Young Scientists (Part II)

Dr. Ying ZHOU

Multiplex Metal-Detection based Assay (MMDA) platform for COVID-19 diagnosis and identification of disease severity biomarkers

Ying Zhou#, Shuofeng Yuan#, Kelvin Kai-Wang To, Xiaohan Xu, Hongyan Li, Jian-Piao Cai, Cuiting Luo, Ivan Fan-Ngai Hung, Kwok-Hung Chan, Kwok-Yung Yuen, Yufeng Li, Jasper Fuk-Woo Chan*, Hongzhe Sun*

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health worldwide.1-3 Accurate diagnosis and understanding of the antibody response is essential for the control of this highly transmissible disease.4 Herein, by using metal-tagged antibodies as reporting probes, we developed a novel multiplex metal-detection based assay (MMDA) platform for serologic profiling of anti-SARS-CoV-2 antibodies including anti-SARS-CoV-2-spike (S) and -nucleocapsid (N) IgA, IgM, and IgG. The MMDA platform exhibits significantly higher sensitivity and specificity than ELISA for the detection of anti-SARS-CoV-2 antibodies. We classified patients into different subgroups based on distinct antibody landscape through in-depth analysis of multiplex data by high dimensional data exploration/visualization tool (tSNE) and machine learning algorithms. We unbiasedly identified the anti-SARS-CoV-2-N IgG and IgA as the most potently induced types of antibodies for COVID-19 diagnosis, and anti-SARS-CoV-2-S IgA as a biomarker for disease severity stratification. MMDA represents a useful platform not only for the diagnosis and disease severity stratification for the ongoing COVID-19 pandemic, but also for vaccine selection and development. 

Reference

1.   J. F. Marcelletti, B. I. Sikic, L. D. Cripe, E. Paietta. Cytom. B Clin. Cytom., 2019, 96 (1), 57-66.

2.   K. Swerts, B. De Moerloose, C. Dhooge, G. Laureys, Y. Benoit, J. Philippé. Eur. J Cancer, 2006, 42 (3), 295-309.

3.   S. C. Bendall, E. F. Simonds, P Qiu, D. A. El-ad, P. O. Krutzik, R. Finck, R. V. Bruggner, R. Melamed, A. Trejo, O. I. Ornatsky. Science, 2011, 332 (6030), 687-696.

4.   P. Theunissen, E. Mejstrikova, L. Sedek, A. J. van der Sluijs-Gelling, G. Gaipa, M. Bartels, E. Sobral da Costa, M. Kotrová, M. Novakova, E. Sonneveld. Blood, 2017, 129 (3), 347-357.

Ms. Mengdan TIAN

ScsBC Proteins Promote Anaerobic Tetrathionate Respiration by Functionally Interacting with Fe-S Cluster-Containing Respiration Enzymes in S. Typhimurium

Iron-sulfur (Fe-S) clusters are inorganic cofactors that have been shown to play important roles in key biological processes including respiration, TCA cycle. Anaerobic respiration depends on a functional Fe-S cluster based on the requirement of quinones as electron carriers. Salmonella enterica serovar Typhimurium is a foodborne pathogen that infects both humans and animals. S. Typhimurium can utilize tetrathionate produced in the inflamed gut for anaerobic respiration (1). During this process, Fe-S cluster of tetrathionate reductase (Ttr) transfers electron to the molybdopterin guanine dinucleotide cofactor (MGD) for the reduction of tetrathionate to thiosulfate. The product thiosulfate can be cleaved to sulfite and sulfide by the thiosulfate reductase (Phs). The metabolic product sulfite is then further reduced by sulfite reductase (Asr), and the final product, sulfide, is generated (2).  H2S has been found to act as a stress signal to modulate protein activities through modifying specific protein cysteine (Cys) thiols by forming protein S-sulfhydration posttranslational modification (PTM). However, it remained unknown about the physiological consequence caused by H2S and the detoxify strategies in salmonella during anaerobic tetrathionate respiration. Our quantitative proteomic analysis revealed that the production of two proteins ScsBC which are predicted to contain thioredoxin folds and catalytic cysteines similar to the enzymes catalyzing disulfide bond formation and isomerization (Dsb proteins) is upregulated more than 30-fold under anaerobic tetrathionate condition. ΔscsBC in S. Typhimurium 14028s  led to reduced viability under anaerobic tetrathionate respiration condition. To investigate the role of Scs proteins during this physiological process,  we performed affinity chromatography with GST-ScsC-coated beads against total cell lysates of S. Typhimurium under anaerobic tetrathionate condition. 163 proteins that only retained by GST-ScsC rather than GST were identified by LC/MSMS. Clusters of Orthologous Groups annotation showed that most of these proteins (25/163)  are involved in energy production and conversion including TtrAB, NapA, NirB, Dms-like STM14_1809, frdAB, Ccp-like STM14_4612 which are involved in anaerobic respiration using alternative electron acceptors (tetrathionate, nitrate, nitrite, DMSO, fumarate, H2O2). Subsequent GST pull-down assay with purified ScsC, TtrA, TtrB proteins  and bacteria two-hybrid assay confirmed the direct interactions between ScsC with these two proteins. Together, these studies reveal a novel function of the Dsb-like protein ScsBC proteins to promote anaerobic tetrathionate respiration by functionally interacting with Fe-S cluster-containing respiratory enzymes in S. Typhimurium.

 

References

 

  1. Winter, S. E., Thiennimitr, Nature 2010, 467, 426-429.
  2. Price-Carter, Journal of bacteriology 2001, 183, 2463-2475.

Dr. Jacky C.H. CHU

Facile One-Pot Synthesis of Cyclic Peptide-Conjugated Photosensitizers for Targeted Antitumoral and Antibacterial Photodynamic Therapy

Jacky C. H. Chu,a Wing-Ping Fong,b Mamie Hui,c Clarence T. T. Wong,a and Dennis K. P. Ng*,a

A novel synthetic strategy for in situ cyclization of peptides and conjugation with photosensitizers through a bifunctional linker was developed. The bifunctional linkers are incorporated with a bis(bromomethyl)benzene unit and an azide or a cyclopentadiene moiety, which can facilitate the cyclization of peptides with two cysteine residues through site-selective alkylation, followed by coupling with a series of bicyclo[6.1.0]non-4-yne-functionalized boron dipyrromethenes (BODIPYs) via strain-promoted azide-alkyne cycloaddition or a maleimide-substituted phthalocyanine via Diels-Alder reaction, respectively. With this methodology, a series of cyclic peptide-conjugated photosensitizers were prepared readily. One of the cyclic RGD peptide-conjugated BODIPYs exhibited high and selective affinity toward the αvβ3 integrin-overexpressed cell lines and induced high photocytotoxicity.1 The phthalocyanine conjugated with an epidermal growth factor receptor (EGFR)-targeted cyclic peptide displayed superior features as an advanced photosensitizer for killing EGFR-overexpressed colorectal carcinoma cells, both in vitro and in vivo.2 In addition, a phthalocyanine conjugated with a cyclic antimicrobial peptide was also prepared which exhibited a synergistic chemo-photodynamic cytotoxic effect against a spectrum of Gram-positive and Gram-negative bacterial strains, including ATCC-type and clinical isolates of multidrug-resistant bacterial strains.3

This work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (Project No. 14304817).

 

References

 

  1. Chu, J. C. H.; Yang, C.; Fong, W.-P.; Wong, C. T. T.; Ng, D. K. P. Chem. Commun. 2020, 56, 11941-11944.
  2. Chu, J. C. H.; Fong, W.-P.; Wong, C. T. T.; Ng, D. K. P. J. Med. Chem. 2021, 64, 2064-2076.
  3. Chu, J. C. H.; Chin, M. L.; Wong, C. T. T.; Hui, M.; Lo, P.-C.; Ng, D. K. P. Adv. Therap. 2021, 4, 2000204.

Ms. Shu CHEN

The Influence of Carbonate Ligands on the Stability, Reduction, and Cytotoxicity of Platinum(IV) Prodrugs

Pt(IV) complexes bearing axial carbonate linkages have drawn much attention recently.1,2 A synthetic method behind it allows to attach the hydroxyl group of bioactive ligands to the available hydroxyl group of Pt(IV) complexes, and the rapid release of free drugs is achieved after the reduction of carbonate-linked Pt(IV) complexes. Further understanding on the properties of Pt(IV) carbonates such as hydrolytic stability and reduction profiles, however, is hindered by limited research. Herein, six mono-carbonated Pt(IV) complexes in which the carbonate axial ligands possess various electron-withdrawing powers were synthesized, and the corresponding mono-carboxylated analogues were also prepared as references to highlight

the different properties. The influence of the coordination environment towards the hydrolysis

and reduction rate of Pt(IV) carbonates and carboxylates was explored. The mono-carbonated

Pt(IV) complexes are both less stable and reduced faster than the corresponding monocarboxylated ones. Moreover, the hydrolysis and reduction profiles are dependent not only on the electron-withdrawing ability of the carbonates but also on the nature of the opposite axial ligands. Besides, the exploration of the hydrolytic pathway for Pt(IV) carbonates suggests that the process proceeds by an attack of 18OH¯ on the carbonyl carbon, followed by elimination, which is different from that of Pt(IV) carboxylates.3-5 This study provides new information on the influence of axial carbonate ligands with different electron-withdrawing abilities on the properties of the Pt(IV) center, which may inspire new thoughts on the design of “multi-action” Pt(IV) prodrugs.

 

References

  1. Yempala, T.; Babu, T.; Karmakar, S.; Nemirovski, A.; Ishan, M.; Gandin, V.; Gibson, D., Angew. Chem. Int. Ed. 2019, 58, 18218-18223.
  2. Babu, T.; Sarkar, A.; Karmakar, S.; Schmidt, C.; Gibson, D., Inorg. Chem. 2020, 59, 5182-5193.
  3. Xu, Z.; Tang, W. K.; Zhou, Q.; Chen, S.; Siu, C.-K.; Zhu, G., Inorg. Chem. Front. 2021, 8, 3794-3802.
  4. Wexselblatt, E.; Yavin, E.; Gibson, D., Angew. Chem. Int. Ed. 2013, 52, 6059-6062.
  5. Kastner, A.; Poetsch, I.; Mayr, J.; Burda, J. V.; Roller, A.; Heffeter, P.; Keppler, B. K.; Kowol, C. R., Angew. Chem. Int. Ed. 2019, 58, 7464-7469.

Ms. Yuen-Ting WONG

Gold-based Agents Effectively Inhibit B1 and B2 Subclass of Metallo-β-lactamase

Yuen-Ting Wonga, Runming Wanga,Pak-Leung Hob, Richard YT Kaob, Hongyan Lia,Hongzhe Suna

Antimicrobial resistance (AMR) has been spotlighted as one of the top threats to global health. The production of metallo-β-lactamase (MBLs) accounts for resistance to β-lactam antibiotics including penicillins and carbapenems.1

Previously, we have discovered that Bi(III)-based drugs serve as effective inhibitors to B1 subclass MBLs.2 In this study, we identified Au(I)-based agents including auranofin (AUR), as broad-spectrum inhibitors of MBLs including B1 subclass MBL (e.g., NDM-1+)3, and B2 subclass MBL (e.g., CphA+ and Sfh-I+). Enzymatic assays demonstrated that the activities of all studied MBLs were strongly inhibited in the presence of Au(I) in a dose-dependent fashion, with the displacement of Zn(II) with Au(I) in their active sites. AUR acted synergistically with meropenem (MER) to reduce the minimum inhibitory concentration (MIC) of MER by 64-folds against NDM-1+ and Sfh-I+ bacteria whereas 8-folds against CphA+ bacteria, and enormously diminish the respective bacterial loads within 24 hours. Additionally, MER-AUR co-treatment yielded a significant decrease in bacterial load in the mammalian cells infected with CphA+ bacteria, and at the same time increased cell viability by over 14-folds, compared to the untreated group. The antimicrobial activity of MER-AUR co-treatment was further translated to murine peritonitis infection model, with significant reduction in CphA+ Aeromonas Sobria loading in mouse spleen and kidney in comparison to antibiotic alone. We conclude that AUR as well as related Au(I) drugs as broad-spectrum inhibitors of MBLs would largely broaden the therapeutic options in treating the infections caused by multidrug-resistant superbugs.

We thank the Research Grants Council (RGC) and Innovative Technology Commission of Hong Kong, the University of Hong Kong and Norman & Celia Yip Foundation for financial support.

Reference

  1. Brown E.D.; Wright G.D. Nature 2016, 529,336-343.
  2. Wang R.; Lai T.P.; Gao P.; Zhang H.; Ho P.L.; Woo P.C.Y.; Ma G.; Kao R.Y.T.; Li H.; Sun H. Commun., 2018, 9, 439.
  3. Sun H.; Zhang Q.; Wang R.; Wang H.; Wong Y.T.; Wang M.; Hao Q; Yan A.; Kao R.; Ho P.L.; Li H. Commun., 2020, 11, 5263.

Mr. Wai Yin Anthony YAU

Extracellular Catecholamine Sensing by Streptavidin-Biotin Strategy

Catecholamines are important in many physiological and behavioral processes like emotion and motor control.1-4 It was reported that abnormal level of catecholamines is associated with neurodegenerative diseases.1-7 Parkinson’s disease, for instance, is characterized by a significant reduction of dopamine level in the brain.5-7  In this work, we report the development of an extracellular catecholamines detection method featuring streptavidin-biotin binding. An N3S-CuII complex, attached on a glass surface, is developed as a selective catecholamine trigger to release a caged biotin.8-9 Streptavidin-Cy5 was then introduced to conjugate to the released biotin to produce a fluorescent response. Level of extracellular catecholamines can then be revealed by fluorescence imaging of the bound Cy5 on the surface.  

References

1.       R. A. Webster, Neurotransmitters, Drugs and Brain Function, John Wiley&Sons, New York, 2001.

2.       D. Purves, G. J. Augustine, D. Fitzpatrick, W. C. Hall, A. S. LaMantia, J. O. McNamara and S. M. Williams, Neuroscience, Sinauer Associates, U.S.A., 2004.

  1. C. Rangel-Barajas, I. Coronel and B. Flor ́an, Aging Dis., 2015, 6, 349-368.

4.       F. Sun, J. Zeng, M. Jing, J. Zhou, J. Feng, S. F. Owen, Y. Luo, F. Li, H. Wang, T. Yamaguchi and Z. Yong, Cell, 2018, 174, 481-496.

  1. M. Perez de la Mora, C. Hernandez-Mondragon, M. Crespo-Ramirez, J. Rejon-Orantes, D. O. Borroto-Escuela and K. Fuxe, Neuroscience, 2020, 439, 301-318.
  2. R. Balestrino and A.H.V. Schapira, European Journal of Neurology, 2020, 27, 27-42.
  3. W. Poewe, K. Seppi, C. M. Tanner, G. M. Halliday, P. Brundin, J. Volkmann, A. E. Schrag and A. E. Lang, Nat Rev Dis Primers, 2017, 3, 17013.

8.       K. Y. Tong, J. Zhao, C. W. Tse, P. K. Wan, J. Rong and H. Y. Au-Yeung, Chem. Sci., 2019, 10, 8519-8526.

9.       Y. P. Wu, C. Y. Chew, T. N. Li, T. H. Chung, E. H. Chang, C. H. Lam and K. T. Tan, Chem. Sci., 2018, 9, 770-776.

Mr. Abhisek Dev BHATTARAI

Photon Counting Spectral Computed Tomography for Localization and Quantitative Assessment of Adipose Tissue and Vasculature

Abhisek B*, Kelvin Tan*, Vince Vardhanabhuti*

As a potential treatment for obesity, the thermogenic property of brown adipose tissues (BAT) has drawn much attention. Globally, obesity has reached epidemic levels, especially amongst children and adolescents, increasing the risk of cardiovascular disease 1, sleep apnoea, musculoskeletal problems, glucose intolerance, and fatty liver disease 2–5. PET imaging has been used to detect the presence of BAT and function. PET imaging, however, is limited by their availability, use of radioactive labels, and low-resolution images. Adipose tissue studies may benefit from advanced imaging techniques such as photon-counting spectral computed tomography (PC-SCT). PC-SCT allows for a simultaneous decomposition and imaging of multiple materials, as well as dose reduction, improved spatial resolution, and reduced metal artifacts. White adipose tissue (WAT) stores excess energy as triglycerides, and BAT dissipates stored energy in the form of heat 6,7. BAT has a different water-to-fat ratio than WAT. This study aims to assess the potential for PC-SCT scanning in localizing BAT in a mice and visualizing changes to vascularization after stimulation. The hypothesis is PC-SCT can localize and quantify BAT and WAT depots in mice based on the fat water contents in adipose tissue 8. Cold/stress causes BAT to undergo changes in lipid content and vasculature 9. We hypothesize high-spectral sensitivity of the scanner enables differentiation of activated and non-activated BAT by observing the changes in vasculature within the adipose tissue using a Gd-based contrast agent. Based on our preliminary results, PC-SCT determined lipid content correlated (R2 = 0.98, 0.97) with known lipid and water mixture in ratios of 10/90, 20/80, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20, 90/10 (Figure 1). According to preliminary data, Gd-based contrast agents can be visualized at concentrations as low as 0.15 mgGd/ml (Figure 2) and with high material decomposition accuracy at concentrations of 1.57 mgGd/ml and above (Figure 3). By tagging more Gd atoms to contrast agent molecules, the result could be improved, making it a useful probe for imaging, and assessing the effectiveness of targeted therapy. Results from the preliminary measurements demonstrate the potential of PC-SCT in localizing fat depots and assessing changes in BAT vasculature with Gd probe in mice model. The study is ongoing, we will now conduct in vivo measurements on WAT, BAT, and activated BAT in a mouse model.

References
1. Cote, A. T., Harris, K. C., Panagiotopoulos, C., Sandor, G. G. S. & Devlin, A. M. Childhood
Obesity and Cardiovascular Dysfunction. J. Am. Coll. Cardiol. 62, 1309–1319 (2013).
2. Africa, J. A., Newton, K. P. & Schwimmer, J. B. Lifestyle Interventions Including Nutrition,
Exercise, and Supplements for Nonalcoholic Fatty Liver Disease in Children. Dig. Dis. Sci. 61, 1375–1386 (2016).
3. Cypess, A. M. et al. Identification and importance of brown adipose tissue in adult humans. N. Engl. J. Med. 360, 1509–1517 (2009).
4. Narang, I. & Mathew, J. L. Childhood obesity and obstructive sleep apnea. J. Nutr. Metab.
2012, 134202 (2012).
5. Pollock, N. K. Childhood obesity, bone development, and cardiometabolic risk factors. Mol.
Cell. Endocrinol. 410, 52–63 (2015).
6. Zhang, F. et al. An Adipose Tissue Atlas: An Image-Guided Identification of Human-like
BAT and Beige Depots in Rodents. Cell Metab. 27, 252-262.e3 (2018).
7. van Marken Lichtenbelt, W. D. et al. Cold-Activated Brown Adipose Tissue in Healthy Men.
N. Engl. J. Med. 360, 1500–1508 (2009).
8. Bateman, C. J. et al. MARS-MD: rejection based image domain material decomposition. J.
Instrum. 13, P05020–P05020 (2018).
9. Baba, S., Jacene, H. A., Engles, J. M., Honda, H. & Wahl, R. L. CT Hounsfield Units of
Brown Adipose Tissue Increase with Activation: Preclinical and Clinical Studies. J. Nucl. Med. 51, 246 LP – 250 (2010).

Mr. Dohyun AHN

Structure-Activity Relationships of Novel Indoloquinoline Ligands and their Copper(II) Complexes as Anticancer Agents

Dohyun Ahn, Maria V. Babak*

Indoloquinolines are naturally occurring plant alkaloids found in the West African Shrub Cryptolepis Sanguinolenta with antimalarial and anticancer activity. The heterocyclic structures of indoloquinolines provided them with many derivatives, in which some derivatives are only obtainable synthetically. In particular, the have been biological activity of the synthetic alkaloid, indolo-[2,3-c]-quinoline, have been seldom investigated Herein, we report a series of novel indolo-[2,3-c]-quinoline-based ligands decorated with Schiff bases and substituent groups, and their copper(II) complexes. The synthesized compounds were characterized by ESI-mass spectrometry, 1H and 13C NMR spectroscopy, and single crystal X-ray diffraction analysis. The compounds demonstrated high cytotoxicity, ranging from nanomolar activity to low micromolar activity against breast cancer and normal human cell lines. The results showed that the position of the bromine substituent on the indoloquinoline backbone and the methylation of the Schiff bases group can affect the cytotoxicity of both the ligands and the complexes. In general, the indolo-[2,3-c]-quinoline derivatives are twice as cytotoxic compared to that observed for the indolo-[3,2-c]-quinoline-based compounds.

Figure 1. Chemical structures of (A) Cu(II) indolo-[2,3-c]-quinoline complex and (B) Cu(II) indolo-[3,2-c]-quinoline complex

Reference:

  1. Primik, M. F., et al. (2010). “Structure−Activity Relationships of Highly Cytotoxic Copper(II) Complexes with Modified Indolo[3,2-c]quinoline Ligands.” Inorganic Chemistry 49(23): 11084-11095.
  2. Van Miert, S., et al. (2005). “Isoneocryptolepine, a Synthetic Indoloquinoline Alkaloid, as an Antiplasmodial Lead Compound.” Journal of Natural Products 68(5): 674-677.