1,5-Disubstituted tetrazoles as PD-1/PD-L1 antagonists.

The progress in cancer survival and treatment has witnessed a remarkable transformation through the innovative approach of targeting the inhibitory immune checkpoint protein PD-1/PD-L1 complex by mAbs, e.g. pembrolizumab (Keytruda). While generating 17.2 billion U.S. dollars in revenue in 2021, the true significance of these developments lies in their ability to enhance cancer patient outcomes. Despite the proven efficacy of mAbs in inhibiting the PD-1/PD-L1 signaling pathways, they face significant challenges, including limited response rates, high production costs, missing oral bioavailability, and extended half-lives that can lead to immune-related adverse effects. A promising alternative approach involves the use of small molecules acting as PD-1/PD-L1 antagonists to stimulate PD-L1 dimerization. However, the precise mechanisms of action of these molecules remain partially understood, posing challenges to their development. In this context, our research focuses on the creation of a novel scaffold based on the Ugi tetrazole four-component reaction (UT-4CR) to develop low-molecular-weight inhibitors of PD-L1. Employing structure-based methods, we synthesized a library of small compounds using biphenyl vinyl isocyanide, leading to the discovery of a structure-activity relationship among 1,5-disubstituted tetrazole-based inhibitors. Supported by a cocrystal structure with PD-L1, these inhibitors underwent biophysical testing, including HTRF and protein NMR experiments, resulting in the identification of potent candidates with sub-micromolar PD-L1 affinities. This finding opens opportunities to the further development of a new class of PD-L1 antagonists, holding promise for improved cancer immunotherapy strategies.

Solubilizer Tag Effect on PD-L1/Inhibitor Binding Properties for m-Terphenyl Derivatives.

Although heavily studied, the subject of anti-PD-L1 small-molecule inhibitors is still elusive. Here we present a systematic overview of the principles behind successful anti-PD-L1 small-molecule inhibitor design on the example of the m-terphenyl scaffold, with a particular focus on the neglected influence of the solubilizer tag on the overall affinity toward PD-L1. The inhibitor developed according to the proposed guidelines was characterized through its potency in blocking PD-1/PD-L1 complex formation in homogeneous time-resolved fluorescence and cell-based assays. The affinity is also explained based on the crystal structure of the inhibitor itself and its costructure with PD-L1 as well as a molecular modeling study. Our results structuralize the knowledge related to the strong pharmacophore feature of the m-terphenyl scaffold preferential geometry and the more complex role of the solubilizer tag in PD-L1 homodimer stabilization.

A combined approach of structure-based virtual screening and NMR to interrupt the PD-1/PD-L1 axis: Biphenyl-benzimidazole containing compounds as novel PD-L1 inhibitors.

Immunotherapy has emerged as a game-changing approach for cancer treatment. Although monoclonal antibodies (mAbs) targeting the programmed cell death protein 1/programmed cell death protein 1 ligand 1 (PD-1/PD-L1) axis have entered the market revolutionizing the treatment landscape of many cancer types, small molecules, although presenting several advantages including the possibility of oral administration and/or reduced costs, struggled to enter in clinical trials, suffering of water insolubility and/or inadequate potency compared with mAbs. Thus, the search for novel scaffolds for both the design of effective small molecules and possible synergistic strategies is an ongoing field of interest. In an attempt to find novel chemotypes, a virtual screening approach was employed, resulting in the identification of new chemical entities with a certain binding capability, the most versatile of which was the benzimidazole-containing compound 10. Through rational design, a small library of its derivatives was synthesized and evaluated. The homogeneous time-resolved fluorescence (HTRF) assay revealed that compound 17 shows the most potent inhibitory activity (IC50 ) in the submicromolar range and notably, differently from the major part of PD-L1 inhibitors, exhibits satisfactory water solubility properties. These findings highlight the potential of benzimidazole-based compounds as novel promising candidates for PD-L1 inhibition.

Discovery of Inhibitory Fragments That Selectively Target Spire2-FMN2 Interaction.

Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2-FMN2 but not the Spire1-FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1H-15N HSQC nuclear magnetic resonance. Extensive structure-activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2-FMN2 interaction.

Structural and biological characterization of pAC65, a macrocyclic peptide that blocks PD-L1 with equivalent potency to the FDA-approved antibodies.

Recent advances in immuno-oncology have opened up new and impressive treatment options for cancer. Notwithstanding, overcoming the limitations of the current FDA-approved therapies with monoclonal antibodies (mAbs) that block the PD-1/PD-L1 pathway continues to lead to the testing of multiple approaches and optimizations. Recently, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD-L1 ranging from sub-micromolar to micromolar. In this study, we present the most potent non-antibody-based PD-1/PD-L1 interaction inhibitor reported to date. The structural and biological characterization of this macrocyclic PD-L1 targeting peptide provides the rationale for inhibition of both PD-1/PD-L1 and CD80/PD-L1 complexes. The IC50 and EC50 values obtained in PD-L1 binding assays indicate that the pAC65 peptide has potency equivalent to the current FDA-approved mAbs and may have similar activity to the BMS986189 peptide, which entered the clinical trial and has favorable safety and pharmacokinetic data. The data presented here delineate the generation of similar peptides with improved biological activities and applications not only in the field of cancer immunotherapy but also in other disorders related to the immune system.

Machine learning model for predicting late recurrence of atrial fibrillation after catheter ablation.

Late recurrence of atrial fibrillation (LRAF) in the first year following catheter ablation is a common and significant clinical problem. Our study aimed to create a machine-learning model for predicting arrhythmic recurrence within the first year since catheter ablation. The study comprised 201 consecutive patients (age: 61.8 ± 8.1; women 36%) with paroxysmal, persistent, and long-standing persistent atrial fibrillation (AF) who underwent cryoballoon (61%) and radiofrequency ablation (39%). Five different supervised machine-learning models (decision tree, logistic regression, random forest, XGBoost, support vector machines) were developed for predicting AF recurrence. Further, SHapley Additive exPlanations were derived to explain the predictions using 82 parameters based on clinical, laboratory, and procedural variables collected from each patient. The models were trained and validated using a stratified fivefold cross-validation, and a feature selection was performed with permutation importance. The XGBoost model with 12 variables showed the best performance on the testing cohort, with the highest AUC of 0.75 [95% confidence interval 0.7395, 0.7653]. The machine-learned model, based on the easily available 12 clinical and laboratory variables, predicted LRAF with good performance, which may provide a valuable tool in clinical practice for better patient selection and personalized AF strategy following the procedure.

Design, Synthesis, and Biological Evaluation of 2-Hydroxy-4-phenylthiophene-3-carbonitrile as PD-L1 Antagonist and Its Comparison to Available Small Molecular PD-L1 Inhibitors.

In search of a potent small molecular PD-L1 inhibitor, we designed and synthesized a compound based on a 2-hydroxy-4-phenylthiophene-3-carbonitrile moiety. Ligand's performance was tested in vitro and compared side-by-side with a known PD-L1 antagonist with a proven bioactivity BMS1166. Subsequently, we modified both compounds to allow 18F labeling that could be used for PET imaging. Radiolabeling, which is used in drug development and diagnosis, was applied to investigate the properties of those ligands and test them against tissue sections with diverse expression levels of PD-L1. We confirmed biological activity toward hPD-L1 for this inhibitor, comparable with BMS1166, while holding enhanced pharmacological properties.

Design, Synthesis, and Antitumor Activity Evaluation of 2-Arylmethoxy-4-(2,2'-dihalogen-substituted biphenyl-3-ylmethoxy) Benzylamine Derivatives as Potent PD-1/PD-L1 Inhibitors.

Novel 2-arylmethoxy-4-(2,2'-dihalogen-substituted biphenyl-3-ylmethoxy) benzylamine derivatives were designed, synthesized, and evaluated in vitro and in vivo against cancers as PD-1/PD-L1 inhibitors. Through the computer-aided structural optimization and the homogeneous time-resolved fluorescence (HTRF) assay, compound A56 was found to most strongly block the PD-1/PD-L1 interaction with an IC50 value of 2.4 ± 0.8 nM and showed the most potent activity. 1H NMR titration results indicated that A56 can tightly bind to the PD-L1 protein with KD < 1 µM. The X-ray diffraction data for the cocrystal structure of the A56/PD-L1 complex (3.5 Å) deciphered a novel binding mode in detail, which can account for its most potent inhibitory activity. Cell-based assays further demonstrated the strong ability of A56 as an hPD-1/hPD-L1 blocker. Especially in an hPD-L1 MC38 humanized mouse model, A56 significantly inhibited tumor growth without obvious toxicity, with a TGI rate of 55.20% (50 mg/kg, i.g.). In conclusion, A56 is a promising clinical candidate worthy of further development.

Exploring the Surface of the Ectodomain of the PD-L1 Immune Checkpoint with Small-Molecule Fragments.

Development of small molecules targeting the PD-L1/PD-1 interface is advancing both in industry and academia, but only a few have reached early-stage clinical trials. Here, we take a closer look at the general druggability of PD-L1 using in silico hot spot mapping and nuclear magnetic resonance (NMR)-based characterization. We found that the conformational elasticity of the PD-L1 surface strongly influences the formation of hot spots. We deconstructed several generations of known inhibitors into fragments and examined their binding properties using differential scanning fluorimetry (DSF) and protein-based nuclear magnetic resonance (NMR). These biophysical analyses showed that not all fragments bind to the PD-L1 ectodomain despite having the biphenyl scaffold. Although most of the binding fragments induced PD-L1 oligomerization, two compounds, TAH35 and TAH36, retain the monomeric state of proteins upon binding. Additionally, the presence of the entire ectodomain did not affect the binding of the hit compounds and dimerization of PD-L1. The data demonstrated here provide important information on the PD-L1 druggability and the structure-activity relationship of the biphenyl core moiety and therefore may aid in the design of novel inhibitors and focused fragment libraries for PD-L1.

Preliminary Studies of Antimicrobial Activity of New Synthesized Hybrids of 2-Thiohydantoin and 2-Quinolone Derivatives Activated with Blue Light.

Thiohydantoin and quinolone derivatives have attracted researchers' attention because of a broad spectrum of their medical applications. The aim of our research was to synthesize and analyze the antimicrobial properties of novel 2-thiohydantoin and 2-quinolone derivatives. For this purpose, two series of hybrid compounds were synthesized. Both series consisted of 2-thiohydantoin core and 2-quinolone derivative ring, however one of them was enriched with an acetic acid group at N3 atom in 2-thiohydantoin core. Antibacterial properties of these compounds were examined against bacteria: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The antimicrobial assay was carried out using a serial dilution method to obtain the MIC. The influence of blue light irradiation on the tested compounds was investigated. The relative yield of singlet oxygen (1O2*, 1Δg) generation upon excitation with 420 nm was determined by a comparative method, employing perinaphthenone (PN) as a standard. Antimicrobial properties were also investigated after blue light irradiation of the suspensions of the hybrids and bacteria placed in microtitrate plates. Preliminary results confirmed that some of the hybrid compounds showed bacteriostatic activity to the reference Gram-positive bacterial strains and a few of them were bacteriostatic towards Gram-negative bacteria, as well. Blue light activation enhanced bacteriostatic effect of the tested compounds.

Mechanism of MyD88S mediated signal termination.

BACKGROUND: A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)-the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited. METHODS: Luciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD). RESULTS: We show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation). CONCLUSIONS: Our results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction. Video abstract.

PD-L1 Inhibitors: Different Classes, Activities, and Mechanisms of Action.

Targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) interaction has become an established strategy for cancer immunotherapy. Although hundreds of small-molecule, peptide, and peptidomimetic inhibitors have been proposed in recent years, only a limited number of drug candidates show good PD-1/PD-L1 blocking activity in cell-based assays. In this article, we compare representative molecules from different classes in terms of their PD-1/PD-L1 dissociation capacity measured by HTRF and in vitro bioactivity determined by the immune checkpoint blockade (ICB) co-culture assay. We point to recent discoveries that underscore important differences in the mechanisms of action of these molecules and also indicate one principal feature that needs to be considered, which is the eventual human PD-L1 specificity.

Beneficial effects of empagliflozin on hematocrit levels in a patient with severe anemia.

INTRODUCTION: Sodium-glucose cotransporter (SGLT2) inhibitors may additionally benefit patients with diabetes by improving their erythropoiesis followed by the elevation of hemoglobin and hematocrit levels. REASON FOR THE REPORT: In the case described, severe normocytic normochromic anemia was resolved when empagliflozin had been introduced to the therapy. A 78-year-old male patient was admitted to our hospital with a non-ST-segment elevation myocardial infarction. His past medical history included diabetes, right coronary artery angioplasty, myocardial infarction and paroxysmal atrial fibrillation which required anticoagulant treatment. When examined, severe normocytic normochromic anemia was also diagnosed. About two years prior to his admission, the patient began suffering from persistent anemia despite the modification of his anticoagulant therapy with warfarin, rivaroxaban and dabigatran. An extensive evaluation failed to provide an explanation for his anemia. OUTCOME: Eventually, only the introduction of empagliflozin successfully increased the values of hemoglobin and hematocrit. Therefore, it transpires that SGLT2 enhances erythropoietin (EPO) secretion which subsequently raises hematocrit levels in patients with severe anemia.

Structural Characterization of a Macrocyclic Peptide Modulator of the PD-1/PD-L1 Immune Checkpoint Axis.

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.

Patients with Non-Obstructive Coronary Artery Disease Require Strict Control of All Cardiovascular Risk Factors: Results from the Polish Local Population Medical Records.

The aim of the project was to compare patients treated with percutaneous transluminal coronary angioplasty (PTCA), who also had undergone PTCA in the past, with a group of people who had had no angiographic stenosis in the lumen of the coronary arteries in the past, and who also required PTCA during index hospitalization. The secondary aim was to compare the obtained data with the characteristics of a group of people who had undergone angiography twice and for whom no significant stenosis had been found in their coronary arteries. The study used registry data concerning 3085 people who had undergone at least two invasive procedures. Acute coronary syndrome (ACS) was significantly more often observed (Non-ST-segment elevation myocardial infarction (NSTEMI) OR 2.76 [1.91-3.99] and ST-segment elevation myocardial infarction (STEMI) OR 2.35 [1.85-2.99]) in patients with no significant coronary stenosis in the past (who required coronary angioplasty at the time of the study), compared to patients who had already had PTCA. They also demonstrated more frequent occurrence of 'multivessel disease'. This was probably most likely caused by inadequate control of cardiovascular risk factors, as determined by higher total cholesterol levels ([mg/dL] 193.7 ± 44.4 vs. 178.2 ± 43.7) and LDL (123.4 ± 36.2 vs. 117.7 ± 36.2). On the other hand, patients in whom no significant stenosis was found in two consecutive angiographies were more likely to be burdened with chronic obstructive pulmonary disease, atrial fibrillation and chronic kidney disease.

Terphenyl-Based Small-Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein-Protein Interaction.

We describe a new class of potent PD-L1/PD-1 inhibitors based on a terphenyl scaffold that is derived from the rigidified biphenyl-inspired structure. Using in silico docking, we designed and then experimentally demonstrated the effectiveness of the terphenyl-based scaffolds in inhibiting PD-1/PD-L1 complex formation using various biophysical and biochemical techniques. We also present a high-resolution structure of the complex of PD-L1 with one of our most potent inhibitors to identify key PD-L1/inhibitor interactions at the molecular level. In addition, we show the efficacy of our most potent inhibitors in activating the antitumor response using primary human immune cells from healthy donors.

Application of bioorthogonal hetero-Diels-Alder cycloaddition of 5-arylidene derivatives of 1,3-dimethylbarbituric acid and vinyl thioether for imaging inside living cells.

New bioorthogonal cycloaddition of 5-arylidene derivatives of 1,3-dimethylbarbituric acid as 1-oxa-1,3-butadienes and vinyl thioether as a dienophile has been applied to imaging inside living cells. The reaction is high yielding, selective, and fast in aqueous media. The proposed 1-oxa-1,3-butadiene derivative conjugated to a FITC fluorochrome selectively and rapidly labels the cancer cells pretreated with the dienophile-taxol. The second order rate constants k2 for various proposed bioorthogonal cycloadditions were estimated to be in the range from 0.9 × 10-2 M-1 s-1 to 1.4 M-1 s-1, which is much better than in the case of the first generation TQ-ligation (o-quinolinone quinone methide and vinyl thioether ligation, k2 = 1.5 × 10-3 M-1 s-1) and comparable or better to that for the second generation TQ-ligation (k2 = 2.8 × 10-2 M-1 s-1). The reaction rate constants k2 of proposed ligation reactions are in the range of the rate constants k2 for tetrazines and norbornenes or tetrazines and cyclopropenes. These findings indicate that this chemistry is suitable for in vitro imaging experiments.

Design, Synthesis, and Biological Evaluation of Imidazopyridines as PD-1/PD-L1 Antagonists.

The PD-1/PD-L1 axis has proven to be a highly efficacious target for cancer immune checkpoint therapy with several approved antibodies. Also, small molecules based on a biphenyl core can antagonize PD-1/PD-L1, leading to the in vitro formation of PD-L1 dimers. However, their development remains challenging, as we do not yet fully understand their mode of action. In this work, we designed a new scaffold based on our previously solved high-resolution structures of low-molecular-weight inhibitors bound to PD-L1. A small compound library was synthesized using the Groebke-Blackburn-Bienaymé multicomponent reaction (GBB-3CR), resulting in the structure-activity relationship of imidazo[1,2-a]pyridine-based inhibitors. These inhibitors were tested for their biological activity using various biophysical assays giving potent candidates with low-micromolar PD-L1 affinities. An obtained PD-L1 cocrystal structure reveals the binding to PD-L1. Our results open the door to an interesting bioactive scaffold that could lead to a new class of PD-L1 antagonists.

Human and mouse PD-L1: similar molecular structure, but different druggability profiles.

In the development of PD-L1-blocking therapeutics, it is essential to transfer initial in vitro findings into proper in vivo animal models. Classical immunocompetent mice are attractive due to high accessibility and low experimental costs. However, it is unknown whether inter-species differences in PD-L1 sequence and structure would allow for human-mouse cross applications. Here, we disclose the first structure of the mouse (m) PD-L1 and analyze its similarity to the human (h) PD-L1. We show that mPD-L1 interacts with hPD-1 and provides a negative signal toward activated Jurkat T cells. We also show major differences in druggability between the hPD-L1 and mPD-L1 using therapeutic antibodies, a macrocyclic peptide, and small molecules. Our study indicates that while the amino acid sequence is well conserved between the hPD-L1 and mPD-L1 and overall structures are almost identical, crucial differences determine the interaction with anti-PD-L1 agents, that cannot be easily predicted in silico.

Composite Nitride Nanoceramics in the System Titanium Nitride (TiN)-Aluminum Nitride (AlN) through High Pressure and High Temperature Sintering of Synthesis-Mixed Nanocrystalline Powders.

Presented is a study on the original preparation of individual and in situ intimately mixed composite nanocrystalline powders in the titanium nitride-aluminum nitride system, Ti:Al = 1:1 (at.), which were used in high pressure (7.7 GPa) and high temperature (650 and 1200 °C) sintering with no binding additives for diverse individual and composite nanoceramics. First, variations in precursor processing pathways and final nitridation temperatures, 800 and 1100 °C, afforded a pool of mixed in the nanosized regime cubic TiN (c-TiN) and hexagonal AlN (h-AlN) composite nanopowders both with varying average crystallite sizes. Second, the sintering temperatures were selected either to preserve initial powder nanocrystallinity (650 °C was lower than both nitridation temperatures) or promote crystal growth and recrystallization (1200 °C was higher than both nitridation temperatures). Potential equilibration towards bimetallic compounds upon solution mixing of the organometallic precursors to nanopowders, monomeric Ti[N(CH3)2]4 and dimeric {Al[N(CH3)2]3}2, was studied with 1H and 13C NMR in C6D6 solution. The powders and nanoceramics, both of the composites and individual nitrides, were characterized if applicable by powder XRD, FT-IR, SEM/EDX, Vicker's hardness, and helium density. The Vicker's hardness tests confirmed many of the composite and individual nanoceramics having high hardnesses comparable with those of the reference h-AlN and c-TiN ceramics. This is despite extended phase segregation and, frequently, closed microsized pore formation linked mainly to the AlN component. No evidence was found for metastable alloying of the two crystallographically different nitrides under the applied synthesis and sintering conditions. The high pressure and high temperature sintering of the individual and in situ synthesis-mixed composite nanopowders of TiN-AlN was demonstrated to yield robust nanoceramics.