An alternative conformation of the N-terminal loop of human dihydroorotate dehydrogenase drives binding to a potent antiproliferative agent.

Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.

Biochemical characterization of Mycobacterium tuberculosis dihydroorotate dehydrogenase and identification of a selective inhibitor.

Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full-length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in-house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 µm, paving the way to hit-to-lead process.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Aryloxyaryl Moiety.

In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.

The integration of AlphaFold-predicted and crystal structures of human trans-3-hydroxy-l-proline dehydratase reveals a regulatory catalytic mechanism.

Computational methods for protein structure prediction have made significant strides forward, as evidenced by the last development of the neural network AlphaFold, which outperformed the CASP14 competitors by consistently predicting the structure of target proteins. Here we show an integrated structural investigation that combines the AlphaFold and crystal structures of human trans-3-Hydroxy-l-proline dehydratase, an enzyme involved in hydroxyproline catabolism and whose structure had never been reported before, identifying a structural element, absent in the AlphaFold model but present in the crystal structure, that was subsequently proved to be functionally relevant. Although the AlphaFold model lacked information on protein oligomerization, the native dimer was reconstructed using template-based and ab initio computational approaches. Moreover, molecular phasing of the diffraction data using the AlphaFold model resulted in dimer reconstruction and straightforward structure solution. Our work adds to the integration of AlphaFold with experimental structural and functional data for protein analysis, crystallographic phasing and structure solution.

Expectations, experiences and preferences of patients and physicians in the informed consent process for clinical trials in oncology.

PURPOSE: The aim of the present study was to explore (1) informed consent (IC) representations, level of understanding, needs, and factors that influence the willingness of cancer patients to participate in randomized controlled trials (RCTs) (phase I) and (2) representations, experiences, and critical issues of physicians involved in the same process (phase II). METHODS: Semi-structured interviews were conducted with 20 cancer patients who had been asked to enroll in a phase II/III RCT (phase I). Two focus groups were conducted with 13 physicians enrolled in the same process (phase II). The content produced was analyzed through a thematic analysis. RESULTS: The themes that emerged in the first phase I were grouped into six categories: IC representation, randomization, experimentation, meeting with the physician, factors that influence the willingness to participate, and trial participants' needs. The themes emerged in the phase II were grouped into four: IC representation, critical issues of the IC, relationship, and recruitment of trial participants. Each theme is articulated into sub-themes and deeply discussed. CONCLUSION: This study highlights (1) the gap between what is ethically demanded in a RCT consultation and the reality of the situation and (2) the difference in perceptions between patients and physicians with reference to the meaning, objectives, and level of understanding of IC.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

A Selective Competitive Inhibitor of Aldehyde Dehydrogenase 1A3 Hinders Cancer Cell Growth, Invasiveness and Stemness In Vitro.

Aldehyde dehydrogenase 1A3 (ALDH1A3) belongs to an enzymatic superfamily composed by 19 different isoforms, with a scavenger role, involved in the oxidation of a plethora of aldehydes to the respective carboxylic acids, through a NAD+-dependent reaction. Previous clinical studies highlighted the high expression of ALDH1A3 in cancer stem cells (CSCs) correlated to a higher risk of cancer relapses, chemoresistance and a poor clinical outcome. We report on the structural, biochemical, and cellular characterization of NR6, a new selective ALDH1A3 inhibitor derived from an already published ALDH non-selective inhibitor with cytotoxic activity on glioblastoma and colorectal cancer cells. Crystal structure, through X-Ray analysis, showed that NR6 binds a non-conserved tyrosine residue of ALDH1A3 which drives the selectivity towards this isoform, as supported by computational binding simulations. Moreover, NR6 shows anti-metastatic activity in wound healing and invasion assays and induces the downregulation of cancer stem cell markers. Overall, our work confirms the role of ALDH1A3 as an important target in glioblastoma and colorectal cells and propose NR6 as a promising molecule for future preclinical studies.

Structure of Thermococcus litoralis Δ1-pyrroline-2-carboxylate reductase in complex with NADH and L-proline.

L-Hydroxyproline (L-Hyp) is a nonstandard amino acid that is present in certain proteins, in some antibiotics and in the cell-wall components of plants. L-Hyp is the product of the post-translational modification of protein prolines by prolyl hydroxylase enzymes, and the isomers trans-3-hydroxy-L-proline (T3LHyp) and trans-4-hydroxy-L-proline (T4LHyp) are major components of mammalian collagen. T4LHyp follows two distinct degradation pathways in bacteria and mammals, while T3LHyp is metabolized by a two-step metabolic pathway that is conserved in bacteria and mammals, which involves a T3LHyp dehydratase and a Δ1-pyrroline-2-carboxylate (Pyr2C) reductase. In order to shed light on the structure and catalysis of the enzyme involved in the second step of the T3LHyp degradation pathway, the crystal structure of Pyr2C reductase from the archaeon Thermococcus litoralis DSM 5473 complexed with NADH and L-proline is presented. The model allows the mapping of the residues involved in cofactor and product binding and represents a valid model for rationalizing the catalysis of Pyr2C reductases.

Mycobacterium tuberculosis Pathogenesis, Infection Prevention and Treatment.

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis (MTB) and it represents a persistent public health threat for a number of complex biological and sociological reasons. According to the most recent Global Tuberculosis Report (2019) edited by the World Health Organization (WHO), TB is considered the ninth cause of death worldwide and the leading cause of mortality by a single infectious agent, with the highest rate of infections and death toll rate mostly concentrated in developing and low-income countries. We present here the editorial section to the Special Issue entitled "Mycobacterium tuberculosis Pathogenesis, Infection Prevention and Treatment" that includes 7 research articles and a review. The scientific contributions included in the Special Issue mainly focus on the characterization of MTB strains emerging in TB endemic countries as well as on multiple mechanisms adopted by the bacteria to resist and to adapt to antitubercular therapies.

Progress in the Field of Aldehyde Dehydrogenase Inhibitors: Novel Imidazo[1,2-a]pyridines against the 1A Family.

Members of the aldehyde dehydrogenase 1A family are commonly acknowledged as hallmarks of cancer stem cells, and their overexpression is significantly associated with poor prognosis in different types of malignancies. Accordingly, treatments targeting these enzymes may represent a successful strategy to fight cancer. In this work we describe a novel series of imidazo[1,2-a]pyridines, designed as aldehyde dehydrogenase inhibitors by means of a structure-based optimization of a previously developed lead. The novel compounds were evaluated in vitro for their activity and selectivity against the three isoforms of the ALDH1A family and investigated through crystallization and modeling studies for their ability to interact with the catalytic site of the 1A3 isoform. Compound 3f emerged as the first in class submicromolar competitive inhibitor of the target enzyme.

Targeting NAD-dependent dehydrogenases in drug discovery against infectious diseases and cancer.

Dehydrogenases are oxidoreductase enzymes that play a variety of fundamental functions in the living organisms and have primary roles in pathogen survival and infection processes as well as in cancer development. We review here a sub-set of NAD-dependent dehydrogenases involved in human diseases and the recent advancements in drug development targeting pathogen-associated NAD-dependent dehydrogenases. We focus also on the molecular aspects of the inhibition process listing the structures of the most relevant molecules targeting this enzyme family. Our aim is to review the most impacting findings regarding the discovery of novel inhibitory compounds targeting the selected NAD-dependent dehydrogenases involved in cancer and infectious diseases.

Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase Inhibitors: Novel Chemotypes to Target Glioblastoma Stem Cells.

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.

Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activity.

Changes in nicotinamide adenine dinucleotide (NAD+) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD+ levels also affect DNA repair capacity as NAD+ is a substrate for PARP-enzymes (mono/poly-ADP-ribosylation) and sirtuins (deacetylation). The ecto-5'-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular NAD+ levels by processing NAD+ and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular NAD+ content and NAD+-dependent DNA repair capacity. Reduced intracellular NAD+ levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD+ reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through NAD+ or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular NAD+ bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD+. A positive correlation between CD73 expression and intracellular NAD+ content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD+ when supplemented with NAD+ or NMN.

Structure of Thermococcus litoralis trans-3-hydroxy-l-proline dehydratase in the free and substrate-complexed form.

Hydroxyprolines (Hyp) are non-standard amino acids derived from the post-translational modification of proteins by prolyl hydroxylase enzymes. Some plants and bacteria produce Hyp, and the isomers trans-3-Hydroxy-l-proline (T3LHyp) and trans-4-Hydroxy-l-proline (T4LHyp) are major components of mammalian collagen. While T4LHyp is metabolised following distinct degradative pathways in mammals and bacteria, T3LHyp metabolic pathway is conserved in bacteria, plants and mammals, and involves a T3LHyp dehydratase (T3LHypD) in the first degradation step. We report here the crystal structure of T3LHypD from the archaea Thermococcus litoralis in the free and substrate-complexed form. The model shows an "open" and a "closed" conformation depending on the presence (or absence) of the substrate in the catalytic site and allows the mapping of the residues involved in ligand recognition. Moreover, the structure highlights the presence of a water molecule interacting with the hydroxy group of the substrate and potentially involved in catalysis. The structure here reported is the first of its family to be elucidated, and represents a valid model for rationalising the substrate specificity and catalysis of T3LHyp dehydratases.

Synthesis and Structure-Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH.

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.

The Synthesis of Kynurenic Acid in Mammals: An Updated Kynurenine Aminotransferase Structural KATalogue.

Kynurenic acid (KYNA) is a bioactive compound that is produced along the kynurenine pathway (KP) during tryptophan degradation. In a few decades, KYNA shifted from being regarded a poorly characterized by-product of the KP to being considered a main player in many aspects of mammalian physiology, including the control of glutamatergic and cholinergic synaptic transmission, and the coordination of immunomodulation. The renewed attention being paid to the study of KYNA homeostasis is justified by the discovery of selective and potent inhibitors of kynurenine aminotransferase II, which is considered the main enzyme responsible for KYNA synthesis in the mammalian brain. Since abnormally high KYNA levels in the central nervous system have been associated with schizophrenia and cognitive impairment, these inhibitors promise the development of novel anti-psychotic and pro-cognitive drugs. Here, we summarize the currently available structural information on human and rodent kynurenine aminotransferases (KATs) as the result of global efforts aimed at describing the full complement of mammalian isozymes. These studies highlight peculiar features of KATs that can be exploited for the development of isozyme-specific inhibitors. Together with the optimization of biochemical assays to measure individual KAT activities in complex samples, this wealth of knowledge will continue to foster the identification and rational design of brain penetrant small molecules to attenuate KYNA synthesis, i.e., molecules capable of lowering KYNA levels without exposing the brain to the harmful withdrawal of KYNA-dependent neuroprotective actions.

Expanding Benzoxazole-Based Inosine 5'-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure-Activity As Potential Antituberculosis Agents.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 µM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

Crystal structure of a thermophilic O6-alkylguanine-DNA alkyltransferase-derived self-labeling protein-tag in covalent complex with a fluorescent probe.

The self-labeling protein tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-tag® technology under harsh reaction conditions. Here we present the crystal structure of SsOGT-H5 in complex with the fluorescent probe SNAP-Vista Green® (SsOGT-H5-SVG) that reveals the conformation adopted by the protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the protein active site. We gained new insights in the conformational changes possibly occurring to the OGT proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H5-SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H5-SVG structure, highlighting the role of the fluorescent ligand in keeping the protein stable after the trans-alkylation reaction.