Selective molecular recognition between proteins is a fundamental event in biological processes that governs cellular growth, function, survival, and differentiation. The immune system, for example, is a complex network of cellular processes regulated by protein-protein interactions (PPIs) between cells, receptors, and secreted molecules. Generating and maintaining an appropriate immune response and regulation requires coordination across many cell types and components, while dysregulation of these interactions can lead to disease. A major obstacle in small molecule therapy development towards these PPIs is their restriction to small protein-protein interfaces and a well-defined hydrophobic pocket. Most PPIs have large contact surface areas and lack traditional binding pockets making them historically challenging for the development of potent small-molecule modulators. To address this limitation, we utilized two binding-based approaches, a unique peptidomimetic fragment library and high-throughput small-molecule microarrays to design and discover molecules that target three important immunoregulatory PPIs: the DQ8-insulin complex, the KEAP1-Nrf2 complex, and the IL-4/IL-4R receptor complex. Many autoimmune diseases involve the ternary PPI complex between immunogenic peptides presented to T cell receptors through the major histocompatibility complex (MHC). Inhibiting this interaction may provide a therapeutic approach for delaying or preventing disease. To target type 1 diabetes, we developed a unique library consisting of 125 fragment-sized molecules that mimic glutamic acid and tyrosine residues from the immunogenic insulin B:9-23 peptide responsible for CD4+ T cell activation. Screening of our library after generation of the MHC class II protein responsible for insulin B:9-23 presentation, DQ8, has resulted in identification of 15 lead fragment compounds to date. Application of our fragment library towards pharmaceutically validated target for inflammation and neurogenerative diseases, the Kelch like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), resulted in a 30% hit rate. These are promising results for the further development of selective compounds to inhibit these interactions. For treating inflammatory diseases, such as asthma or cancer, we report the identification of a first-in-class small molecule inhibitor to the cytokine Interleukin-4 (IL-4). The PPI between IL-4 and its receptor complex (IL-4Rα) contains no conventional binding pockets and binding is driven through clusters of complementary residues. Through the combination of small-molecule microarrays and cell-based assays we identified the lead compound, Nico-52, with micromolar inhibitory potency and micromolar affinity. A library of 60 analogs of Nico-52 was synthesized and preliminary structure activity relationships suggest amenability of the p-fluorophenyl substituent and importance of the diol substituent to retain binding potency. These studies resulted in development of a more potent inhibitor to IL-4 with a p-aniline substituent, which could be developed into a targeting ligand to deliver additional therapeutic payloads to an IL-4 enriched microenvironment. In summary, we have developed a peptidomimetic small molecule fragment library as a toolkit for screening against challenging PPI targets with applications towards type 1 diabetes and developed a first-in-class small molecule inhibitor towards IL-4 with applications towards inflammatory diseases. / 2025-09-21T00:00:00Z

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop. In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.