The hunt for an HIV vaccine – unique insights from an inspiring Cohort of women in South Africa

“We have been privileged to have worked with community members who are so committed to the research that could one day realize our shared vision of a world without AIDS.” 

Professor Salim Abdool Karim, Director of CAPRISA, South Africa.

Since HIV was found to be the cause of acquired immune deficiency syndrome (AIDS) in 1983, scientists have been working endlessly towards the development of an effective vaccine to end the global HIV pandemic which has claimed more than 32 million lives[1] and impacted millions more. Unfortunately, the best vaccine candidate we have had to date was from the famous Thai RV144 trial which only resulted in 31.2% efficacy (Rerks-Ngarm et al., 2009[2]). However, hope remains and more than 30 years after the discovery of HIV, we have uncovered many vulnerabilities of the virus which could lead to the development of an effective HIV vaccine to solve one of the big global challenges of our age.

One of the keys to a successful vaccine is the use of broadly neutralizing antibodies (bNAbs). These special antibodies bind to the HIV envelope protein and prevent the virus from infecting host cells. What makes them even more special is that they can bind numerous mutated versions of the virus and therefore overcome the problem of the HIV variability. Understanding the diverse ways that antibodies use to target the HIV envelope is important to the development of an HIV vaccine which can produce such unique and unusual antibodies and effectively protect vaccinated individuals from HIV infection.

My name is Dr Thandeka Moyo and I am a GCRF START grant-funded Postdoctoral Research Fellow at South Africa’s National Institute for Communicable Diseases (NICD), affiliated to the University of the Witwatersrand (Wits). Over the past few years, my colleagues and I have gained new insights into bNAbs in chronic HIV infection, insights which contribute significantly to the worldwide hunt for an HIV vaccine. Most recently, with access to the UK’s national synchrotron – Diamond Light Source (Diamond) – facilitated by the GCRF START grant, we were able to solve the structure for one member of a family of antibodies which has revealed a uniquely long loop in the light chain of the antibody – a loop up to three times longer than other published anti-HIV antibodies![3]  Such insights are exciting, providing opportunities not only to expand my skills and knowledge as an early career scientist but also to inspire further hope that we will one day have all the necessary information to design an effective vaccine to end the global HIV pandemic.

South Africa has the biggest HIV epidemic in the world, with an estimated 7.5 million people infected nationally according to UNAIDS[4]. In this article, I will outline some of the insights achieved over several years of investigating broadly neutralizing antibodies through women participating in the CAPRISA Cohort – a research programme based in the KwaZulu-Natal province of South Africa. Without these women enabling us to study their donated samples, we would still have many unanswered questions.

“The experiences of the CAPRISA bnAb cohort studies epitomize the strength of the relationship between CAPRISA researchers and the local communities – a relationship of respect and equality.”

Professor Salim Abdool Karim, Director of CAPRISA, South Africa.
The rural town of Vulindlela in KwaZulu-Natal, South Africa, where the CAPRISA Vulindlela Clinical Research Clinic is based. Photo credit: Dean Demos

Investigating broadly neutralizing antibodies in chronic HIV infection

A subset of individuals who are infected with HIV develop broadly neutralizing antibodies (bNAbs) in chronic HIV infection. Unfortunately, these special antibodies are very unusual with characteristics not found in most other antibodies that we have in our bodies. For example, these antibodies are highly mutated and have longer “arms” that reach out and bind to the virus. Most antibodies do not have these characteristics, so researchers have spent years studying these unique bNAbs to get a better sense of how to produce them with an HIV vaccine.

In our laboratory at the NICD, we study antibodies in HIV-infected women from KwaZulu-Natal who participate in the CAPRISA Cohort.  Established in 2003, this Cohort has tracked women over several years from before HIV infection, through to when a subset of them were just infected (acute stage), and years after infection (chronic stage). Throughout the course of the study, the women received healthcare and HIV counselling through the Cohort and have continued to participate in it for years. Blood samples were taken at various time points and from these samples we have been able to track the evolution of the viruses in these women as well as the way their antibodies have adapted throughout infection – with some women developing these special bNAbs.

The CAPRISA Cohort has been invaluable in providing us with novel information on how bNAbs develop as the virus mutates, as well as how we can engineer a vaccine strategy more widely to make these antibodies. This has helped us understand the development of bNAbs and how we can use these antibodies for an effective HIV vaccine. Outlined below are examples from three women in the Cohort (referred to as CAP256, CAP248 and CAP314 respectively) who have developed special antibodies.

CAPRISA Cohort participants CAP256 and CAP248

CAPRISA Cohort participant CAP256 became infected with HIV during the study and then re-infected with another variant form of HIV 15 weeks after initial infection – a phenomenon referred to as superinfection. A related virus to the superinfecting virus changed the immune response in this woman and she developed bNAbs after this event.  Scientists in the USA isolated antibodies from the blood donated by CAP256[5] and discovered that the best antibody isolated bound to the apex (the top region) of the HIV envelope protein. This antibody is the most potent antibody isolated to date that binds to this target and the exact mode of binding for this antibody was not understand until recently. Led by our collaborators at the National Institute of Health in the USA, a study of the high-resolution structure of this antibody bound to the HIV envelope using cryo-electron microscopy revealed that it uses two distinct mechanisms to bind to this region (Gorman et al., 2020[6]). The use of these diverse strategies is likely the cause of its extremely high potency. The antibodies from CAP256 are currently in an HIV prevention clinical trial with results expected in the next few years.

Another CAPRISA Cohort participant, CAP248, also developed bNAbs. Researchers isolated an antibody from this participant which was unusual in the HIV envelope site that it targeted. Using negative stain electron microscopy (Scarff et al., 2018[7]), they showed that this antibody from CAP248 bound to a target proximal to the viral membrane and parts of the antibody interacted directly with viral membrane (Wibmer et al., 2017[8]). This mode of binding is unique and represents a novel way an antibody can bind to the HIV envelope protein. This novel binding mechanism may provide insight into the design of an HIV vaccine candidate that can produce this type of antibody response.

Solving a unique antibody structure with the help of the GCRF START grant – CAPRISA Cohort participant CAP314

The last participant to highlight in this article is CAP314. CAP314 developed bNAbs within two years of infection which is a relatively short time for HIV-infected individuals to develop these special antibodies. We isolated antibodies from three families of antibodies that developed in this individual. One family of antibodies mutated over time in response to the mutating HIV variants circulating in CAP314 at the same time. We were able to solve the structure for one member of this antibody family by X-ray crystallography on Diamond’s i03 beamline, in 2019, with Dr Dave Hall as our local contact in the UK for the beamline session and access provided by the GCRF START grant. Solving the structure of this antibody has revealed its uniqueness in that it has an extremely long light chain loop. The loops of antibodies are like arms which reach out and attach to their target on the HIV Envelope. Most anti-HIV antibodies have long loops on the heavy chain of the antibody, but this antibody has a uniquely long loop – up to three times longer than other published anti-HIV antibodies – in the light chain of the antibody. This long light chain loop reaches into the HIV envelope protein and makes the necessary contacts for this antibody to bind to the virus and stop it from infecting cells. Novel and unique binding mechanisms of antibodies like these give us important insights which could help us in our quest to design an HIV vaccine.

Aerial view of the UK’s national synchrotron, Diamond Light Source, located at the Harwell Campus in Oxfordshire, UK. ©Diamond Light Source

Global solidarity, shared responsibility through phenomenal CAPRISA Cohort and collaborations  

We are very grateful to the women in the CAPRISA Cohort who make our vital work towards the goal of HIV prevention possible, and to our collaboration with the GCRF-funded START grant. START is a phenomenal initiative supporting capacity development of structural biologists throughout Africa by providing improved access to world class synchrotron equipment, mentoring and expertise. I have been fortunate to have found extremely supportive mentors in START Co-Investigator’s (CoI’s), Prof. Penny Moore and Prof. Lynn Morris, who have encouraged my independence and supported me throughout my Postdoctoral studies.

“Over the last decade, we have learned an incredible amount about how some HIV infected women make broadly neutralizing antibodies. These insights have significantly contributed to HIV vaccine design. This has only been possible because of the extraordinary commitment shown by CAPRISA Cohort donors who come back again and again, and the clinical staff who care for them. We are truly indebted to them.” 

GCRF START Co-I, Prof. Penny Moore, University of the Witwatersrand and the National Institute for Communicable Diseases.

The UN states that a core principle of the 17 Sustainable Development Goals (SDGs), and of the AIDS response, is that “no one should be left behind. The AIDS epidemic cannot be ended without “the needs of people living with and affected by HIV, and the determinants of health and vulnerability, being addressed.”– UNAIDS[9]

Read more here about HIV/AIDS and the UN Sustainable Development Goals.

Read more about World AIDS Day 2020 here.

World AIDS Day 2020 – Global solidarity, shared responsibility. Photo credit: UNAIDS

More about Dr Thandeka Moyo

 GCRF START Postdoctoral Research Fellow, Dr Thandeka Moyo, holds a BSc with distinctions in Biochemistry and Microbiology and a BSc (Hons) in Biochemistry from Rhodes University. She went on to obtain a MSc (Med) and PhD in Clinical Science and Immunology from the University of Cape Town where she looked at the mechanisms used by various HIV strains to gain resistance to broadly neutralizing antibody responses. Based at South Africa’s National Institute for Communicable Diseases and affiliated to the University of the Witwatersrand, Thandeka’s postdoctoral research involves understanding the structure and function of HIV neutralizing antibodies by X-ray crystallography. More recently she has added SARS-CoV-2 to her research focus, developing serological assays to measure humoral responses to infection and vaccination. 

Dr Thandeka Moyo, GCRF START Postdoctoral Research Fellow at the National Institute for Communicable Diseases, affiliated to the University of the Witwatersrand, South Africa. ©Diamond Light Source

More about Prof. Penny Moore

GCRF START Co-I, Prof. Penny Moore, is a Reader and DST/NRF South African Research Chair of Virus-Host Dynamics at the University of the Witwatersrand and the National Institute for Communicable Diseases. She holds a joint appointment as Honorary Senior Scientist in Virus-Host Dynamics at the Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu-Natal.  Moore co-directs a team of more than 15 scientists and 10 graduate students, with the team’s research focused predominantly on HIV neutralizing antibodies and their interplay with the evolving virus.

Prof. Penny Moore, GCRF START Co-I, Reader and DST/NRF South African Research Chair of Virus-Host Dynamics at the University of the Witwatersrand and the National Institute for Communicable Diseases, South Africa.
©Diamond Light Source

Footnotes

[1] https://www.unaids.org/en/resources/fact-sheet accessed November 2020

[2] RERKS-NGARM, S., PITISUTTITHUM, P., NITAYAPHAN, S., KAEWKUNGWAL, J., CHIU, J., PARIS, R., PREMSRI, N., NAMWAT, C., DE SOUZA, M. & ADAMS, E. 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. New England Journal of Medicine, 361, 2209-2220; doi: 10.1056/NEJMoa0908492.

[3] The structure was solved by Dr Thandeka Moyo from the NICD, South Africa, and Taylor Sicard and Dr Jean-Philippe Julien from the University of Toronto, Canada.

[4] http://aidsinfo.unaids.org/ accessed November 2020.

[5] The donor was identified by Prof. Penny Moore in research that commenced in 2005.

[6] GORMAN, J., CHUANG, G.-Y., LAI, Y.-T., SHEN, C.-H., BOYINGTON, J. C., DRUZ, A., GENG, H., LOUDER, M. K., MCKEE, K. & RAWI, R. 2020. Structure of Super-Potent Antibody CAP256-VRC26. 25 in Complex with HIV-1 Envelope Reveals a Combined Mode of Trimer-Apex Recognition. Cell Reports, 31, 107488, https://www.cell.com/cell-reports/pdf/S2211-1247(20)30366-1.pdf.

[7] SCARFF, C. A., FULLER, M. J., THOMPSON, R. F. & IADANZA, M. G. 2018. Variations on negative stain electron microscopy methods: tools for tackling challenging systems. JoVE (Journal of Visualized Experiments), e57199. https://www.jove.com/t/57199/variations-on-negative-stain-electron-microscopy-methods-tools-for.

[8] WIBMER, C. K., GORMAN, J., OZOROWSKI, G., BHIMAN, J. N., SHEWARD, D. J., ELLIOTT, D. H., ROUELLE, J., SMIRA, A., JOYCE, M. G., NDABAMBI, N., DRUZ, A., ASOKAN, M., BURTON, D. R., CONNORS, M., ABDOOL KARIM, S. S., MASCOLA, J. R., ROBINSON, J. E., WARD, A. B., WILLIAMSON, C., KWONG, P. D., MORRIS, L. & MOORE, P. L. 2017. Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape. PLoS Pathog, 13, e1006074. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006074.

[9] https://www.unaids.org/en/AIDS_SDGs accessed November 2020.

Taking energy materials to the next level

Investigating lithium-ion battery cathode materials for new generation improvements in sustainable energy solutions

“Ensuring access to affordable, reliable, sustainable and modern energy for all will open a new world of opportunities for billions of people through new economic opportunities and jobs, empowered women, children and youth, better education and health, more sustainable, equitable and inclusive communities, and greater protections from, and resilience to, climate change.”

UN Sustainable Development Goal 7 – Energy[1].

Lithium-ion batteries are used around the world in everyday portable electronics, in electric vehicles as well as in small power grids.  Scientists from the Energy Materials Research Group at the University of the Witwatersrand (Wits), South Africa, study the materials needed to improve the performance, safety, affordability and environmental footprint of lithium-ion batteries in line with important sustainable development goals (SDGs). A range of different cathode materials, as well as battery chemistry is studied to increase the understanding of the materials themselves: how various synthetic routes introduce impurity phases in these materials and how this can be avoided, and what the effects are of intentional doping and co-doping of these materials[2] to explore the impact they have on the structure, performance and impurities formed.

In this work, however, laboratory-based measurements do not often reveal a clear picture of the overall structure of the material, particularly when it contains lower concentrations of impurity phases which are either below the limit at which it can be detected or cannot be resolved from the major phase. It is therefore important to firstly, employ multiple techniques to provide complementary information and secondly, to obtain high-resolution measurements from synchrotron-based techniques which expose far more in terms of other phases in the samples. This is where the GCRF START grant plays a vital part.

“A synchrotron is millions of times more capable than the equipment in our labs in terms of brightness and detail, which makes access through the GCRF START grant to the UK’s national synchrotron, Diamond Light Source (Diamond), so significant.”

GCRF START Co-I, Prof. David Billing[3], Professor in the School of Chemistry and Co-PI of the Energy Materials Research Group at Wits.

Thus far we have data from various synchrotrons for high resolution X-ray diffraction and total scattering, as well as X-ray absorption spectrometry,” says Group Co-PI, Prof. Caren Billing, lecturer and Associate Professor in the School of Chemistry. “The GCRF START grant provides us with these important experimental opportunities, alongside vital skills training and knowledge exchange for building capacity and training emerging scientists in the energy materials field.”

The ultimate aim of the Group is to address global energy, climate, and health challenges which, amongst other aims, includes enabling better “access to clean and safe cooking fuels and technologies and expanding the use of renewable energy beyond the electricity sector, as well as to increase electrification in sub-Saharan Africa[4]. To this end, improved battery storage solutions are one of a creative mix of options the Group is examining, with the help of their collaborators, including the GCRF START grant.

“GCRF START asks us to consider questions in our research around the global challenges like: am I using components that are sustainable? Are we using elements which are abundant, affordable, and environmentally compatible?”, explains Prof. Dave Billing. “In doing so, we are mindful that the economic and social situations in Europe are different to Africa: different resources, different engineers, and different environments where the solutions have to work.  Everything costs energy, fundamentally, and the whole solution has to fit the community – these are the bigger picture questions that START encourages us to ask.”

Rural village in South Africa. Photo credit: Rebekka Stredwick. ©Diamond Light Source

Some of the Group’s early career scientists focus on various lithium metal ion phosphate materials, where they are studied with the idea of using earth abundant metal ions of benefit for cost effective production and materials with a possible impact on local mining opportunities[5] to support local economies. Two of these scientists – Michelle Thiebaut from South Africa and Michelle Nyoni from Zimbabwe – are PhD research students at Wits working on lithium iron phosphate and lithium vanadium phosphate respectively, examining them as cathode materials. Both students are supervised and mentored by Prof. Dave Billing and Prof. Caren Billing, and are part of a growing number of female scientists in the Energy Materials Research Group at Wits. Michelle Thiebaut and Michelle Nyoni describe the aims, techniques and motivation for their projects in the case studies below.

Michelle Thiebaut’s research: studying Lithium iron phosphate for battery cathode materials

“When Michelle Thiebaut first started in the group, she referred to herself as “an analogue girl in a digital world”. Michelle is now the forerunner in the group in processing XAS data and has been the only chemistry student to operate the Mössbauer spectroscopy instrument in the School of Physics at Wits.” – Prof. Caren Billing, Prof. Caren Billing, University of the Witwatersrand.

Being a new researcher in a field such as energy materials is both daunting and exciting because this field is always changing and improving. One needs to change and improve one’s skill set just as quickly but the key to continue is finding one’s motivation. My main motivation is seeing how people in South Africa and in other developing countries are struggling with everyday tasks, especially people in the rural areas – tasks like coming home and doing their homework. These tasks are things many people take for granted but I think it is unacceptable that people should be struggling to get by without proper, cheap and a long-lasting access to clean energy and electricity.

My second motivation is our planet. Every person has an obligation to the planet and to live an environmentally cleaner life. By pushing science in the energy materials field means also pushing towards a greener tomorrow. Trying to break through in this field as a young female is still a bit tough with people questioning your skill set and abilities but I do think we owe former female scientists a great deal of respect for paving the way for us.

My research field is energy materials, specifically investigating the cathode material LiFePO4 found in lithium-ion batteries. My focus with this material is to find a low cost, low energy synthetic route and to possibly improve the performance. LiFePO4 is a naturally occurring mineral but can also be synthesised in a lab. This naturally occurring mineral is not phase pure[6], meaning that the iron is commonly mixed with other metals such as manganese, magnesium and calcium which lowers the electrochemical performance[7]. Cathode materials are the positive electrodes of batteries and host the mobile ions (in this case lithium). The mobile ions are the ions that are removed from the structure when the battery is being charged and when the battery discharges (depletes) the ions are inserted back into the structure[8]. Current cathode materials are not only expensive to produce but also have some safety issues like overheating and short-circuiting associated with them – challenges we want to overcome.

Compared with other cathode materials, LiFePO4 has the advantage of being environmentally friendly, meaning there are no toxic materials presents, relatively cost efficient (no expensive metals/rare earth metals needed to synthesise the material) and is structurally and thermally stable. This means that the structure does not collapse with the removal of the mobile ions and the structure prevents the battery from overcharging as well as overheating, making this material safer to use[9]. However, one of the main disadvantages of LiFePO4 is the electrochemical performance such as the ionic (movement of ions through the crystal lattice)[10] and electronic (the ability to conduct or resist electric current), which are both important properties for cathode material. The mobile ions are restricted to movement through a 1-dimensional channel. Overcoming these problems has been the main focus for most research groups[11].

In my research, the electrochemical performance can be improved by doping with a selection of different metal ions. Inserting small amounts of metal ions into the structure can improve the battery performance differently depending on the metal. For example, nickel improves the stability of the structure and enhances the movement of lithium through the structure; copper improves the conductivity and improves the reversibility of the lithium ions in the structure; and manganese improves the reversibility as well as the stability of the structure.

Exploring materials through multiple techniques and collaborative efforts

To fully understand my material, it is very important to understand how the structure changes with small changes in my synthetic method and it is the collaborative effort between the Chemistry and Physics Departments at my university – the University of the Witwatersrand – which makes this possible, and through access to world class synchrotron sources to utilise the benefits of synchrotron data to further characterise my materials.  Selected samples were sent to the Synchrotron source at Brookhaven National Laboratory (NSLS-II) in the USA and to Diamond Light Source, the UK’s national synchrotron (Diamond), with access to Diamond provided by the GCRF START grant. We have obtained data from synchrotron X-ray diffraction and total scattering, as well as X-ray absorption spectroscopy. Having remote access to state-of-the-art synchrotron equipment in this COVID-19 travel restricted world is heaven-sent as the research can continue even when no travelling is allowed.

Aerial view of the UK’s national synchrotron, Diamond Light Source, located at the Harwell Campus in Oxfordshire, UK. ©Diamond Light Source

To thoroughly characterise my synthesised materials, I have made use of our lab-based diffractometers in the Chemistry department at Wits as well as the Mössbauer spectrometer and the Raman spectrometer in the Physics department.  Mössbauer spectroscopy and Raman spectroscopy are very useful for identifying crystalline (presence of long-range order of the atoms – regular arrangement of atoms over a longer distance) as well as amorphous (only the presence of short-range order of atoms – regular arrangement of atoms but only over a short distance) species in my samples. It is important to identify all the crystalline and amorphous species in the sample as impurities can occur in both forms and could negatively affect the battery performance.

Mössbauer spectroscopy is also useful for identifying the different local iron (Fe) environments present in my sample and to determine the form of iron – (oxidation state – Fe2+ is the desired state in my samples). Raman spectroscopy aids as a structural fingerprint that can be used to determine the identity of one’s material and is also useful in identifying any impurities present that the lab diffractometers could not detect due poor detection limits or due to the phase being amorphous. Synthesised samples can have a mixture of the desired product as well as impurities. There could be multiple sources for the formation of impurities but the most common causes can be either synthesis related (impurities that are formed due to a specific synthetic route) or impurities formed due to sensitivity to air (being exposed to air could cause some small changes like a change in oxidation state of a metal). Impurities can block the channel and subsequently the movement of the mobile ion and negatively affect the performance of the material and the battery.

Michelle Thiebaut, PhD student at the University of the Witwatersrand, South Africa. ©Diamond Light Source

Michelle Nyoni’s research: investigating Lithium vanadium phosphate for improved battery cathode materials

“Michelle Nyoni is a lady who, against many odds, is striving to obtain her PhD in energy materials. Impacted currently by Covid19 travel restrictions, Michelle is normally an ‘out-of-seat’ student who works full time at Chinhoyi University of Technology (CUT) in Zimbabwe and comes to South Africa for laboratory experiments at the University of the Witwatersrand during her teaching breaks as facilities for her research topic at CUT are limited. She has worked hard during her visits here to gather sufficient data so that she can process it when she returns home and brings with her a great deal of positivity and energy on each visit.” – Prof. Caren Billing, Prof. Caren Billing, University of the Witwatersrand.

While working in the farming sector in my home country of Zimbabwe, I realised that we are blessed with abundant renewable sources of energy – wind and solar – yet hindered by the challenge of how to store this energy effectively.  This is where the subject of batteries came into my life and where my current PhD research area fits in. I am investigating lithium vanadium phosphates as cathode materials for lithium-ion batteries. I began my PhD studies part-time, in 2017, at the University of the Witwatersrand in South Africa under the supervision of Prof. Caren Billing and Prof. Dave Billing, while working as a Chemistry Lecturer at CUT in Zimbabwe.

The inspiration for my research is the fact that South Africa is one of the biggest vanadium producers in the world and Zimbabwe is one of the biggest lithium producers in the world. Therefore, if the raw materials are locally available it will hopefully mean reduced cost of battery production. My research is directly linked to Sustainable Development Goal (SDG) 7 concerning affordable and clean energy but by contributing to SDG 7, my research also contributes to achieving SDGs 1-6 and 8-9.

The aim of my research is to do much of the material characterisation by focussing on understanding what is happening at the atomic level, asking questions like: what is happening with the structure and the material? Lithium vanadium phosphate materials have been made but is this synthesis method reproducible? Does it work for upscaling to commercial levels? How do slight changes within the synthesis (preparation method) affect the material? How does adding a dopant manipulate the electrochemical properties of my material?

Lithium vanadium phosphates are potentially effective because their various properties are attractive – they have a high thermodynamic and kinetic stability, and studies have shown they possess the potential to have very good electrochemical properties, which means they will have high specific energy, high working voltage, and good cycle stability. Normally, as batteries get older, the cycling gets poorer and poorer but lithium vanadium phosphate materials have good cycle stability and a lower price tag so they are not as expensive as some of the alternative materials that can be used.   There are also other advantages. Lithium vanadium phosphate materials provide improved safety, phosphates are more environmentally friendly than some other materials[12], and the vanadium contributes to the energy density as well as the voltage of the cells – in fact, our Lithium vanadium phosphate materials can reach voltages of over 4 volts! A key application is in electric vehicles which will benefit from increased length of travel due to the cycle stability of Lithium vanadium phosphate materials and the higher specific energy density, amongst other improvements due to the advantages described above.

The cathode material determines the voltage and capacity of a battery and the cathode in a lithium-ion battery is the positive electrode, which is normally a metal oxide that is responsible for being the source of lithium ions that carry the electric current when a battery is in use (discharging)[13]. There are various components that contribute to the cost of the battery with the cathode material within the battery usually one of the biggest costs, along with the separator[14]. The lithium vanadium phosphate materials that I am working with are cathode materials, therefore if we can source these locally within the SADC region of Africa, which includes 15 member states, this would make them a lot more affordable and accessible – which is the goal I am driving at.

Cutting edge techniques to determine material characterisation and impact

The techniques I will use in my PhD studies aim to test the lithium vanadium phosphate materials in depth so that I can contribute to research that is already available to help find viable products that can be used in Africa. Techniques include powder X-ray diffraction for the phase identification and Raman spectroscopy to enable me to determine the structural finger-print to ensure I am making the same product each time so that when I do change a parameter, the resulting effect will be clear. The GCRF START grant enables us to use the Diamond synchrotron for variable temperature experiments. Therefore, I would want to look out for how the material changes when we vary the temperature. I would also use XAFS– X-ray absorption fine structure spectroscopy at the B18 beamline at Diamond to study the changes in the neighbourhoods of particular atoms.

Another technique is transition electron microscopy – the determination of particle size and the distribution of those particles as well as the general morphology of the fine particles within the material. Additionally, I want to use STA – Simultaneous Thermal Analysis – to look at the thermal stability to ask a series of questions: how stable is my material and what happens under temperature changes? Does it break down or decompose? How does this effect the overall electrochemical properties because when we use these batteries they will heat up? What is the impact, for example, if I were to leave my phone device with a battery using these materials in the sun – how would the warmth of the sun affect it? Would the structure and performance be impacted? Therefore, I would do extensive electrochemical testing which includes cyclic voltammetry and electrochemical impedance tests, amongst others, to ensure the batteries with these materials are viable in the varied environmental conditions found across Africa, including very warm environments.

Michelle Nyoni, part time PhD student at the University of the Witwatersrand, South Africa, and Chemistry lecturer at
the Chinhoyi University of Technology (CUT), Zimbabwe. ©Diamond Light Source

The GCRF START grant: a bridge to sustainable growth and life-changing possibility

“Being part of the GCRF START collaboration has certainly taken our work in energy materials in South Africa to the next level!”

Prof. Caren Billing, University of the Witwatersrand

Many of the Group’s research projects are now at the point where data has been measured and obtained and the next learning curve of how to process the data is underway. Progress has been made, Prof. Caren Billing reports, which, without funding from the GCRF START Grant, would have been an even larger hurdle to overcome.The resultis cutting-edge science and capacity building, knowledge exchange and access to the latest techniques and technology, and a new generation of gifted scientists working towards the shared vision of developing novel, green and affordable energy solutions to inspire life-changing possibility in Africa and beyond.

“This is where the GCRF START grant comes in,” says Prof. David Billing, “it provides that bridge. Yes, there’s a skills gap here in Africa but for me that gap is possibly smaller than others; as long as we are staying current on the XRD side we can transition easily and tackle the more challenging newer techniques – there’s a whole suite of them but that will grow – and START gets us there! This is also what you need to get to the higher impact journals; it also to gets us closer to current answers and future possibilities rather than just ‘the best we can do’ with 30-year-old technology.”

“In terms of energy solutions, take the scenario of load shedding (electricity cuts) which poses a huge challenge across countries in Africa. The thought of being able to go off grid is vital. If you think about a rural village which is cooking using wood or charcoal and lighting in the form of paraffin or candles – this is energy poverty. If you can find a cheap, clean, sustainable source of energy to replace these – that would be life changing!”

GCRF START Co-I, Prof. David Billing, Professor in the School of Chemistry and Co-PI of the Energy Materials Research Group at Wits.
Prof. Caren Billing, Lecturer and Associate Professor in the School of Chemistry and Co-PI in the
Energy Materials Research Group at the University of the Witwatersrand, South Africa. ©Diamond Light Source
Prof. Dave Billing, Professor in the School of Chemistry and Assistant Dean in the Faculty of Science at the University of the Witwatersrand,
South Africa; and Co-PI of the Energy Materials Research Group and GCRF START Co-I. ©Diamond Light Source

Footnotes

[1] United Nations Sustainable Development Goals: Energy for Sustainable Development, https://sdgs.un.org/topics/energy

[2] Introducing small amounts of other metal ions into the structure during synthesis without changing the structure of the material

[3] Prof. David Billing is also Assistant Dean in the Faculty of Science at the University of the Witwatersrand, South Africa.

[4] https://www.un.org/sustainabledevelopment/energy/

[5] https://www.gcis.gov.za/sites/default/files/docs/resourcecentre/yearbook/yb1919-16-Mineral-Resources.pdf

[6] An easy way to picture this is in terms of ores. Generally, an ore will contain a mixture of minerals and hence is not ‘phase pure’.

[7] Information on the natural occurring triphylite (mineral data):  https://www.mindat.org/min-4020.html as well as an electrochemical comparison: https://www.sciencedirect.com/science/article/pii/S0378775301007273?casa_token=zcBRvEsb5SMAAAAA:Lrk2oIjOY-9W73OBWasFIrxQaP7mhWNfQ4HrYjzx7Ib_r95Pq4ix8eORm0IBm29G-izoI18

[8] How Lithium batteries work in: https://www.explainthatstuff.com/how-lithium-ion-batteries-work.html

[9] Advantages and disadvantages of Lithium-iron-phosphate v lithium ion: https://blog.epectec.com/lithium-iron-phosphate-vs-lithium-ion-differences-and-advantages

[10] See Figure 1. How the lithium ions move in a battery in: https://www.spectroscopyonline.com/view/techniques-raman-analysis-lithium-ion-batteries; see also: The channels through which lithium has to move in LiFePO4  in the paper:  Yi, T., Li, X., Liu, H. et al. Recent developments in the doping and surface modification of LiFePO4 as cathode material for power lithium ion battery. Ionics 18, 529–539 (2012). https://doi.org/10.1007/s11581-012-0695-y

[11] Jingkun Li, Zi-Feng. Past and Present of LiFePO4: From Fundamental Research to Industrial Applications. Chem. Volume 5, Issue 1, 10 January 2019, Pages 3-6 (2019), Elsevier. https://doi.org/10.1016/j.chempr.2018.12.012;

V.S.L. Satyavani,A. Srinivas Kumar,P.S.V. Subba Rao.Methods of synthesis and performance improvement of lithium iron phosphate for high rate Li-ion batteries: A review. Engineering Science and Technology 19, Issue 1, March 2016, Pages 178-188. Elsevier. https://doi.org/10.1016/j.jestch.2015.06.002;

Yi, T., Li, X., Liu, H. et al. Recent developments in the doping and surface modification of LiFePO4 as cathode material for power lithium ion battery. Ionics 18, 529–539 (2012). https://doi.org/10.1007/s11581-012-0695-y

[12] Hameed, S.A., Reddy, M.V., Sakar, N., Chowdari, B.V.R. & Vittal, J.J.; Royal Society of Chemistry Advances 2015, 5, 60630-60637

[13] See: ‘The four components of a Lithium battery’: https://www.samsungsdi.com/column/technology/detail/55272.html?pageIndex=1&idx=55272&brdCode=001&listType=list&searchKeyword=

[14] See: Figure 3. ‘Total material costs of all 10 considered cell chemistries plus Panasonic NCA Use Case differentiated in combined CAM cost, anode cost, and secondary material costs’ in: Wentker, M.; Greenwood, M.; Leker, J. A Bottom-Up Approach to Lithium-Ion Battery Cost Modeling with a Focus on Cathode Active Materials. Energies 201912, 504. https://doi.org/10.3390/en12030504

Focus on fungal oxidoreductases for infectious disease drug targets

“The atomic structure of proteins provides an intimate insight into these magnificent macromolecules. This knowledge is crucial to truly understand how they function; whether it is to answer a burning question or to manipulate them – either to enhance if their reactions are desirable, or to inhibit if they are harmful.”   

Prof. Dirk Opperman, University of the Free State, South Africa 

Our research studies the structures of bacterial and fungal oxidoreductases (enzymes) which are possible drug targets to combat infectious disease. The current focus is fungal drug targets for fungal infectious diseases which can be very serious, especially for immune-compromised patients, such as those who are HIV/AIDS positive, organ transplant receivers, patients undergoing chemotherapy, and many more. This research is performed at the University of the Free State’s (UFS’s) Department of Microbial, Biochemical and Food Biotechnology1 and is led by the two Principal Investigators (PIs) of the Biocatalysis group at UFS, Prof. Dirk Opperman, who is a GCRF START Co-Investigator (Co-I), and Prof. Martie Smit. In our laboratory, we have solved the structures of a number of bacterial and fungal enzymes by X-ray crystallography over the past few years, two of the most recent – solving the structures of fungal cytochrome P450 reductase (Dec 2019) and Baeyer-Villiger monooxygenase (Feb 2020) – were assisted by the GCRF START grant and published in the journals Scientific reports2 and Catalysts3 respectively, as described later in this article.   

Dr Carmien Tolmie conducting molecular biology experiments at the University of the Free State’s Department of Microbial,
Biochemical and Food Biotechnology. Photo credit: Rodolpho do Aido Machado. ©Diamond Light Source 

The scale of the Fungal infection and drug resistance challenge  

Currently, there are three classes of anti-fungal drugs that are used to combat infectious fungal disease, but there is an increasing number of drug resistant (and even multi-drug resistant) fungi against these drugs meaning that these pathogenic fungi have become or are becoming resistant to the current medication used to treat patients. If no drugs are effective against invasive opportunistic fungi, the prognosis for immune-compromised patients is very poor, and many people will die. 

“It is therefore imperative that we search for and develop new antifungal compounds to address the growing challenge [of drug-resistance to opportunistic fungi], which impacts countries across Africa, as well as globally. This is especially urgent if the world is to meet the UN’s Sustainable Development Goals of Health and Wellbeing and Food Security by 2030.” 

Dr Carmien Tolmie, University of the Free State, South Africa 

Fungal infections are often underreported and because of this the extent of the situation is not fully known.  In South Africa, this is of particular concern because of our high incidence of HIV/AIDS. For example, one group4 reported that 90 % of HIV/AIDS positive patients on prolonged treatment contract oropharyngeal candidiasis (also called Thrush), an infection caused by a yeast, which is a type of fungus called Candida  (Dos Santos Abrantes, McArthur and Africa 2014). However, this is only one statistic, and the problem is much wider. In another example, Cryptococcal meningitis is a deadly brain infection caused by the soil-dwelling fungus Cryptococcus. Worldwide, nearly 220,000 new cases of cryptococcal meningitis occur each year, resulting in 181,000 deaths, most of which occur in sub-Saharan Africa (CDC, 2020). 

Using the powerful beams of Diamond’s synchrotron light to determine protein structures 

Our quest to find new drug targets involves examining the chemical processes that happen in the fungal cell in order to keep the cell alive. We choose an enzyme – a special type of protein involved in these processes – which will be a good target for anti-fungal medication. The experiments we do to produce our protein crystals include molecular biology, protein expression, purification and crystallisation.  We clone the gene that encodes the enzyme and insert it into a suitable host to produce the protein, such as the bacterium Escherichia coli, by protein expression methods. Escherichia coli is easy to manipulate and inexpensive to culture in large volumes. We then isolate the enzyme by protein purification methods which exploit the physicochemical properties of the enzyme to separate the target from the host proteins, and crystallise it before we examine it by X-ray crystallography.  

Dr Carmien Tolmie purifying proteins using an AKTA chromatography system at the University of the Free State’s Department of Microbial,
Biochemical and Food Biotechnology, South Africa. Photo credit: Rodolpho do Aido Machado. ©Diamond Light Source 

We use an in-house crystallisation robot at UFS to prepare the crystallisation trials and we regularly collect crystal diffraction data via remote access at the macromolecular beamlines of the UK’s national synchrotron –  Diamond Light Source (Diamond). This is a great help as we can control the beamline equipment from our offices, so we don’t incur the expense of travelling to the UK to use synchrotron techniques essential for our research. While we have X-ray diffractometers at the University of the Free State, they are not nearly as powerful as the beamlines of the Diamond synchrotron. Diamond’s beamline hardware has been developed to such an advanced stage that data collection can proceed very rapidly, enabling us to collect data much faster (minutes) than at our home sources (days). This high throughput is essential when searching for and identify tiny molecules that might potentially bind to the protein and possibly act as inhibitors, as large libraries of fragments must be screened. 

“The brilliant light generated by Diamond (10 billion times brighter than the sun!)  enables us to determine the structure of the proteins to extremely high resolutions, as well as structures from small or weakly diffracting crystals that we cannot study with our own laboratory techniques.” 

Dr Carmien Tolmie, University of the Free State, South Africa 

 We use the protein structure to search for small molecules that will bind to the enzyme and possibly stop it from working (act as inhibitors). In Biocatalysis, knowledge of the protein structure can identify ways in which one can change, or mutate, the enzyme to perform the specific reactions desired.  If the protein is a drug target, the structure can be used in Structure-Based Drug Discovery to develop new medications. This process can also be used for other applications like developing new pesticides for agriculture. Therefore, the next step in the research process is to use fragment screening methods  to identify lead compounds that can be further developed into inhibitors, thus helping develop a next generation drug. The fragment screening is done in collaboration with Prof. Frank von Delft and the XChem group on the I04-1 beamline at Diamond through the GCRF START grant and will be undertaken once Covid19 travel restrictions are lifted.  

Beamline I04-1 Experimental Hutch – Sample changer and sample environment at the UK’s national synchrotron, Diamond Light Source. ©Diamond Light Source Ltd 

Solving the structures of fungal cytochrome P450 reductase and Baeyer-Villiger monooxygenase  

Recently, we were able to solve and gain new insights into the structures and mechanisms of the fungal cytochrome P450 reductase (CPR) from Candida tropicalis and the Baeyer-Villiger monooxygenase BVMOAFL210 from Aspergillus flavus, research that was made possible by the GCRF START grant. The results were published in the journals Scientific reports and Catalysts respectively, and the research on the CPR was done in collaboration with scientists from the University of Cape Town5, who are also part of the START project. The publications were co-authored by START PDRAs Ana Ebrecht (first author on CPR paper) and Rodolpho do Aido Machado (co-author on BVMOAFL210).  

The CPR plays a pivotal role in primary and secondary metabolism of different species, from bacteria to animals and plants. In fungi, it supplies electrons to enzymes that are vital for the survival of the organism. The CPR mechanism is complex and involves conformational changes that need to be finely tuned to optimise the process. The structural characterisation of the CPR helps to understand how this process occurs and what are the differences with the human homolog, opening the possibility to use it as a drug target. The structure and mutation data of BVMOAFL210 allowed us to better understand the role of the amino acid at a specific position in the enzyme, in terms of regioselectivity (the position in the substrate where the oxygen atom is inserted) as well as the sulfoxidation (the number of oxygen atoms inserted in a sulfur-containing compound). This residue may be used in future studies for directed evolution experiments to evolve the enzyme to catalyse a desired reaction.  

In order to achieve the results, we first needed to produce pure protein. The proteins were crystallised by the vapour-diffusion method with the Douglas Oryx Nano crystallization robot located in our crystallography lab in our department. In these experiments, a library of 384 crystallisation conditions were screened and a few conditions yielded crystals. These crystals were cryo-cooled and shipped at liquid nitrogen conditions in a specialised container to Diamond where we collected data on the macromolecular crystallography beamlines through remote access. We processed the data and solved the structure with programs from the CCP4 suite of macromolecular data processing software. The proteins were characterised further by investigating their kinetic properties with several spectrophotometric assays using a UV/Visible light spectrophotometer.  

Dr Carmien Tolmie using a Douglas Oryx Nano crystallisation robot to set up protein crystallisation trials at the University of the Free State’s Department of Microbial,
Biochemical and Food Biotechnology, South Africa. Credit Rodolpho do Aido Machado. ©Diamond Light Source 

For BVMOAFL210, we created mutations at a specific position and determined how these mutations alter the biocatalytic profile of the enzyme using whole-cell biotransformation experiments, followed by Gas-Chromatography Mass Spectrometry (GC-MS) analyses. In terms of the fungal cytochrome P450 reductase (CPR), the next steps will be to use the CPR for fragment screening to gain further, more detailed insights. This method uses protein crystals of the target enzyme to identify small molecule fragments that bind to the enzyme. These fragments are then elaborated into larger molecules with higher potency, which will hopefully not only inhibit the specific enzyme, but also the growth of pathogenic fungi.  

Benefitting from increased research capacity through the GCRF START grant 

The grant has contributed greatly to the research capacity of the Biocatalysis and Structural Biology Research group, making it possible to appoint a START Postdoctoral Research Assistant (PDRA) who focused on structural biology research, the research itself partially funded by START. START also helped to develop the skills of the researchers in the group by funding workshops, as well as workshop attendance, and a research exchange of the START PDRA to the UK in 2019. In addition, START has introduced us to world-class scientists at Diamond and other institutions who we can consult if we need advice on our experiments.  

“With this sharing of knowledge, capacity building and cutting-edge research enabled by the GCRF START grant, it is our fervent aim to make a lasting, positive impact in terms of sustainable health, well-being and food security solutions now, and well into the future.”

Dr Carmien Tolmie, University of the Free State, South Africa 

Click here to read more about the UN’s Sustainable Development Goals  

Acknowledgements  

We would like to thank Prof. Trevor Sewell from the START Centre of Excellence at the University of Cape Town’s Aaron Klug Centre for Imaging and Analysis for the pivotal role he has played both in GCRF START and Structural Biology in South Africa. The START Centre of Excellence is a collaborative, shared resource where participants in the START programme can access everything they need to get started with their research, such as advice and the necessary equipment which may not be available in their own laboratories elsewhere. This also includes the technological support and expertise to access the UK’s national synchrotron – Diamond Light Source – through the GCRF START grant (such as support with sample preparation, shipping, and remote access experiments). 

Dr Carmien Tolmiehttps://orcid.org/0000-0001-9095-3048 

Dr Carmien Tolmie, researcher in the Department of Microbial, Biochemical and Food Biotechnology
at the University of the Free State, South Africa. ©Diamond Light Source 

Prof.Dirk Oppermanhttps://orcid.org/0000-0002-2737-8797 

Prof. Dirk Opperman, researcher in the Department of Microbial, Biochemical 
and Food Biotechnology at the University of the Free State, South Africa. ©Diamond Light Source 

Footnotes

[1] Research in the Department falls broadly into three main areas: (i) production of safe and novel food products, (ii) biocatalytic production of chemicals or bioremediation of chemical pollution, and (iii) improvement of human and animal health. Our Biocatalysis Group focuses heavily on biocatalysis which involves the use of one or more enzymes, either as cell-free enzymes or enzymes in whole cells, to convert a substrate into a value-added product. This includes converting alkanes, alcohols, fatty acids or monoterpenes into value added building blocks of pharmaceuticals, bio-plastics, cosmetics, flavours and/or fragrances.

[2] Ebrecht AC, Van der Bergh N, Harrison STL, Smit MS, Sewell BT and DJ Opperman (2019). Biochemical and structural insights into the cytochrome P450 reductase from Candida tropicalis. Scientific Reports 9:20088. doi: 10.1038/s41598-019-56516-6: https://www.nature.com/articles/s41598-019-56516-6

[3] Tolmie C,Do Aido Machado R, Ferroni FM, Smit MS and DJ Opperman (2020). Natural variation in the ‘control loop’ of BVMOAFL210 and its influence on regioselectivity and sulfoxidation. Catalysts 10(3): 339. doi: 10.3390/catal10030339: https://www.mdpi.com/2073-4344/10/3/339

[4] Dos Santos Abrantes PM, McArthur CP, Africa CWJ. Multi-drug resistant oral Candida species isolated from HIV-positive patients in South Africa and Cameroon. Diagn Microbiol Infect Dis 2014;79:222–7.

[5] https://www.news.uct.ac.za/article/-2020-03-12-sa-women-take-the-lead-in-structural-biology

New generation solutions to Neglected Tropical Diseases and Nosocomial ESKAPE Infections

“Africa is a bank when it comes to diseases. The beauty of the GCRF START grant is that it offers us the opportunity to deal with these challenges in Africa, while enabling international collaboration and access to amazing synchrotron facilities with world class results.”

Dr Ikechukwu Anthony Achilonu, Protein Structure-Function Research Unit (PSFRU), University of the Witwatersrand, South Africa 

Neglected Tropical Diseases (NTD’s) and Antimicrobial Resistance (AMR) are major challenges threatening the world’s sustainability and development efforts across the spectrum of the UN’s Sustainable Development Goals, causing millions of deaths each year1. My name is Dr Ikechukwu Achilonu and I work in the PSFRU at the University of the Witwatersrand, in South Africa, where I focus on rational design and discovery of new generation anthelminthic and anti-bacterial drug targets to tackle NTD’s and Nosocomial infections by ESKAPE2 pathogens.  

Dr Ikechukwu Anthony Achilonu, Protein Structure-Function Research Unit (PSFRU), School of Molecular and Cell Biology,
Faculty of Science, University of the Witwatersrand. ©Diamond Light Source 

To design inhibitors to serve as potential drugs against these pathogens, high-resolution 3D protein structures are needed to test the ability of certain drugs to interact with and inhibit the properties of the proteins being studied. This is made possible with the GCRF START grant which provides us with access to state-of-the-art synchrotron techniques, equipment and expertise at the UK’s world class national synchrotron, Diamond Light Source (Diamond), enabling the X-ray diffraction from protein crystals generated in our research. The GCRF START grant is currently our only conduit to the UK to enable us to conduct these ultra-high-quality experiments.  

My recent research success is solving the Schistosomiasis joponicum Glutathione S-transferase (GST) enzyme has been recently published and is a tribute to the value of collaborating with Diamond and GCRF START. The solved structure is among the crystal structures with the highest resolution in the global protein database for this enzyme with two different ligands. Of the authors, Dr Ramesh Pandian is a GCRF START postdoctoral fellow (Host: Prof. Yasian Sayed), and Dr Sylvia Fanucchi, Prof. Yasien Sayed, Prof. Heine W. Dirr, and I – Dr Ikechukwu Achilonu – are collaborators on the GCRF START grant.

The resulting publication emphasises the need to exploit the unique structural diversity between Schistosoma GST and other human GSTs for a rational approach to design new generation anthelminthics. This research, outlined later in this article, will pave the way for the next exciting step in the structural validation of druggable targets for this debilitating and life-threatening Schistosomiasis (Bilharzia), a NTD which infects large numbers of people across several countries each year4.  

Aerial view of Diamond Light Source, which is the UK’s national synchrotron. ©Diamond Light Source 2020

The extent of the challenge: NTD’s and ESKAPE pathogens 

AMR is described by the World Health Organization5 as “one of the biggest threats to global health, food security, and development today…compromising our ability to treat infectious diseases, as well as undermining many other advances in health and medicine.  

NTD’s are a diverse group of communicable diseases caused by a variety of pathogens prevalent in tropical and subtropical conditions, such as viruses, bacteria, protozoa and parasitic worms (helminths). More than a billion people in over 149 countries are impacted by NTD’s with huge costs to developing economies of billions of US dollars every year. My research on NTD’s focuses on Schistosoma and Wuchereria (Wuchereria is a human parasitic worm (Filariworm) that is the major cause of lymphatic filariasis) – both of which display AMR. Populations living in poverty, without adequate sanitation and in close contact with infectious vectors and domestic animals and livestock, are those worst affected by NTD’s6.  

The six nosocomial ESKAPE pathogens that exhibit multidrug resistance and virulence are often acquired in health care settings like hospitals (Health Care Acquired Infection/HAI) and feature on the World Health Organization’s ‘Priority Pathogens list’7. The persistent use of antibiotics has provoked the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria, which can make even the most effective drugs ineffective. These bacteria have built-in abilities to find new ways to become resistant and can pass on genetic materials which enable other bacteria to become drug resistant. Like NTD’s, this situation is made worse by limited or non-existent infection prevention and control (IPC) programmes, combined with an inadequate water supply, poor sanitation, and a weak hygiene infrastructure in health facilities, which make the burden of HAI several fold higher in low- and middle-income countries than in high-income ones.  

World class results! Solving the Schistosomiasis japonicum GST enzyme  

Over the past three years, I have been working on the Schistosoma GST enzyme, which is a protein that is involved in detoxification of foreign molecules in the Schistosome parasite that causes the disease Schistosomiasis/Bilharzia. This enzyme very critical to the survival of the parasite. If the enzyme is not functioning properly, the parasite cannot move from egg stage to lava stage. Therefore, if the enzyme could be effectively inhibited, one would have a drug against the parasite. 

In my research, I used a molecule which is a natural product found in pomegranate juice called Ellagic Acid. One of my MSc. students, Ms Blessing Akumadu, carried out the study I had conceptualised, showing that this molecule can inhibit the activity of the Schistosoma GST by 66%. The concept was based on an earlier study that ellagic acid and its derivative inhibits Plasmodium GST (duly acknowledged in the forthcoming paper).   To be more confident about making extrapolations from the structure of the Schistosoma japonicum GST enzyme, we needed to see that our wet lab results had meaning in 3D high resolution. Using the I038 and I04 beamlines at Diamond, we were able to solve the structure of the 26 kilodalton GST at 1.53 Å resolution and were amazed when we saw the results! We had achieved the highest resolution of any of the several variants of the Schistosoma japonicum GST in the global protein database!   

What we observed is the Schistosoma 26 kilodalton GST enzyme (Sj26GST), which is a dimeric protein (it has two similar subunits joined together) with an interface where the subunits are joined. The unique thing about this protein is that the dimer interface can be exploited for drug interaction, unlike other GST’s in humans. The results have enabled us to see where the Ellagic Acid molecule (the potential drug) can bind to the protein. In this parasite we only have two classes of GSTs (in humans there are several) – the 26 kilodalton (kDa) GST and the 28 kDa GST. Having studied the 26 kDa GST, we are now currently working on the 28 kDa GST across the three species of the Schistosoma parasite that infect humans (mansoni, haematobium and japonicum) to see if the Ellagic Acid molecule can also inhibit the 28 kDa GST of the Schistosoma parasite.  

The implications of these results mean we can go on and redesign this potential drug molecule because we can see the type of interaction this drug is making with the protein in the 3D images. The more the drug interacts, the stronger the binding. Therefore, we can start to redesign this drug for better binding so that the binding and the efficiency is improved beyond the 66% to maybe 75% or even to 99 % in order to completely shut down the activity of the enzyme at a very low amounts of the drug. Without the high-resolution 3D crystal structure solved using Diamond, we could not do this. We can now exploit this interaction by adding a bond here or a functional group there to improve the strength of the interaction and potency of the drug, and produce new high resolution structures until we have a drug that shuts down the enzyme and produces a new drug target to which the Schistosoma parasite is not resistant to. 

The benefit of studying multiple NTD and ESKAPE pathogens simultaneously – the case of nicotinamide adenine transferase enzyme in klebsiella pneumonia and Enterococcus faecium. 

In my experience, the techniques one applies to studying one protein can also be applied to the others, which is why I study a range of druggable proteins from several human NTD and ESKAPE pathogens simultaneously. For example, my students and I are examining the nicotinate-nucleotide adenylyltransferaseenzyme which is important in the redox metabolism in both klebsiella pneumonia and Enterococcus faeciumKlebsiella pneumonia is a Gram-negative bacterium resistant to multiple drugs and increasingly resistant to most available antibiotics (it has a thin cell wall surrounded by a protective cell membrane which makes it harder for antibodies to penetrate). In health care settings, patients who require devices like ventilators or intravenous catheters, and patients who are taking long courses of certain antibiotics are most at risk for Klebsiella infections.   

The Gram-positive Enterococcus faecium is a human pathogen that causes nosocomial bacteremia, surgical wound infection, endocarditis, and urinary tract infections (it has thick cell walls but no outer membrane). Studying both simultaneously has led to us noticing that the Gram-negative function of the nicotinamide adenine transferase enzyme performs differently from the Gram-positive function of the same enzyme which is exciting because this enables us to infer structural and functional differences between this enzymes from different bacteria that are supposed to perform similar activities in these bacterial pathogens. This has serious implication in the design of a broad-spectrum antibiotics that will target this enzyme in different organisms.  

The process of solving protein structures – systematic integration of multi-facted ‘new generation’ theoretical and empirical studies 

The approaches we use in the research outlined above are multi-faceted and multi-disciplinary involving the integration of sophisticated ‘new generation’ theoretical and empirical studies and, as already indicated, a range of cutting-edge equipment. To design drugs against disease pathogens, the three-dimensional architecture of the druggable protein is needed. Druggable proteins are proteins that are key to the survival of the pathogen which enables it to replicate inside the human host, either intracellularly or extracellularly. In the case of bacteria, for example, they need to manufacture their own energy and without the enzyme (protein) that makes this possible, the bacteria would not survive. Therefore, if we know the protein’s DNA sequence, we should be able to predict that we can synthesis this protein and develop the druggable target.  

To elucidate the crystal structure of these druggable proteins it is possible to use home X-ray sources. Ultimately, however, there is a major advantage of using a powerful synchrotron light source like Diamond over most home X-ray sources because the amount of inferences made from a protein crystal with resolutions in the range of 1.3 – 1.7 Å is unprecedented. Such crystals can be further studied using computational algorithms and can also serve as models for replacement when solving the crystal structure of a protein that is very similar in structure. 

In order to generate the recombinant proteins that are druggable targets, we need large amounts of these proteins. For this we use E. coli bacteria like a factory to synthesize our target proteins and carry out a rigorous ‘quality assurance’ process to ensure the proteins we make are pure, stable and homogenous for successful crystallisation experiments.  If the protein is an enzyme, it should be able to catalyze the same function it does naturally in an organism (in vivo), inside a test tube (in vitro), which means that whatever we send off to Diamond for X-ray diffraction experiments, we need to be sure that the biological function is intact. To do this, the proteins are assayed (measured) for catalytic activity in vitro (if they are enzymes) and are further characterised using several biophysical techniques to test their structure and the function to the point we are satisfied that the proteins are active, properly folded and can perform the function they are supposed to perform properly (biological activity is maintained).  

We purify the proteins to homogeneity using an ensemble of protein chromatography techniques. For ‘quality assurance’, we check the secondary structure content (Far-UV circular dichroism), the tertiary structure (intrinsic and extrinsic fluorescence spectroscopy); and the quaternary structure (using size exclusion HPLC). Every physical interaction between two biomolecules generates heat or absorbs heat as part of the function of the protein to interact with another molecule (molecular recognition). Therefore, to estimate the thermodynamics of protein-ligand interaction (interaction ability), we use isothermal titration calorimetry.  

Once we are satisfied that the proteins are of good quality and quantity, we begin protein crystallisation. I like to use the analogy that structural biologists are akin to photographers and using X-ray diffraction is like taking a snapshot of the protein which has been forced to assemble as repeating units in a crystalline form. There are several approaches we use. We can either conduct crystallisation manually, or we can do it robotically using our in-house Douglas Instrument Oryx 8 crystallography robot for high throughput protein crystallisation.  

To solve the protein structure – sometimes in complex with potential drugs – X-ray light is invaluable. However, X-rays have very short wavelengths and if you keep bombarding a protein crystal with X-rays, it will heat it up and gradually fall apart because of X-ray damage. By changing the protein from solution to a crystal, and then embedding it in a liquid able to resist the intense bombardment of X-ray (cryo-protection) by freezing it at the temperature of liquid nitrogen (-196 degrees Celsius), the sample stays stable and the X-rays will last 100x longer in the X-ray beam! Our crystal samples are therefore sent to Diamond in special containers which keep the samples crystals frozen. GCRF START makes it very easy to ship our crystals to the UK without lots of red tape. We use DHL and we have an easy system which means it only takes a few days to get to Diamond ready for the experiments; the GCRF START grant helps this process at no cost to us.  

We can use an X-ray diffractometer such as a small in-house diffractometer to solve the protein structure, or the ultimate technique for X-ray crystallography – a massive synchrotron like Diamond! Diamond’s I03 and I04 beamlines are used to fire X-rays onto our crystal samples. The X-ray will be scattered based on the structure of the protein/molecule (X-ray diffraction). Then, using mathematical modelling and other techniques, the 3D structure of the protein is resolved, either on its own or in a complex with a drug or other biomolecules such as DNA.   

My theoretical (computational) studies involve several molecular modelling techniques to model the atomistic behaviour of our proteins of interest, especially how the dynamics of these proteins are altered upon drug binding. We also screen various in silico (virtual) libraries (including PubChem, ZINC and Drug Bank databases) for potential hits that we further extend to the theoretical studies, including high-throughput virtual screening, induced-fit ligand docking, molecular dynamic simulation and free binding energy calculations. 

Sometimes, however, the process provides a few surprises, which was the case in our journey to solving the structure of the Schistosoma japonicum GST enzymeWe usually send our crystals to Diamond at the same time as the other groups in South Africa because we have the same slots for data collection. Two weeks before we were supposed to ship our crystals, we found that none were ready yet to ship.  This wasn’t good. Therefore, to hurry things along, I asked one of my MSc students, Jessica Olfsen, who knows the crystallization robot very well to generate the crystals we needed within one week. This she did, but they were very ugly looking crystals! I did not think we would get any data out of these crystals but at least we had crystals to send. I so doubted their usefulness that when I collected the data, I didn’t even want to look at it, let alone solve the protein structure. In my disappointment I asked one of our new post-docs (Dr Pandian) who needed a task to look at it. The resulting 3D high resolution structure blew him away! When we both looked at it, we saw just how beautiful it was. I had written the paper already, but it had needed more experimental data for publishing so now I had it! The structure was solved at unprecedented resolution and the paper was accepted for publishing. 

As a structural biologist, drug discovery and protein biochemistry are completely intertwined and multidisciplinary in the sense that I cannot do without a synthetic chemist (organic chemist). These are the people who will synthesize the drug. For example, once we add the extra functional groups, it will improve the binding capacity of the drug. The synthetic chemist will then go into the laboratory, resynthesize the drug before I will return to my lab and see if the inhibition improves. When an improvement in the inhibition is apparent, we go through the whole process again: recrystallize the protein with the proposed drug, and then use Diamond again to produce another high-resolution 3D structure of the re-modelled drug molecule and protein interaction.  

“This whole drug discovery process can take some time, so it is vital that we have access to world class synchrotron facility like Diamond for the lifetime of this research we until we have the results needed to tackle the NTD’s and ESKAPE pathogens which impact countless lives.” 

Dr Ikechukwu Anthony Achilonu, Protein Structure-Function Research Unit (PSFRU), University of the Witwatersrand, South Africa 

Read more here about the UN’s Sustainable Development Goals. 

More about Dr Achilonu 

Dr Ikechukwu Anthony Achilonu is Senior Researcher and the Interim South African Research Chair (NRF/SARChI) in Protein Biochemistry and Structural Biology at the Protein Structure-Function Research Unit (PSFRU), School of Molecular and Cell Biology, Faculty of Science, University of the Witwatersrand in South Africa.  

Publications: https://pubmed.ncbi.nlm.nih.gov/?term=Achilonu+I&cauthor_id=30183110 

Footnotes:

[1] Transforming our world: the 2030 Agenda for Sustainable Development – A/RES/70/1. New York, USA: United Nations; 2015.

[2] ESKAPE is an acronym for the group of bacteria, encompassing both Gram-positive and Gram-negative species, made up of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. 

[3] Authors: Blessing O. Akumadu, Ramesh Pandian, Jessica Olfsen, Roland Worth, Monare Thulo, Tshireletso Mentor, Sylvia Fanucchi, Yasien Sayed, Heini W. Dirr, Ikechukwu Achilonu. Title: Molecular basis of inhibition of Schistosoma japonicum glutathione transferase by ellagic acid: insights into biophysical and structural studies. Publication: Molecular and Biochemical Parasitology (2020);Elsevier. https://doi.org/10.1016/j.molbiopara.2020.111319

[4] https://www.who.int/news-room/fact-sheets/detail/schistosomiasis

[5] World Health Organization Global action plan on AntiMicrobial Resistance (AMR). Geneva: World Health Organization; 2015.  https://www.who.int/antimicrobial-resistance/global-action-plan/en/

[6] https://www.who.int/neglected_diseases/resources/9789241515450/en/

[7] https://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed

SA women take the lead in structural biology

A team of researchers from the University of Cape Town (UCT) and University of the Free State (UFS) has achieved a remarkable feat in the field of structural biology by determining the structure of an enzyme that could be a key component in producing valuable commodity chemicals in greener, sustainable processes.

Known as cytochrome P450 reductase (CPR), the enzyme has received much attention – not only for its ability to perform difficult chemistry, but also for its role as drug target.

“The task was enormous,” said team member Naadia van der Bergh, a PhD student in UCT’s Centre for Bioprocess Engineering Research (CeBER). “CPR is a massive enzyme. It contains 679 amino acids and there were two molecules in the asymmetric unit. Added to that, our initial structure was determined and solved on the basis of a low-resolution map. Interpreting this structure was a truly gruelling effort.”

The results of the research were published on 27 December 2019 in the journal Scientific Reports (9:20088) by the Nature Publishing Group.

International exposure

With support from the Global Challenges Research Fund’s (GCRF) Synchrotron Techniques for African Research and Technology (START) programme, the team was given the opportunity to conduct parts of their research at the Diamond Light Source synchrotron in Oxfordshire in the United Kingdom (UK)

Read more on the University of Cape Town News.

Image: Ana Ebrecht (left) and Naadia van der Bergh are part of a team of researchers from UCT and UFS that achieved a remarkable feat in the field of structural biology.