Kuang-Lung Hsueh

DNA-encoded library (DEL) technology, especially when combined with machine learning (ML), is a powerful method to discover novel inhibitors. DEL-ML can expand a larger chemical space and boost cost-effectiveness during hit finding. Heme oxygenase-1 (HO-1), a heme-degrading enzyme, is linked to diseases such as cancer and neurodegenerative disorders. The discovery of five series of new scaffold HO-1 hits is reported here, using a DEL-ML workflow, which emphasizes the model's uncertainty quantification and domain of applicability. This model exhibits a strong extrapolation ability, identifying new structures beyond the DEL chemical space. About 37% of predicted molecules showed a binding affinity of K-D < 20 mu M, with the strongest being 141 nM, amd 14 of those molecules displayed >100-fold selectivity for HO-1 over heme oxygenase-2 (HO-2). These molecules also showed structural novelty compared to existing HO-1 inhibitors. Docking simulations provided insights into possible selectivity rationale.

Biological products are increasingly important, and therefore the industry has begun to adopt quality by design, as recommended by the ICH and the U.S. FDA. Smaller companies, however, have faced difficulties in employing full-scale experiments or the quality by design strategy. Thus, this study provides an alternative way to build a model from existing data with experimental software that does not require full-scale experiments. This empirical study hopes to provide a practical way to improve the efficiency of smaller biopharmaceutical companies and researchers. Moreover, the models provided here can be applied to process characterization in recombinant protein production.

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe2+) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen-or iron-sensitive coordination of the Fe-S cluster.

We report nuclear magnetic resonance (NMR) data indicating that the Rieske protein from the cytochrome bc complex of Thermus thermophilus (TtRp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. To test models concerning the mechanism by which TtRp transfers between different sites on the complex, we monitored (1)H, (15)N, and (13)C NMR signals as a function of the redox state and molar ratio of added ligand. Our studies of full-length TtRp were conducted in the presence of dodecyl phosphocholine micelles to solvate the membrane anchor of the protein and the hydrophobic tail of the ligand (hydroubiquinone). NMR data indicated that hydroubiquinone binds to TtRp and stabilizes an altered protein conformation. We utilized a truncated form of the Rieske protein lacking the membrane anchor (trunc-TtRp) to investigate redox-state-dependent conformational changes. Local chemical shift perturbations suggested possible conformational changes at prolyl residues. Detailed investigations showed that all observable prolyl residues of oxidized trunc-TtRp have trans peptide bond configurations but that two of these peptide bonds (Cys151-Pro152 and Gly169-Pro170 located near the iron-sulfur cluster) become cis in the reduced protein. Changes in the chemical shifts of backbone signals provided evidence of redox-state- and ligand-dependent conformational changes localized near the iron-sulfur cluster. These structural changes may alter interactions between the Rieske protein and the cytochrome b and c sites and provide part of the driving force for movement of the Rieske protein between these two sites.

The Rieske protein component of the cytochrome bc complex contains a [2Fe-2S] cluster ligated by two cysteines and two histidines. We report here the pK(a) values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Thermus thermophilus (TtHp) as determined by NMR spectroscopy. Knowledge of these pK(a) values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated Q-cycle. Although we earlier had observed the pH dependence of a N-15 NMR signal from each of the two ligand histidines in oxidized TtRp (Lin, I. J.; Chen, Y.; Fee, J. A.; Song, J.; Westler, W. M.; Markley, J. L. J. Am. Chem. Soc. 2006, 128, 10672-10673), the strong paramagnetism of the [2Fe-2S] cluster prevented the assignment of these signals by conventional methods. Our approach here was to take advantage of the unique histidine leucine (His134-Leu135) sequence and to use residue-selective labeling to establish a key sequence-specific assignment, which was then extended. Analysis of the pH dependence of assigned C-13', C-13(alpha), and N-15(epsilon 2) signals from the two histidine cluster ligands led to unambiguous assignment of the pK(a) values of oxidized and reduced TtRp. The results showed that the pK(a) of His134 changes from 9.1 in oxidized to similar to 12.3 in reduced TtRp, whereas the pK(a) of His154 changes from 7.4 in oxidized to similar to 12.6 in reduced TtRp. This establishes His154, which is close to the quinone when the Rieske protein is in the cytochrome b site, as the residue experiencing the remarkable redox-dependent pK(a) shift. Secondary structural analysis of oxidized and reduced TtRp based upon our extensive chemical shift assignments rules out a large conformational change between the oxidized and reduced states. Therefore, TtRp likely translocates between the cytochrome b and cytochrome c sites by passive diffusion. Our results are most consistent with a mechanism involving the coupled transfer of an electron and transfer of the proton across the hydrogen bond between the hydroquinone and His154 at the cytochrome b site.