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.

Bacteria depend on the ferrous iron transport (Feo) system for the uptake of ferrous iron (Fe2+). The Feo system is crucial for colonization and virulence of pathogens. In γ-proteobacteria, the system consists of FeoA, FeoB, and FeoC. The function of FeoA remains unknown. FeoB likely forms the channel, whose regulation has been suggested to involve its GTPase domain (part of its NFeoB domain). FeoC from Klebsiella pneumonia was found to contain a [4Fe4S] cofactor, whose presence was speculated to enhance the GTPase activity of FeoB (Hsueh, K.-L., et al., J. Bacteriol. 2013 195(20): 4726–34). We present results here that support and extend that hypothesis. We monitored the GTPase activity of FeoB by NMR spectroscopy and found that the presence of 7% FeoC-[4Fe-4S]3+ (the highest level of cofactor achieved in vitro) increased the GTPase rate of NFeoB by 3.6-fold over NFeoB. The effect depends on the oxidation state of the cluster; with reduction of the cluster to [4Fe-4S]2+ the GTPase greatly decreased the GTPase rate. From the effects of point mutations in FeoC on GTPase rates, we conclude that Lys62 and Lys68 on FeoC each contribute to increased GTPase activity on NFeoB. Mutation of Thr37 of NFeoB to Ser nearly abolished the GTPase activity. The GTPase activity of the isolated K. pneumoniae NFeoB-FeoC complex (NFeoBC) was found to be higher in KCl than in NaCl solution. We solved the X-ray structure of the NFeoBC crystallized from KCl and compared it with a prior X-ray structure crystalized from NaCl. We propose a hypothesis, consistent with these results, to explain the factors that influence the GTPase activity. Bacteria may use the oxygen-sensitive cluster as a sensor to up-regulate the gate closing speed.
•We demonstrated that when 7% [4Fe-4S]3+-FeoC (holo-FeoC) is bound to FeoB, the GTPase rate could be enhanced by ∼3.6-fold. This is the first Fe-S protein to regulate G protein.•We tried to perturb the [4Fe-4S]3+ cluster with DTT and found that the DTT-reduced cluster ([4Fe-4S]2+) could no longer activate the GTPase. Also, the key local positive charges (K62 and K68) around the cluster of FeoC were found to be essential to the rate enhancement.•We solved a crystal structure in KCl with missing the secondary structures that support the putative activation mechanism.•Also, we purified a threonine to serine (T37S) mutant FeoB protein and found that the GTPase rate of T37S-FeoB dropped by more than 85%. Last, we summarize the structural evidences to explain the putative activation mechanism.

Simple Summary Grenz rays are low energy X-rays and have been used for dermatological radiotherapy for decades. This study calculates the relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) for DNA double-strand break (DSB) induction and cell survival under hypoxic conditions for Grenz-ray therapy (GT). The RBE values for cell survival for cells irradiated by 15 kV, 10 kV and 10 kVp and titanium K-shell X-rays (4.55 kV) relative to Co-60 gamma-rays are 1.0-1.6 at an aerobic condition (21% O-2) and further increase to 1.2-2.1 in conditions of severe hypoxia (0.1% O-2). The OER values for DSB induction relative to Co-60 gamma-rays are ~2.4 for GT, but the OER for cell survival is 2.8-2.0 as photon energy decreases from 15 kV to 4.55 kV. Grenz-ray therapy (GT) is commonly used for dermatological radiotherapy and has a higher linear energy transfer, relative biological effectiveness (RBE) and oxygen enhancement ratio (OER). GT is a treatment option for lentigo maligna and lentigo maligna melanoma. This study aims to calculate the RBE for DNA double-strand break (DSB) induction and cell survival under hypoxic conditions for GT. The yield of DSBs induced by GT is calculated at the aerobic and hypoxic conditions, using a Monte Carlo damage simulation (MCDS) software. The RBE value for cell survival is calculated using the repair-misrepair-fixation (RMF) model. The RBE values for cell survival for cells irradiated by 15 kV, 10 kV and 10 kVp and titanium K-shell X-rays (4.55 kV) relative to Co-60 gamma-rays are 1.0-1.6 at the aerobic conditions and moderate hypoxia (2% O-2), respectively, but increase to 1.2, 1.4 and 1.9 and 2.1 in conditions of severe hypoxia (0.1% O-2). The OER values for DSB induction relative to Co-60 gamma-rays are about constant and ~2.4 for GT, but the OER for cell survival is 2.8-2.0 as photon energy decreases from 15 kV to 4.55 kV. The results indicate that GT results in more DSB induction and allows effective tumor control for superficial and hypoxic tumors.

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.

IscR is an Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mossbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S](+) cluster in vivo. However, upon anaerobic isolation of IscR, some clusters became oxidized to the [2Fe-2S](+) form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that the cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mossbauer, and nuclear magnetic resonance spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved His107 residue is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status.

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.