51情报站

Biological evaluations of N-based Ligand frameworks and their corresponding Re(I)-Tricarbonyl complexes

University of the Western Cape supervisor: Dr. Orbett Alexander
51情报站-Kansas City host: Dr. Justin Randall
UMSAEP coordinator: Prof. Rodney Uphoff
Visiting researchers/students: Ms. Brithney Snymann, Ms. Boitumelo Nthapo

The one-month research visit of two MSc students from the University of the Western Cape (UWC) to the laboratory of Prof. J. Randal at the 51情报站-Kansas City (UMKC) provided a highly valuable international training opportunity that significantly enhanced both their scientific development and the broader complementary collaborative relationship between the two institutions.

A major benefit of the visit was the students' exposure to a specialised biological research environment that complements the synthetic chemistry focus of their MSc projects at UWC. Before being permitted to conduct experiments independently, the students were required to undergo formal laboratory safety and technical training, which resulted in certification for biological laboratory work. This experience exposed them to internationally recognised standards of laboratory practice, biosafety procedures, data management, and experimental design within a biological setting.

The mobility programme further enabled the students to acquire hands-on experience in biological assays and advanced experimental techniques that are not routinely accessible within their home laboratory environment. The complementary expertise available at UMKC allowed the students to strengthen the application component of their research, bridging the gap between compound synthesis and biological evaluation. Such interdisciplinary training is essential for developing well-rounded scientists capable of translating fundamental chemical discoveries into practical biomedical applications.

Beyond the technical skills gained, the experience provided significant personal and professional growth. Living and working in a foreign country exposed the students to different scientific cultures, research philosophies, and problem-solving approaches, while simultaneously strengthening their confidence, independence, adaptability, and communication skills. The opportunity to interact with international researchers and students also expanded their professional networks and broadened their perspectives regarding future research and career opportunities.

The exchange aligns strongly with several United Nations Sustainable Development Goals (SDGs), particularly SDG 3 (Good Health and Well-Being) through research directed toward the development and biological evaluation of novel therapeutic agents aimed at addressing global health challenges; SDG 4 (Quality Education) through advanced skills development, experiential learning, and international research training; SDG 8 (Decent Work and Economic Growth) through the cultivation of highly skilled graduates equipped for the knowledge economy; SDG 9 (Industry, Innovation and Infrastructure) through the promotion of scientific innovation, technology development, and research capacity building; and SDG 17 (Partnerships for the Goals) through meaningful international collaboration, knowledge exchange, and the strengthening of sustainable institutional partnerships.

Importantly, the successful placement of the students has further strengthened the growing partnership between UWC and UMKC.

The socioeconomic relevance of this project lies in its potential to contribute to the development of new antimicrobial agents at a time when antibiotic resistance is a growing global health and economic burden. Infectious diseases caused by resistant bacteria place significant strain on healthcare systems, particularly in low- and middle-income countries, where treatment options are limited and hospital stays are prolonged, increasing overall healthcare costs. By investigating novel metal-based compounds with antibacterial activity, this work supports the long-term goal of expanding the pipeline of effective therapeutic agents.

From a broader perspective, the project also contributes to skills development and capacity building through advanced training in synthetic chemistry, bioinorganic chemistry, and microbiological evaluation. This enhances human capital development and prepares highly skilled graduates who can contribute to both academic and industrial sectors. Such training is particularly important in strengthening local scientific expertise and reducing reliance on external technologies. In addition, the international collaboration embedded in this work promotes knowledge exchange, research networking, and access to advanced methodologies, which are essential for scientific innovation and competitiveness. This aligns with broader socioeconomic development goals by fostering partnerships that enhance research output and innovation capacity.

Overall, the project supports improved public health outcomes, contributes to economic sustainability by addressing antimicrobial resistance, and strengthens scientific capacity and collaboration within and beyond the region.

Aims

The aim of this study is to design and synthesize an array of pyridyl-based ligands and subsequently coordinate them to rhenium(!) metal centres to generate a series of Re(I) complexes, followed by comprehensive structural and photophysical characterization. The work further seeks to explore their electronic properties using advanced spectroscopic techniques, including flash photolysis studies, in order to gain insight into their excited-state behaviour, stability, and reactivity. Ultimately, this research aims to establish clear structure-property relationships that will guide the rational development of Re(I)-based systems with enhanced photophysical performance and potential applicability in biological and therapeutic contexts.

Objectives

The objectives of this study are centred on the design, synthesis, and characterization of pyridyl-based ligand frameworks and their corresponding rhenium(!) metal complexes. Following successful preparation and structural elucidation of these compounds, the study further extends to biological evaluation through a series of in vitro assays aimed at investigating their potential antibacterial activity. Collectively, these objectives aim to correlate structural features with both biological performance and photochemical properties, ultimately guiding the development of functional metal-based compounds with potential antimicrobial applications.

• Antimicrobial Evaluation - MIC Assay: To assess the antimicrobial potential of all synthesized compounds by determining Minimum Inhibitory Concentration (MIC) values against a panel of clinically relevant bacterial strains.
• Cytotoxicity Assessment - HEK293 Cell Assay: To evaluate the cytotoxicity of all compounds against the Human Embryonic Kidney 293 (HEK293) cell line, using a propidium iodide (Pl)-based fluorescence assay, to determine their selectivity and preliminary safety profiles.
• Hemolytic Activity Assessment - Red Blood Cells (RBC's) Hemolysis Assay: Determine the hemolytic activity of all compounds against human red blood cells (RBC's) at a concentration of 256 碌g/mL to identify compounds with acceptable hemocompatibility for potential systemic application.
• In Vivo Acute Toxicity and Efficacy Studies: To progress selected lead compounds into preliminary in vivo studies using the Galleria mellonella infection model, determining the lethal dose (LDso) and evaluating therapeutic efficacy against Methicillin-Resistant Staphylococcus Aureus (MRSA) USAl 00 infection benchmarked against Vancomycin.

Results

Biological studies results (student 1)

Minimum Inhibitory Concentration (MIC) Assay

The Minimum Inhibitory Concentration (MIC) assay is a fundamental microbiological method used to determine the lowest concentration of a compound that completely inhibits visible microbial growth. This assay was performed to evaluate the antimicrobial potential of all synthesized ligands and metal complexes against a panel of clinically relevant bacterial strains.

The assay was performed using a standard 96-well plate. Overnight bacterial cultures were first prepared in Mueller-Hinton (MH) or Roswell Park Memorial Institute (RPMI) media. The plate was set up and the compounds and controls were added to the plate. The compounds were then serially diluted across the wells of the plate and inoculated with the bacterial suspensions. The plate was sealed with parafilm and incubated overnight at 37 掳C. Following incubation, the wells were inspected for turbidity, where the absence of visible growth indicated inhibition and the presence of turbidity indicated bacterial growth. The MIC value was recorded as the lowest concentration at which no visible growth was observed.

To validate the assay, two controls were used such as:

• Negative control consisting ofDMSO and MH media without bacteria.
• Positive control consisting of Carbenicillin with MH media and bacteria.

Table 1 shows that most of the compounds (1-12) dissolved well in DMSO, except for compounds 11 and 12, which only dissolved partly, likely because they are less soluble and less compatible with the solvent. The compounds were then tested against two bacteria: a Gram-negative strain (E. Coli W3110) and a Gram-positive strain (B. subtilis PY79). Only compound 7 showed activity against E. coli, while compounds 4 and 7 were active against B. subtilis, with compound 7 showing stronger activity. Based on these results, compounds 4 and 7 were selected for further testing against additional bacteria. 

 Table 2 shows the antibacterial results for compounds 4 and 7 tested against four bacteria: two Gram-negative (E. coli ATCC 25922 and A. baumannii AB5075) and two Gram-positive (S. epidermidis ATCC 12228 and S. aureus USA100). Vancomycin was used as a reference antibiotic for comparison. The compounds were tested in two growth conditions (MH and RPMI media), with A. baumannii assessed after 72 hours in RPMI due to slow growth. 

Compound 7 performed better than compound 4 across all bacteria tested. For the Gram-negative strains, compound 7 showed moderate activity in MH media but much stronger activity in RPMI media, while compound 4 showed little to no activity in MH and only moderate activity in RPMI. For the Gram-positive strains, compound 7 showed good activity against S. epidermidis and very strong activity against S. aureus, with effectiveness close to vancomycin. In contrast, compound 4 showed only moderate to weak activity. 

Overall, compound 7 demonstrated the most promising antibacterial activity, particularly against S. aureus USA100, and was therefore selected for further biological evaluation. 

Biological studies results (Student 2)

Given the substantial number of compounds evaluated, a preliminary screening was undertaken to identify which compounds showed activity at the highest concentration.

The initial screening data revealed that from the series, compounds 25 and 26 exhibited moderate but promising antibacterial activity against B. subtilis PY79 across all replicates. These preliminary findings indicate an overall selective trend towards Gram-positive bacteria. Guided by these results, the two most promising compounds were advanced for expanded MIC determination across several bacterial species for a more thorough evaluation.

The MIC assays were employed to determine the lowest concentration of each compound to inhibit visible microbial growth. The MIC was conducted using a 96-well plate using the following standardized protocol:

1. Preparation of bacterial overnight cultures in Mueller-Hinton (MH) or RPMI media.
2. Set up of 96-well plates with compound serial dilutions, positive control (Carbenicillin + MH + bacteria), and negative control (DMSO + MH, no bacteria).
3. Incubation at 36 degrees C. 
4. Visual reading of results: growth vs. no growth to determine the MIC endpoint. 

Both compounds 25 and 26 showed no activity against Gram-negative bacteria (Escherichia coli and Acinetobacter baumannii) in MH media (MIC >256 碌g/mL), but moderate activity was observed in RPMI media. In particular, both compounds inhibited A. baumannii (MIC = 32 碌g/mL), while weaker effects were seen against E. coli (MIC = 64 碌g/mL for compound 25 and 128 碌g/mL for compound 26). Against Gram-positive bacteria, both compounds showed moderate inhibition of Staphylococcus epidermidis ATCC 12228 in MH media (MIC = 128 碌g/mL), with no data available in RPMI due to lack of bacterial growth. For Staphylococcus aureus USA100, improved activity was observed in RPMI compared to MH, with MIC values of 16 碌g/mL and 32 碌g/mL for compounds 25 and 26, respectively.

Overall, both compounds demonstrated selective activity toward Gram-positive bacteria, with their effectiveness also influenced by the growth medium used.

Conclusion and evaluation of results

The overall findings from this study demonstrate that the synthesized compounds exhibit varying degrees of antibacterial activity that are strongly dependent on both their structural features and the growth conditions used during testing. Across the different sets of compounds evaluated, a consistent trend emerges: most compounds show limited or no activity against Gram-negative bacteria in standard MH media, while improved performance is observed in RPMI media. This suggests that environmental conditions play a significant role in influencing the apparent biological activity of these compounds.

A key outcome of the study is the identification of compounds with selective antibacterial behaviour. In particular, certain compounds (notably compound 7, and to a lesser extent compounds 4, 25, and 26) demonstrate stronger activity against Gram-positive bacteria, especially Staphylococcus aureus USA100. Compound 7, in particular, stands out as the most promising candidate, showing activity that approaches that of vancomycin under RPMI conditions. This highlights its potential as a lead structure for further optimisation. In contrast, compounds such as 25 and 26 display moderate, but more limited, activity and appear to benefit less from structural or environmental modulation.

Overall, the results suggest a clear structure activity relationship where subtle differences in ligand design significantly influence antibacterial potency and selectivity. The enhanced activity observed in RPMI medium further indicates that physiological-like conditions may better reflect the true therapeutic potential of these compounds compared to rich media alone.

Disclaimer: Further experiments were undertaken to build on the initial objectives of this study and to gain deeper insight into the most promising lead compounds. These additional investigations were designed to further evaluate and validate the biological performance and properties of the selected candidates under more detailed experimental conditions. However, the resulting data could not be included in this report due to intellectual property (IP) considerations and associated confidentiality restrictions. Despite the exclusion of these findings, the extended work supports and strengthens the overall conclusions of the study, particularly regarding the identification of lead compounds with enhanced antibacterial activity. The additional experiments contributed valuable supplementary evidence for prioritising specific compounds for further development and optimisation. Importantly, the absence of this data does not compromise the scientific integrity of the study, but rather reflects ongoing efforts toward potential translation and protection of novel findings.

Future dissemination of these results will be considered once appropriate IP processes have been finalised, allowing for a more comprehensive understanding of the compounds' full biological and chemical profiles.

Acknowledgements

We wish to express their sincere gratitude to the UMSAEP program and to all individuals and institutions (UWC and UMKC) who contributed to the successful collaboration and the exchange program. Special appreciation is extended to Prof. Uphoff, Dr. Bawa and the UWC IRO office for their exceptional leadership and sustained commitment in championing the
programme over an extended period. Their vision and dedication were instrumental in enabling this collaboration, particularly through their thoughtful selection and support of our project, which made this research exchange possible.

We further acknowledge their generous and considerate decision to provide special financial support by subsidising the participation of two students, Ms. Brithnet Snyman and Ms. Boitumelo Nthako, thereby significantly strengthening the capacity and impact of the collaborative training experience. Their support not only facilitated meaningful scientific exchange but also contributed to the development of emerging researchers within the programme.

Finally, special thanks are extended to Prof. J. Randall and his research group for their outstanding hospitality and support throughout the collaboration. His group provided a welcoming and intellectually stimulating environment, which greatly enriched the research experience and contributed to the successful completion of the work.

We are deeply appreciative of all the guidance, trust, and collaborative spirit that made this project possible.