Clinical Score and Machine Learning-Based Model to Predict Diagnosis of Primary Aldosteronism in Arterial Hypertension

Primary aldosteronism (PA) is the cause of arterial hypertension in 4% to 6% of patients, and 30% of patients with PA are affected by unilateral and surgically curable forms. Current guidelines recommend screening for PA approximate to 50% of patients with hypertension on the basis of individual factors, while some experts suggest screening all patients with hypertension. To define the risk of PA and tailor the diagnostic workup to the individual risk of each patient, we developed a conventional scoring system and supervised machine learning algorithms using a retrospective cohort of 4059 patients with hypertension. On the basis of 6 widely available parameters, we developed a numerical score and 308 machine learning-based models, selecting the one with the highest diagnostic performance. After validation, we obtained high predictive performance with our score (optimized sensitivity of 90.7% for PA and 92.3% for unilateral PA [UPA]). The machine learning-based model provided the highest performance, with an area under the curve of 0.834 for PA and 0.905 for diagnosis of UPA, with optimized sensitivity of 96.6% for PA, and 100.0% for UPA, at validation. The application of the predicting tools allowed the identification of a subgroup of patients with very low risk of PA (0.6% for both models) and null probability of having UPA. In conclusion, this score and the machine learning algorithm can accurately predict the individual pretest probability of PA in patients with hypertension and circumvent screening in up to 32.7% of patients using a machine learning-based model, without omitting patients with surgically curable UPA

Albumin-Binding PSMA Ligands: Optimization of the Tissue Distribution Profile

The prostate-specific membrane antigen (PSMA) has emerged as an attractive prostate cancer associated target for radiotheragnostic application using PSMA-specific radioligands. The aim of this study was to design new PSMA ligands modified with an albumin-binding moiety in order to optimize their tissue distribution profile. The compounds were prepared by conjugation of a urea-based PSMA-binding entity, a DOTA chelator, and 4-(<i>p</i>-iodophenyl)butyric acid using multistep solid phase synthesis. The three ligands (PSMA-ALB-02, PSMA-ALB-05, and PSMA-ALB-07) were designed with varying linker entities. Radiolabeling with ¹⁷⁷Lu was performed at a specific activity of up to 50 MBq/nmol resulting in radioligands of >98% radiochemical purity and high stability. In vitro investigations revealed high binding of all three PSMA radioligands to mouse (>64%) and human plasma proteins (>94%). Uptake and internalization into PSMA-positive PC-3 PIP tumor cells was equally high for all radioligands. Negligible accumulation was found in PSMA-negative PC-3 flu cells, indicating PSMA-specific binding of all radioligands. Biodistribution and imaging studies performed in PC-3 PIP/flu tumor-bearing mice showed enhanced blood circulation of the new radioligands when compared to the clinically employed ¹⁷⁷Lu-PSMA-617. The PC-3 PIP tumor uptake of all three radioligands was very high (76.4 ± 2.5% IA/g, 79.4 ± 11.1% IA/g, and 84.6 ± 14.2% IA/g, respectively) at 24 h post injection (p.i.) resulting in tumor-to-blood ratios of ∼176, ∼48, and ∼107, respectively, whereas uptake into PC-3 flu tumors was negligible. Kidney uptake at 24 h p.i. was lowest for ¹⁷⁷Lu-PSMA-ALB-02 (10.7 ± 0.92% IA/g), while ¹⁷⁷Lu-PSMA-ALB-05 and ¹⁷⁷Lu-PSMA-ALB-07 showed higher renal retention (23.9 ± 4.02% IA/g and 51.9 ± 6.34% IA/g, respectively). Tumor-to-background ratios calculated from values of the area under the curve (AUC) of time-dependent biodistribution data were in favor of ¹⁷⁷Lu-PSMA-ALB-02 (tumor-to-blood, 46; tumor-to-kidney, 5.9) when compared to ¹⁷⁷Lu-PSMA-ALB-05 (17 and 3.7, respectively) and ¹⁷⁷Lu-PSMA-ALB-07 (39 and 2.1, respectively). The high accumulation of the radioligands in PC-3 PIP tumors was visualized on SPECT/CT images demonstrating increasing tumor-to-kidney ratios over time. Taking all of the characteristics into account, ¹⁷⁷Lu-PSMA-ALB-02 emerged as the most promising candidate. The applied concept may be attractive for future clinical translation potentially enabling more potent and convenient prostate cancer radionuclide therapy

Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties To Improve Prostate Cancer Therapy

The treatment of metastatic castration-resistant prostate cancer (mCRPC) remains challenging with current treatment options. The development of more effective therapies is, therefore, urgently needed. Targeted radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting ligands has revealed promising clinical results. In an effort to optimize this concept, it was the aim of this study to design and investigate PSMA ligands comprising different types of albumin binders. PSMA-ALB-53 and PSMA-ALB-56 were designed by combining the glutamate-urea-based PSMA-binding entity, a DOTA chelator and an albumin binder based on the 4-(<i>p</i>-iodophenyl)-moiety or <i>p</i>-(tolyl)-moiety. The compounds were labeled with ¹⁷⁷Lu (50 MBq/nmol) resulting in radioligands of high radiochemical purity (≥98%). Both radioligands were stable (≥98%) over 24 h in the presence of l-ascorbic acid. The uptake into PSMA-positive PC-3 PIP tumor cells in vitro was in the same range (54–58%) for both radioligands; however, ¹⁷⁷Lu-PSMA-ALB-53 showed a 15-fold enhanced binding to human plasma proteins. Biodistribution studies performed in PC-3 PIP/flu tumor-bearing mice revealed high tumor uptake of ¹⁷⁷Lu-PSMA-ALB-53 and ¹⁷⁷Lu-PSMA-ALB-56, respectively, demonstrated by equal areas under the curves (AUCs) for both radioligands. The increased retention of ¹⁷⁷Lu-PSMA-ALB-53 in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios when compared to ¹⁷⁷Lu-PSMA-ALB-56. Kidney clearance of ¹⁷⁷Lu-PSMA-ALB-56 was faster, and hence, the tumor-to-kidney AUC ratio was 3-fold higher than in the case of ¹⁷⁷Lu-PSMA-ALB-53. Due to the more favorable tissue distribution profile, ¹⁷⁷Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3 PIP tumor-bearing mice. The tumor growth delay after application of ¹⁷⁷Lu-PSMA-ALB-56 and ¹⁷⁷Lu-PSMA-617 applied at the same activities (2 or 5 MBq per mouse) revealed better antitumor effects in the case of ¹⁷⁷Lu-PSMA-ALB-56. As a consequence, the survival of mice treated with ¹⁷⁷Lu-PSMA-ALB-56 was prolonged when compared to the mice, which received the same activity of ¹⁷⁷Lu-PSMA-617. Our results demonstrated the superiority of ¹⁷⁷Lu-PSMA-ALB-56 over ¹⁷⁷Lu-PSMA-ALB-53 indicating that the <i>p</i>-(tolyl)-moiety was more suited as an albumin binder to optimize the tissue distribution profile. ¹⁷⁷Lu-PSMA-ALB-56 was more effective to treat tumors than ¹⁷⁷Lu-PSMA-617 resulting in complete tumor remission in four out of six mice. This promising results warrant further investigations to assess the potential for clinical application of ¹⁷⁷Lu-PSMA-ALB-56

Synthesis, Radiolabeling, and Characterization of Plasma Protein-Binding Ligands: Potential Tools for Modulation of the Pharmacokinetic Properties of (Radio)Pharmaceuticals

The development of (radio)­pharmaceuticals with favorable pharmacokinetic profiles is crucial for allowing the optimization of the imaging or therapeutic potential and the minimization of undesired side effects. The aim of this study was, therefore, to evaluate and compare three different plasma protein binders (PPB-01, PPB-02, and PPB-03) that are potentially useful in combination with (radio)­pharmaceuticals to enhance their half-life in the blood. The entities were functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator via a l-lysine and β-alanine linker moiety using solid-phase peptide chemistry and labeled with ¹⁷⁷Lu (<i>T</i>_1/2 = 6.65 days), a clinically established radiometal. The binding capacities of these radioligands and ¹⁷⁷Lu–DOTA were evaluated using human plasma and solutions of human serum albumin (HSA), human α₁-acid glycoprotein (α₁-AGP), and human transthyretin (hTTR) by applying an ultrafiltration assay. ¹⁷⁷Lu–DOTA–PPB-01 and ¹⁷⁷Lu–DOTA–PPB-02 bound to a high and moderate extent to human plasma proteins (>90% and ∼70%, respectively), whereas the binding to hTTR was considered negligible (<10%). ¹⁷⁷Lu–DOTA–PPB-03 showed almost complete binding to human plasma proteins (>90%) with a high fraction bound to hTTR (∼50%). Plasma protein binding of the ¹⁷⁷Lu–DOTA complex, which was used as a control, was not observed (<1%). ¹⁷⁷Lu–DOTA–PPB-01 and ¹⁷⁷Lu–DOTA–PPB-02 were both displaced (>80%) from HSA by ibuprofen, specific for Sudlow’s binding site II and coherent with the aromatic structures, and >80% by their respective binding entities. ¹⁷⁷Lu–DOTA–PPB-03 was displaced from hTTR by the site-marker l-thyroxine (>60%) and by its binding entity PPB-03* (>80%). All three radioligands were investigated with regard to the in vivo blood clearance in normal mice. ¹⁷⁷Lu–DOTA–PPB-01 showed the slowest blood clearance (<i>T</i>_1/2,β: >15 h) followed by ¹⁷⁷Lu–DOTA–PPB-03 (<i>T</i>_1/2,β: ∼2.33 h) and ¹⁷⁷Lu–DOTA–PPB-02 (<i>T</i>_1/2,β: ∼1.14 h), which was excreted relatively fast. Our results confirmed the high affinity of the 4-(4-iodophenyl)-butyric acid entity (PPB-01) to plasma proteins, while replacement of the halogen by an ethynyl entity (PPB-02) reduced the plasma protein binding significantly. An attractive approach is the application of the transthyretin binder (PPB-03), which shows high affinity to hTTR. Future studies in our laboratory will be focused on the application of these binding entities in combination with clinically relevant targeting agents for diagnostic and therapeutic purposes in nuclear medicine

(S)-Selectivity in Phenylacetyl Carbinol Synthesis Using the Wild- Type Enzyme Acetoin:Dichlorophenolindophenol Oxidoreductase from Bacillus licheniformis

Thiamine diphosphate (ThDP)-dependent enzymes are well known biocatalysts for the asym- metric synthesis of a-hydroxy ketones with preferential (R)-selectivity. Pharmaceutically relevant phenyl- acetyl carbinol (PAC) has been prepared with absolute (S)-configuration only on a few occasions using enzyme variants suitably designed through rational site-directed mutagenesis approaches. Herein, we de- scribe the synthesis of (S)-phenylacetyl carbinol products with extended reaction scope employing the readily available wild-type ThDP-dependent enzyme acetoin :dichlorophenolindophenol oxidore- ductase (Ao:DCPIP OR) from Bacillus lichenifor- mis. On a semipreparative scale, cross-benzoin-like condensations of methylacetoin (donor) and differently substituted benzaldehydes proceed with almost complete chemoselectivity yielding the target (S)-1- hydroxy-1-phenylpropan-2-one derivatives with high conversion efficiencies (up to 95%) and good enan- tioselectivities (up to 99%). Ao:DCPIP OR accepts hydroxy- and nitrobenzaldehydes and also sterically demanding substrates such as 1-naphthaldehyde and 4-(tert-butyl)benzaldehyde, which are typically poor acceptors in enzymatic transformations. The explorative synthesis of (S)-phenylpropionyl carbinol medi- ated by Ao:DCPIP OR via carboligation of benzal- dehyde with 3,4-hexanedione is also reported

Binding Mode Characterization of Novel RNA Polymerase Inhibitors Using a Combined Biochemical and NMR Approach

Bacterial RNA polymerase (RNAP) represents a validated target for the development of broad-spectrum antibiotics. However, the medical value of RNAP inhibitors in clinical use is limited by the prevalence of resistant strains. To overcome this problem, we focused on the exploration of alternative target sites within the RNAP. Previously, we described the discovery of a novel RNAP inhibitor class containing an ureidothiophene-2-carboxylic acid core structure. Herein, we demonstrate that these compounds are potent against a set of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) strains (MIC 2–16 μg mL^–1) and rifampicin-resistant <i>Escherichia coli</i> TolC strains (MIC 12.5–50 μg mL^–1). Additionally, an abortive transcription assay revealed that these compounds inhibit the bacterial transcription process during the initiation phase. Furthermore, the binding mode of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis studies and ligand-based NMR spectroscopy. Competition saturation transfer difference (STD) NMR experiments with the described RNAP inhibitor myxopyronin A (<b>Myx</b>) suggest that the ureidothiophene-2-carboxylic acids compete with <b>Myx</b> for the same binding site in the RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping) experiments and molecular docking simulations provided a binding model in which the ureidothiophene-2-carboxylic acids occupy the region of the <b>Myx</b> western chain binding site and slightly occlude that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic acids are a highly attractive new class of RNAP inhibitors that can avoid the problem of resistance

Cylindrofridins A–C, Linear Cylindrocyclophane-Related Alkylresorcinols from the Cyanobacterium <i>Cylindrospermum stagnale</i>

A rapid and exhaustive one-step biomass extraction as well as an enrichment and cleanup procedure has been developed for HPLC-UV detection and quantification of closely related [7.7]­paracyclophanes and structural derivatives based on a two-phase solvent system. The procedure has been validated using the biomass of the carbamidocyclophane- and cylindrocyclophane-producing cyanobacterium <i>Nostoc</i> sp. CAVN2 and was utilized to perform a screening comprising 102 cyanobacterial strains. As a result, three new cylindrocyclophane-related alkylresorcinols, cylindrofridins A–C (<b>1</b>–<b>3</b>), and known cylindrocyclophanes (<b>4</b>–<b>6</b>) were detected and isolated from <i>Cylindrospermum stagnale</i> PCC 7417. Structures of <b>1</b>–<b>3</b> were elucidated by a combination of 1D and 2D NMR experiments, HRMS, and ECD spectroscopy. Cylindrofridin A (<b>1</b>) is the first naturally occurring [7.7]­paracyclophane-related monomeric derivative. In contrast, cylindrofridins B (<b>2</b>) and C (<b>3</b>) represent dimers related to <b>1</b>. Due to chlorination at the alkyl carbon atom in <b>1</b>–<b>3</b>, the site of [7.7]­paracyclophane macrocycle formation, the cylindrofridins represent linearized congeners of the cylindrocyclophanes. Compounds <b>1</b>–<b>3</b> were not toxic against nontumorigenic HaCaT cells (IC₅₀ values >25 μM) compared to the respective cylindrocyclophanes, but <b>1</b> was the only cylindrofridin showing moderate activity against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) and <i>Streptococcus pneumoniae</i> with MIC values of 9 and 17 μM, respectively

Discovery of Cycloalkyl[<i>c</i>]thiophenes as Novel Scaffolds for Hypoxia-Inducible Factor-2α Inhibitors

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy–aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology

Anaerobic Metabolism of 3-Hydroxybenzoate by the Denitrifying Bacterium Thauera aromatica

The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate–CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate–CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of β-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways