (due to the accumulation of vaccine- and natural-mediated immunity) was confirmed (18). The group received state honors from the President of the Republic in the main national auditorium broadcasted nationwide, and according to a national survey performed by an independent public opinion consultant group, 90% of the population was supportive of the scientific advisory work performed.[8]

Back to the basics: redox biochemistry to disrupt SARS-CoV-2 and new horizons in redox biology

During the work of GACH, our research group began studies on the structural biology of the SARS-CoV-2 spike glycoprotein, a key element on the virus surface needed to bind to the host cell and initiate the infection process. In particular, we explored how the spike glycoprotein structure could be disrupted by redox processes mediated by compounds that can “break” chemical bonds in the protein, i.e. via reduction of protein disulfides. The reduction process leads to instability in the spike receptor binding domain and resulted in decreased capacity to bind to the host-cell plasma membrane receptor (i.e. ACE-2) (Figure 4). The use of the reducing compounds, some of which are already in clinical trials for other lung disease conditions, and the subsequent conformational alteration in spike, decreased viral entry to mammalian host cells . Nicely, this work reflected a new fertile international collaboration that started “at a distance” during the pandemic with USA-based groups; the contribution represents a proof-of-concept to open opportunities for future redox-based therapeutics to treat pulmonary lung infections, including those mediated by coronaviruses.

After finishing the highly demanding scientific advisory activity in the second semester of 2021, I have fully resumed research and academic activities. In this regard, in 2022 I communicated new ideas that may help to further develop the redox field in human health, disease and therapeutics .

Final Comments

The journey described herein illustrates that the paths of basic sciences and their applications can be intertwined and serve major societal challenges. There is a need to foster the connections between state-of-the-art science and leading scientists with their engagement in pressing local, regional and planetary problems. This approach nurtures the ethos of science and helps the society and politicians to envision the potential and strength of a well-cultivated scientific system for the accomplishment of sustainable human development.

References

1.        Murphy, M.P., Bayir, H., Belousov, V., Chang, C.J., Davies, K.J.A., Davies, M.J., Dick, T.P., Finkel, T., Forman, H.J., Janssen-Heininger, Y., Gems, D., Kagan, V.E., Kalyanaraman, B., Larsson, N.-G., Ginger, M.N., Nyström, T., Poulsen, H.E., Radi, R., van Remmen, H., Schumacker, P.T., Thornalley, P.J., Toyokuni, S., Winterbourn, C.C., Yin, H., and Halliwell, B. (2022) Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab. 4, 651-662.

2.        Jones, D.P., and Radi, R. (2014) Redox pioneer: professor Helmut Sies. Antioxidants & redox signaling. 10.1089/ars.2014.6037

3.        Epstein, F.H., Moncada, S., and Higgs, A. (1993) The L-Arginine-Nitric Oxide Pathway. New England Journal of Medicine. 10.1056/nejm199312303292706

4.        Radi, R. (2004) Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci U S A. 101, 4003-4008.

5.        Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., and Freeman, B.A. (1990) Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 10.1073/pnas.87.4.1620

6.        Radi, R., Beckman, J.S., Bush, K.M., and Freeman, B.A. (1991) Peroxynitrite oxidation of sulfhydryls: The cytotoxic potential of superoxide and nitric oxide. Journal of Biological Chemistry. 266, 4244-4250.

7.        Ferrer-Sueta, G., Campolo, N., Trujillo, M., Bartesaghi, S., Carballal, S., Romero, N., Alvarez, B., and Radi, R. (2018) Biochemistry of Peroxynitrite and Protein Tyrosine Nitration. Chem Rev. 10.1021/acs.chemrev.7b00568

8.        Beckman, J.S., and Koppenol, W.H. (1996) Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and the ugly. Am J Physiol Cell Physiol. 10.1152/ajpcell.1996.271.5.c1424

9.        Piacenza, L., Zeida, A., Trujillo, M., and Radi, R. (2022) The superoxide radical switch in the biology of nitric oxide and peroxynitrite. Physiol Rev. 10.1152/physrev.00005.2022

10.      Banerjee, R. (2015) When the good and the bad make the ugly: The discovery of peroxynitrite. Journal of Biological Chemistry. 10.1074/jbc.O115.000001

11.      Radi, R. (2013) Peroxynitrite, a stealthy biological oxidant. Journal of Biological Chemistry. 288, 26464-26472.

12.      Radi, R. (2019) The origins of nitric oxide and peroxynitrite research in Uruguay: 25 years of contributions to the biochemical and biomedical sciences. Nitric Oxide. 10.1016/j.niox.2019.03.003

13.      Moreno, P., Moratorio, G., Iraola, G., Fajardo, Á., Aldunate, F., Pereira-Gómez, M., Perbolianachis, P., Costábile, A., López-Tort, F., Simón, D., Salazar, C., Ferrés, I., Díaz-Viraqué, F., Abin, A., Bresque, M., Fabregat, M., Maidana, M., Rivera, B., Cruces, M.E., Rodríguez-Duarte, J., Scavone, P., Alegretti, M., Nabón, A., Gagliano, G., Rosa, R., Henderson, E., Bidegain, E., Zarantonelli, L., Piattoni, V., Greif, G., Francia, M.E., Robello, C., Durán, R., Brito, G., Bonnecarrere, V., Sierra, M., Colina, R., Marin, M., Cristina, J., Ehrlich, R., Paganini, F., Cohen, H., Radi, R., Barbeito, L., Badano, J.L., Pritsch, O., Fernández, C., Arim, R., Batthyány, C., and Group., I.C.-19 W. (2020) An effective COVID-19 response in South America: the Uruguayan Conundrum. medRxiv.

14.      Taylor, L. (2020) Uruguay is winning against covid-19. This is how. The BMJ. 10.1136/bmj.m3575

15.      Haldane, V., de Foo, C., Abdalla, S.M., Jung, A.S., Tan, M., Wu, S., Chua, A., Verma, M., Shrestha, P., Singh, S., Perez, T., Tan, S.M., Bartos, M., Mabuchi, S., Bonk, M., McNab, C., Werner, G.K., Panjabi, R., Nordström, A., and Legido-Quigley, H. (2021) Health systems resilience in managing the COVID-19 pandemic: lessons from 28 countries. Nat Med. 10.1038/s41591-021-01381-y

16.      Clinton, J., Cohen, J., Lapinski, J., and Trussler, M. (2021) Partisan pandemic: How partisanship and public health concerns affect individuals’ social mobility during COVID-19. Sci Adv. 10.1126/sciadv.abd7204

17.      Gollwitzer, A., Martel, C., Brady, W.J., Pärnamets, P., Freedman, I.G., Knowles, E.D., and van Bavel, J.J. (2020) Partisan differences in physical distancing are linked to health outcomes during the COVID-19 pandemic. Nat Hum Behav. 10.1038/s41562-020-00977-7.

18.      Fiori, M., Bello, G., Wschebor, N., Lecumberry, F., Ferragut, A., and Mordecki, E. (123AD) Decoupling between SARS-CoV-2 transmissibility and population mobility associated with increasing immunity from vaccination and infection in South America. 10.1038/s41598-022-10896-4.

19.      Shi, Y., Zeida, A., Edwards, C.E., Mallory, M.L., Sastre, S., Machado, M.R., Pickles, R.J., Fu, L., Liu, K., Yang, J., Baric, R.S., Boucher, R.C., Radi, R., and Carroll, K.S. (2022) Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein. Proc Natl Acad Sci U S A. 10.1073/pnas.2120419119.

20.      Radi, R. (2022) Interplay of carbon dioxide and peroxide metabolism in mammalian cells. Journal of Biological Chemistry. https://doi.org/10.1016/j.jbc.2022.102358

[1] The term “redox” is utilized to refer to complimentary chemical processes in which a molecule gains (reduction) and another loses (oxidation) electrons. Redox reactions in biochemical contexts involve electron transfer between molecules.

[2] A radical denotes a molecule that contains an unpaired electron in its outer molecular orbital.

[3] I hold MD and PhD degrees from Universidad de la República, 1989 and 1991, respectively.

[4] GACH, Grupo Asesor Científico Honorario.

[5] https://ourworldindata.org/metrics-explained-covid19-stringency-index

[6] Significantly more press space (newspapers, radio, television) is dedicated to science topics, a much larger number of University students decided to begin scientific careers and the general public became aware of the existence of local science and scientists in Uruguay.

[7] In fact, as current President of the Academia Nacional de Ciencias del Uruguay, I participated in several meetings with the President, Ministers and Parliament members to discuss their commitment to increase the public budget for science in 2023 and the following years.

[8] https://www.cifra.com.uy/index.php/2021/07/08/evaluacion-del-grupo-asesor-cientifico-honorario/