Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins.

A novel near-infrared fluorescent probe for β-galactosidase has been developed based on a hemicyanine skeleton, which is conjugated with a D-galactose residue via a glycosidic bond. The probe serves as a substrate of β-galactosidase and displays rapid and sensitive turn-on fluorescent responses to β-galactosidase in aqueous solution. A 12.8-fold enhancement of fluorescence intensity at 703 nm was observed after incubation of 10 nM of β-galactosidase with 5 μM probe for 10 min. The probe can sensitively detect as little as 0.1 nM of β-galactosidase and shows linear responses to the enzyme concentration below 1.4 nM. The kinetic study showed that the probe has high binding affinity to β-galactosidase with Km = 3.6 μM. The probe was used to detect β-galactosidase in living cells by employing the premature cell senescence model. The probe exhibited strong fluorescent signals in senescent cells but not in normal cells, which demonstrats that the probe is able to detect the endogenous senescence-associated β-galactosidase in living cells.

The residue lysine 28 (K28) is known to form an important salt bridge that stabilizes the Aβ amyloid structure, and acetylation of lysine 28 (K28Ac) slows the Aβ42 fibrillization rate but does not affect fibril morphology. On the other hand, acetylation of lysine 16 (K16Ac) residue greatly diminishes the fibrillization property of Aβ42 peptide and also affects its toxicity. This is due to the fact that lysine 16 acetylated amyloid beta peptide forms amorphous aggregates instead of amyloid fibrils. This is likely a result of increased hydrophobicity of the K16-A21 region due to K16 acetylation, as confirmed by molecular dynamic simulation studies. The calculated results show that the hydrophobic patches of aggregates from acetylated peptides were different when compared to wild-type (WT) peptide. K16Ac and double acetylated (KKAc) peptide aggregates show significantly higher cytotoxicity compared to the WT or K28Ac peptide aggregates alone. However, the heterogeneous mixture of WT and acetylated Aβ42 peptide aggregates exhibited higher free radical formation as well as cytotoxicity, suggesting dynamic interactions between different species could be a critical contributor to Aβ pathology.

Insulin aggregates under storage conditions via disulfide interchange reaction. It is also known to form aggregates at the site of repeated injections in diabetes patients, leading to injection amyloidosis. This has fueled research in pharmaceutical and biotechnology industry as well as in academia to understand factors that modulate insulin stability and aggregation. The main aim of this study is to understand the factors that modulate aggregation propensity of insulin under conditions close to physiological and measure effect of "seeds" on aggregation kinetics. We explored the aggregation kinetics of insulin at pH 7.2 and 37 °C in the presence of disulfide-reducing agent dithiothreitol (DTT), using spectroscopy (UV-visible, fluorescence, and Fourier transform infrared spectroscopy) and microscopy (scanning electron microscopy, atomic force microscopy) techniques. We prepared insulin "seeds" by incubating disulfide-reduced insulin at pH 7.2 and 37 °C for varying lengths of time (10 min to 12 h). These seeds were added to the native protein and nucleation-dependent aggregation kinetics was measured. Aggregation kinetics was fastest in the presence of 10 min seeds suggesting they were nascent. Interestingly, intermediate seeds (30 min to 4 h incubation) resulted in formation of transient fibrils in 4 h that converted to amorphous aggregates upon longer incubation of 24 h. Overall, the results show that insulin under disulfide reducing conditions at pH and temperature close to physiological favors amorphous aggregate formation and seed "maturity" plays an important role in nucleation dependent aggregation kinetics.

Background: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase, is a negative regulator of immune cell activation1. Genetic deletion or pharmacological inhibition of Cbl-b resulted in hyper-reactive and co-stimulation independent T cell activation and cytokine production1. In syngeneic tumor models, CD8 T-cell and NK-cell mediated rejection of tumours were observed1. These findings point to Cbl-b as a therapeutic target in cancer immunotherapy. Inhibition of Cbl-b also demonstrated the potential to enhance the efficacy of check-point blockers like anti-PD-1 antibody, an unmet need in the clinic. Methods: Using intuitive medicinal chemistry design supported by computational approaches, we identified a lead Cbl-b inhibitor. SAR was developed using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation and exhaustion assays. In vivo efficacy was demonstrated in syngeneic mouse colon tumor model. Results: Our lead Cbl-b inhibitor demonstrated potent binding to Cbl-b, robust anti-tumor cytokine secretion in human and mouse T cells, whole blood and potent reversal of T cell exhaustion. A strong tumor growth inhibition was demonstrated by the lead compound in a mouse colon tumor model. Compared to single agent, a combination of the lead compound with anti-PD-1 antibody induced enhanced complete tumor rejections. Conclusions: We have identified a novel, potent and orally bioavailable Cbl-b inhibitor that demonstrated robust in vitro and in vivo anti-tumor profiles. Acknowledgements: We thank Sanjib Das, Ajit Patil, Gauri Gawas, Savita Pandita, Priya Yadav, Sneha Pusadkar, Mayura Behere, Subhadip Das, Shravankumar Kolli and Radheshyam Yadav for their contributions to the project References: 1. Clinical and Experimental Immunology, 204: 14-31, 2020 Citation Format: Murugan Chinnapattu, Sandeep Shelke, Prashant Ingale, Nayan Waghmare, Nanasaheb Kadlag, Manoj Pawar, Akshay Kangane, Sachin S. Chaudhari, Jagmohan Saini, Vidya Kattige, Arti Joshi, Colina Dutta, Debjyoti Boral, Sheetal Kadam, Varada Potdar, Jiju Mani, Pooja Sawant, Megha Marathe, Madhavi Mulay, Akshata Virdikar, Sravan Mandadi, Atul Akarte, Anuj Singh, Chandrasekhar Misra, Pandurang Lambade, Chaitanya Tirumalasetty, Raju Patole, Vikas Karande, Dayanidhi Behera, Pankaj Jain, Vishwanath Kurawattimath, Nagaraj Gowda, Pravin S. Iyer. A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 462.