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.

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.

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.

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.

Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

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.

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.

Resveratrol enhances apoptosis induced by the heterocyclic aromatic amines in p53-wt LoVo cells, but not in p53-deficient HaCaT cells.

In the present study, we investigated the influence of resveratrol on PhIP treated human colon cancer cells and compared the effect to HaCaT cells considered as normal, human keratinocytes. Our results show that resveratrol decreases DNA damage in both cell types, it increases the sensitivity of LoVo cells to apoptosis and has no effect on PhIP-treated HaCaT cells. We confirm that PhIP-induced apoptosis is p53 and caspase 3/7 dependent. Interestingly, normal cells such as HaCaT, which lack functional p53 are more resistant to PhIP treatment.

Systematic 'foldamerization' of peptide inhibiting p53-MDM2/X interactions by the incorporation of trans- or cis-2-aminocyclopentanecarboxylic acid residues.

A 'foldamerization' strategy for the discovery of biologically active peptide is evaluated using as an example the peptides that inhibit the p53-MDM2/X interactions. Application of a peptide scan with two constrained ß-residue of trans and cis stereochemistry indicated a substitution pattern that leads to active molecules with enhanced conformational stability and high resistance to proteolysis. This procedure led to the discovery of a peptide that showed subnanomolar inhibition of the p53-MDM2 interaction (Ki = 0.4 nM) with resistance to proteolysis enhanced by ca. two orders of magnitude. Crystallographic analysis and molecular modelling allowed for understanding of these peptide-protein interactions at the molecular level.

Di-bromo-Based Small-Molecule Inhibitors of the PD-1/PD-L1 Immune Checkpoint.

Immune checkpoint blockade is one of the most promising strategies of cancer immunotherapy. However, unlike classical targeted therapies, it is currently solely based on expensive monoclonal antibodies, which often inflict immune-related adverse events. Herein, we propose a novel small-molecule inhibitor targeted at the most clinically relevant immune checkpoint, PD-1/PD-L1. The compound is capable of disrupting the PD-1/PD-L1 complex by antagonizing PD-L1 and, therefore, restores activation of T cells similarly to the antibodies, while being cheap in production and possibly nonimmunogenic. The final compound is significantly smaller than others reported in the literature while being nontoxic to cells even at high concentrations. The scaffold was designed using a structure-activity relationship screening cascade based on a new antagonist-induced dissociation NMR assay, called the weak-AIDA-NMR. Weak-AIDA-NMR finds true inhibitors, as opposed to only binders to the target protein, in early steps of lead compound development, and this process makes it less time and cost consuming.

Anti-CD44 DNA Aptamers Selectively Target Cancer Cells.

CD44 is a type I transmembrane glycoprotein interacting with a number of extracellular components, including hyaluronic acid (HA). CD44-HA axis is involved in a variety of processes, including adhesion, migration, differentiation, trafficking, and others. CD44 is overexpressed in several cancers where binding of HA induces signal transduction leading to activation of antiapoptotic proteins and factors linked to drug resistance. As such, CD44 has been implicated in cancer growth, progression, and metastasis. It has been convincingly demonstrated that blocking CD44-HA interaction decreases cancer cell survival and metastasis. In this study, using in vitro selection, we have developed DNA aptamers recognizing a HA-binding domain of CD44 with high affinity and specificity. The aptamers bind to CD44 with nanomolar affinities and efficiently inhibit the growth of leukemic cancer cells characterized by high expression of CD44. The selectivity is demonstrated by an irrelevant effect on cells characterized by low CD44 levels. The obtained aptamers broaden the existing landscape of potential approaches to the development of antitumor strategies based on inhibition of the CD44 axis.

Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein-Protein Interactions.

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein-protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.

Hitting on the move: Targeting intrinsically disordered protein states of the MDM2-p53 interaction.

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal ''lid'' segment of MDM2 from the proximity of the p53-complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds.

CA-170 - A Potent Small-Molecule PD-L1 Inhibitor or Not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 - a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

Helping the Released Guardian: Drug Combinations for Supporting the Anticancer Activity of HDM2 (MDM2) Antagonists.

The protein p53, known as the "Guardian of the Genome", plays an important role in maintaining DNA integrity, providing protection against cancer-promoting mutations. Dysfunction of p53 is observed in almost every cancer, with 50% of cases bearing loss-of-function mutations/deletions in the TP53 gene. In the remaining 50% of cases the overexpression of HDM2 (mouse double minute 2, human homolog) protein, which is a natural inhibitor of p53, is the most common way of keeping p53 inactive. Disruption of HDM2-p53 interaction with the use of HDM2 antagonists leads to the release of p53 and expression of its target genes, engaged in the induction of cell cycle arrest, DNA repair, senescence, and apoptosis. The induction of apoptosis, however, is restricted to only a handful of p53wt cells, and, generally, cancer cells treated with HDM2 antagonists are not efficiently eliminated. For this reason, HDM2 antagonists were tested in combinations with multiple other therapeutics in a search for synergy that would enhance the cancer eradication. This manuscript aims at reviewing the recent progress in developing strategies of combined cancer treatment with the use of HDM2 antagonists.

A therapeutic patent overview of MDM2/X-targeted therapies (2014-2018).

INTRODUCTION: MDM2 and MDMX proteins provide the inhibition of p53 tumor suppressor, thus allowing for accelerated mutation-driven cancer microevolution. A pharmacological blockade of MDM2/X-p53 interaction results in p53 reactivation in p53wt cells, leading to cancer growth inhibition. Throughout the past 20 years, multiple chemical entities have been proposed to reactivate p53 by antagonizing MDM2/X proteins. AREAS COVERED: This manuscript reviews 2014-2018 therapeutic patents in the field of MDM2/X antagonists and is a continuation of previous reviews on similar matter. The patents covering the use of MDM2/X antagonists in drug combinations are also presented in this review, as they constitute an important trend in the field of cancer treatment with MDM2/X antagonists. EXPERT OPINION: In the years 2014-2018, several previously-known chemical scaffolds have been further developed and disclosed. Importantly, in the same time period, many lead compounds have entered clinical trials for the treatment of cancer patients. Meanwhile, several important reports have pointed to serious limitations of anticancer properties of MDM2 antagonists. As a result, many efforts have been made to seek for positive, synergistic therapeutic effects of combined anti-cancer treatment strategies. One recent example is a dual targeting of MDM2 and additional protein targets by utilizing the PROTAC technology.

A fluorinated indole-based MDM2 antagonist selectively inhibits the growth of p53wt osteosarcoma cells.

The p53 protein is engaged in the repair of DNA mutations and elimination of heavily damaged cells, providing anticancer protection. Dysregulation of p53 activity is a crucial step in carcinogenesis. This dysregulation is often caused by the overexpression of negative regulators of p53, among which MDM2 is the most prominent one. Antagonizing MDM2 with small molecules restores the activity of p53 in p53 wild-type (p53wt ) cells and thus provides positive outcomes in the treatment of p53wt cancers. Previously, we have reported the discovery of a panel of fluoro-substituted indole-based antagonists of MDM2. Here, we demonstrate the biological activity and stereoselectivity of the most active compound from this series. Both enantiomers of the esterified form of the compound, as well as its corresponding carboxylic acids, were found active in fluorescence polarization (FP) assay, nuclear magnetic resonance (NMR) and microscale thermophoresis (MST) assay, with Ki and KD values around 1 µm. From these four compounds, the esterified enantiomer (R)-5a was active in cells, which was evidenced by the increase of p53 levels, the induced expression of p53-target genes (CDKN1A and MDM2), the selective induction of cell cycle arrest, and selective growth inhibition of p53wt U-2 OS and SJSA-1 compared to p53del SAOS-2 cells. The analysis of the crystal structure of human MDM2 in complex with the compound (R)-6a (carboxylic acid of the active (R)-5a compound) revealed the classical three-finger binding mode. Altogether, our data demonstrate the activity of the compound and provide the structural basis for further structure optimization.

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

Identification of small-molecule inhibitors of USP2a.

USP2a is a deubiquitinating protease that rescues its target proteins from destruction by the proteasome by reversing the process of protein ubiquitination. USP2a shows oncogenic properties in vivo and has been found to be a specific activator of cyclin D1. Many types of cancers are addicted to cyclin D1 expression. Targeting USP2a is a promising strategy for cancer therapy but little progress has been made in the field of inhibition of USP2a. Using NMR-based fragment screening and biophysical binding assays, we have discovered small molecules that bind to USP2a. Iterations of fragment combination and structure-driven design identified two 5-(2-thienyl)-3-isoxazoles as the inhibitors of the USP2a-ubiquitin protein-protein interaction. The affinity of these molecules for the catalytic domain of USP2a parallels their ability to interfere with USP2a binding to ubiquitin in vitro. Altogether, our results establish the 5-(2-thienyl)-3-isoxazole pharmacophore as an attractive starting point for lead optimization.