N-methylmorpholine incorporation into the structure of biphenyl leads to the bioactive inhibitor of PD-1/PD-L1 interaction.

We present new small-molecular probes targeting the human PD-L1 protein. The molecules were designed by incorporating a newly discovered N-methylmorpholine substituent into a known biphenyl-based structure. Four prototype derivatives of 4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carbonitrile (STD4), comprising a morpholine substituent fused with a biphenyl core at different orientations were first verified for their potential binding to PD-L1 using the molecular docking method. A more favorable 7-phenyl derivative of STD4 was then equipped with an amide bond, pyridine, and either a tris(hydroxymethyl)aminomethane or serinol tail leading to two final molecules. Among them, compound 1c showed activity in three bioassays, i.e., the homogenous time-resolved fluorescence (HTRF) assay, immune checkpoint blockade (ICB) assay, and T-cell activation (TCA) assay. Our work shows that morpholine can substitute for dioxane and becomes a promising component in PD-L1-targeting molecules. This finding unlocks new avenues for optimizing PD-L1-targeting compounds, presenting exciting prospects for future developments in this field.

C2-Symmetrical Terphenyl Derivatives as Small Molecule Inhibitors of Programmed Cell Death 1/Programmed Death Ligand 1 Protein-Protein Interaction.

The PD-1/PD-L1 complex is an immune checkpoint responsible for regulating the natural immune response, but also allows tumors to escape immune surveillance. Inhibition of the PD-1/PD-L1 axis positively contributes to the efficacy of cancer treatment. The only available therapeutics targeting PD-1/PD-L1 are monoclonal antibody-based drugs, which have several limitations. Therefore, small molecule compounds are emerging as an attractive alternative that can potentially overcome the drawbacks of mAb-based therapy. In this article, we present a novel class of small molecule compounds based on the terphenyl scaffold that bind to PD-L1. The general architecture of the presented structures is characterized by axial symmetry and consists of three elements: an m-terphenyl core, an additional aromatic ring, and a solubilizing agent. Using molecular docking, we designed a series of final compounds, which were subsequently synthesized and tested in HTRF assay and NMR binding assay to evaluate their activity. In addition, we performed an in-depth analysis of the mutual arrangement of the phenyl rings of the terphenyl core within the binding pocket of PD-L1 and found several correlations between the plane angle values and the affinity of the compounds towards the protein.

Correction: Reyes Romero et al. Computer- and NMR-Aided Design of Small-Molecule Inhibitors of the Hub1 Protein. Molecules 2022, 27, 8282.

In the published publication [...].

Nonsymmetrically Substituted 1,1'-Biphenyl-Based Small Molecule Inhibitors of the PD-1/PD-L1 Interaction.

Therapeutic antibodies directed against either programmed cell death-1 protein (PD-1) or its ligand PD-L1 have demonstrated efficacy in the treatment of various cancers. In contrast with antibodies, small molecules have the potential for increased tissue penetration; better pharmacology; and therefore, improved antitumor activity. A series of nonsymmetric C2 inhibitors were synthesized and evaluated for PD-1/PD-L1 interaction inhibition. These compounds induced PD-L1 dimerization and effectively blocked PD-L1/PD-1 interaction in a homogeneous time-resolved fluorescence (HTRF) assay with most inhibitors exhibiting IC50 values in the single-digit nM range and below. Their high inhibitory potency was also demonstrated in a cell-based coculture PD-1 signaling assay where 2 exhibited an EC50 inhibitory activity of 21.8 nM, which approached that of the PD-L1 antibody durvalumab (EC50 = 0.3-1.8 nM). Structural insight into how these inhibitors interact with PD-L1 was gained by using NMR and X-ray cocrystal structure studies. These data support further preclinical evaluation of these compounds as antibody alternatives.

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.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.

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.

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.

Nutlin-3a-aa: Improving the Bioactivity of a p53/MDM2 Interaction Inhibitor by Introducing a Solvent-Exposed Methylene Group.

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.

Computer- and NMR-Aided Design of Small-Molecule Inhibitors of the Hub1 Protein.

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein.

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.

Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction.

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC50 value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.

Imaging of Clear Cell Renal Carcinoma with Immune Checkpoint Targeting Aptamer-Based Probe.

Immune checkpoint targeting immunotherapy has revolutionized the treatment of certain cancers in the recent years. Determination of the status of immune checkpoint expression in particular cancers may assist decision making. Here, we describe the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target engaging aptamers are selected by iterative enrichment from a random ssDNA pool and the binding is characterized biochemically. Specificity and dose dependence is demonstrated in vitro in the cell culture using human kidney tumor cells (786-0), human melanoma cells (WM115 and WM266.4) and human glioblastoma LN18 cancer cells. The utility of the probe in vivo is demonstrated using two mouse tumor models, where we show that the probe exhibits excellent potential in imaging. We postulate that further development of the probe may allow universal imaging of different types of tumors depending on their PD-L1 status, which may find utility in cancer diagnosis.

Analysis tools for single-monomer measurements of self-assembly processes.

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin.

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.

A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity.

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.

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.