Current Immunotherapeutic Strategies Targeting the PD-1/PD-L1 Axis in Non-Small Cell Lung Cancer with Oncogenic Driver Mutations

Treatment strategies targeting programed cell death 1 (PD-1) or its ligand, PD-L1, have been developed as immunotherapy against tumor progression for various cancer types including non-small cell lung cancer (NSCLC). The recent pivotal clinical trials of immune-checkpoint inhibiters (ICIs) combined with cytotoxic chemotherapy have reshaped therapeutic strategies and established various first-line standard treatments. The therapeutic effects of ICIs in these clinical trials were analyzed according to PD-L1 tumor proportion scores or tumor mutational burden; however, these indicators are insufficient to predict the clinical outcome. Consequently, molecular biological approaches, including multi-omics analyses, have addressed other mechanisms of cancer immune escape and have revealed an association of NSCLC containing specific driver mutations with distinct immune phenotypes. NSCLC has been characterized by driver mutation-defined molecular subsets and the effect of driver mutations on the regulatory mechanism of PD-L1 expression on the tumor itself. In this review, we summarize the results of recent clinical trials of ICIs in advanced NSCLC and the association between driver alterations and distinct immune phenotypes. We further discuss the current clinical issues with a future perspective for the role of precision medicine in NSCLC.     

Identification of the FDA-Approved Drug Pyrvinium as a Small-Molecule Inhibitor of the PD-1/PD-L1 Interaction

Immune checkpoint blockade involving inhibition of the PD-1/PD-L1 interaction has provided unprecedented clinical benefits in treating a variety of tumors. To date, a total of six antibodies that bind to either PD-1 or PD-L1 protein and in turn inhibit the PD-1/PD-L1 interaction have received clinical approvals. Despite being highly effective, these expensive large biotherapeutics possess several inherent pharmacokinetic limitations that can be successfully overcome through the use of low-molecular-weight inhibitors. One such promising approach involves small-molecule induced dimerization and sequestration of PD-L1, leading to effective PD-1/PD-L1 inhibition. Herein, we present the discovery of such potential bioactive PD-L1 dimerizers through a structure- and ligand-based screening of a focused library of approved and investigational drugs worldwide. Pyrvinium, an FDA-approved anthelmintic drug, showed the highest activity in our study with IC₅₀ value of ∼29.66 μM. It is noteworthy that Pyrvinium, being an approved drug, may prove especially suitable as a good starting point for further medicinal chemistry efforts, leading to design and development of even more potent structural analogs as selective PD-1/PD-L1 inhibitors. Furthermore, the adopted integrated virtual screening protocol may prove useful in screening other larger databases of lead- and drug-like molecules for hit identification in the domain of small-molecule PD-1/PD-L1 inhibitors.     

Reconstitution of Monocyte Subsets and PD-L1 Expression but Not T Cell PD-1 Expression in Obstructive Sleep Apnea Patients upon PAP Therapy

Obstructive sleep apnea (OSA) is characterized by nocturnal breathing intermissions resulting in oxidative stress and eventually, a low-grade systemic inflammation. The study aimed to investigate the impact of positive airway pressure (PAP) therapy on the inflammatory milieu as measured by monocyte and T cell phenotypic alterations. Participants were assessed for their OSA severity before PAP therapy and about six months later, including patient-reported outcome and therapy usage by telemetry readout. The distributions of the CD14/CD16-characterized monocyte subsets as well as the CD4/CD8-characterized effector T cell subsets with regard to their PD-1 and PD-L1 expression were analyzed by flow cytometry from blood samples. Data of 25 patients revealed a significant reconstitution of the monocyte subset distribution and a decrease in PD-L1 expression on pan-monocytes and CD8⁺ T cells without an association to initial AHI and overweight. The PD-1 expression was still increased on T cell subsets, especially on CD4⁺ TH17/22 cells. We conclude that PAP therapy might have a rapid effect on the monocyte phenotype and overall PD-L1 expression levels. However, T cell immune alterations especially on TH17/22 cells persist longer, indicating an ongoing disturbance of the adaptive immune system.     

PD-L1 Expression in 65 Conjunctival Melanomas and Its Association with Clinical Outcome

Conjunctival melanoma (CM) iss a rare and aggressive tumour that is increasing in frequency. The prognostic value of PD-L1 expression, alone or in combination with CD8 and PD-1 expression and the BRAF and NRAS status, has not been determined in CM to date. We evaluated the expression of PD-L1, CD8, PD-1 in CM and investigated whether there was an association between the expression of these markers and the BRAF and NRAS molecular profile as well as some clinico-pathological criteria. A total of sixty-five CM were assessed for PD-L1, PD-1, and CD8 expression by immunohistochemistry (IHC) and for BRAF and NRAS genomic alterations using molecular biology techniques and anti-BRAF and anti-NRAS antibodies. PD-L1 expression in tumour cells (TC) was very low or absent but detected in tumour-infiltrating immune cells (IC). A correlation was observed between the expression of PD-L1, CD8, and PD-1 in IC. No correlation between PD-L1 expression (in tumour and/or immune cells) and BRAF or NRAS mutations was observed. PD-L1 expression in IC correlated with a higher pTNM stage and PD-L1 expression in TC with worse disease-specific survival. PD-L1 expression is a potential prognostic biomarker that correlates with poor prognosis in CM patients.     

PD-1/PD-L1 Immuno-Mediated Therapy in NAFLD: Advantages and Obstacles in the Treatment of Advanced Disease

Non-alcoholic fatty liver disease (NAFLD) is characterized by an enhanced activation of the immune system, which predispose the evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Resident macrophages and leukocytes exert a key role in the pathogenesis of NAFLD. In particular, CD4+ effector T cells are activated during the early stages of liver inflammation and are followed by the increase of natural killer T cells and of CD8+ T cytotoxic lymphocytes which contribute to auto-aggressive tissue damage. To counteract T cells activation, programmed cell death 1 (PD-1) and its ligand PDL-1 are exposed respectively on lymphocytes and liver cells’ surface and can be targeted for therapy by using specific monoclonal antibodies, such as of Nivolumab, Pembrolizumab, and Atezolizumab. Despite the combination of Atezolizumab and Bevacizumab has been approved for the treatment of advanced HCC, PD-1/PD-L1 blockage treatment has not been approved for NAFLD and adjuvant immunotherapy does not seem to improve survival of patients with early-stage HCC. In this regard, different ongoing phase III trials are testing the efficacy of anti-PD-1/PD-L1 antibodies in HCC patients as first line therapy and in combination with other treatments. However, in the context of NAFLD, immune checkpoints inhibitors may not improve HCC prognosis, even worse leading to an increase of CD8+PD-1+ T cells and effector cytokines which aggravate liver damage. Here, we will describe the main pathogenetic mechanisms which characterize the immune system involvement in NAFLD discussing advantages and obstacles of anti PD-1/PDL-1 immunotherapy.     

The Role of Staphylococcus aureus and Its Toxins in the Pathogenesis of Allergic Asthma

Bronchial asthma is one of the most common chronic diseases worldwide and affects more than 300 million patients. Allergic asthma affects the majority of asthmatic children as well as approximately 50% of adult asthmatics. It is characterized by a Th2-mediated immune response against aeroallergens. Many aspects of the overall pathophysiology are known, while the underlying mechanisms and predisposing factors remain largely elusive today. Over the last decade, respiratory colonization with Staphylococcus aureus (S. aureus), a Gram-positive facultative bacterial pathogen, came into focus as a risk factor for the development of atopic respiratory diseases. More than 30% of the world’s population is constantly colonized with S. aureus in their nasopharynx. This colonization is mostly asymptomatic, but in immunocompromised patients, it can lead to serious complications including pneumonia, sepsis, or even death. S. aureus is known for its ability to produce a wide range of proteins including toxins, serine-protease-like proteins, and protein A. In this review, we provide an overview of the current knowledge about the pathophysiology of allergic asthma and to what extent it can be affected by different toxins produced by S. aureus. Intensifying this knowledge might lead to new preventive strategies for atopic respiratory diseases.     

PD-1/PD-L1, MDSC Pathways,and Checkpoint Inhibitor Therapy in Ph(-) Myeloproliferative Neoplasm: A Review

There has been significant progress in immune checkpoint inhibitor (CPI) therapy in many solid tumor types. However, only a single failed study has been published in treating Ph(-) myeloproliferative neoplasm (MPN). To make progress in CPI studies on this disease, herein, we review and summarize the mechanisms of activation of the PD-L1 promoter, which are as follows: (a) the extrinsic mechanism, which is activated by interferon gamma (IFN γ) by tumor infiltration lymphocytes (TIL) and NK cells; (b) the intrinsic mechanism of EGFR or PTEN loss resulting in the activation of the MAPK and AKT pathways and then stat 1 and 3 activation; and (c) 9p24 amplicon amplification, resulting in PD-L1 and Jak2 activation. We also review the literature and postulate that many of the failures of CPI therapy in MPN are likely due to excessive MDSC activities. We list all of the anti-MDSC agents, especially those with ruxolitinib, IMID compounds, and BTK inhibitors, which may be combined with CPI therapy in the future as part of clinical trials applying CPI therapy to Ph(-) MPN.     

Mechanisms of PD-L1 Regulation in Malignant and Virus-Infected Cells

Programmed cell death protein 1 (PD-1), a receptor on T cells, and its ligand, PD-L1, have been a topic of much interest in cancer research. Both tumour and virus-infected cells can upregulate PD-L1 to suppress cytotoxic T-cell killing. Research on the PD-1/PD-L1 axis has led to the development of anti-PD-1/PD-L1 immune checkpoint blockades (ICBs) as promising cancer therapies. Although effective in some cancer patients, for many, this form of treatment is ineffective due to a lack of immunogenicity in the tumour microenvironment (TME). Despite the development of therapies targeting the PD-1/PD-L1 axis, the mechanisms and pathways through which these proteins are regulated are not completely understood. In this review, we discuss the latest research on molecules of inflammation and innate immunity that regulate PD-L1 expression, how its expression is regulated during viral infection, and how it is modulated by different cancer therapies. We also highlight existing research on the development of different combination therapies with anti-PD-1/PD-L1 antibodies. This information can be used to develop better cancer immunotherapies that take into consideration the pathways involved in the PD-1/PD-L1 axis, so these molecules do not reduce their efficacy, which is currently seen with some cancer therapies. This review will also assist in understanding how the TME changes during treatment, which will provide further rationale for combination therapies.     

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68⁺ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8⁺ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4⁺ and CD8⁺ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8⁺ T cells in HCC.     

Role of ERK Pathway in the Pathogenesis of Atopic Dermatitis and Its Potential as a Therapeutic Target

Atopic dermatitis (AD) is an eczematous skin disorder characterized by type 2 inflammation, barrier disruption, and intense itch. In addition to type 2 cytokines, many other cytokines, such as interferon gamma (IFN-γ), interleukin 17 (IL-17), and interleukin 22 (IL-22), play roles in the pathogenesis of AD. It has been reported that the extracellular signal-regulated kinase (ERK) is downstream of such cytokines. However, the involvement of the ERK pathway in the pathogenesis of AD has not yet been investigated. We examined the expression of p-ERK in mouse and human AD skin. We also investigated the effects of the topical application of an ERK inhibitor on the dermatitis score, transepidermal water loss (TEWL), histological change, and expression of filaggrin, using an AD-like NC/Nga murine model. The effects of an ERK inhibitor on filaggrin expression in normal human epidermal keratinocytes (NHEKs) and on chemokine production from bone marrow-derived dendritic cells (BMDCs) were also evaluated. p-ERK was highly expressed in mouse and human AD skin. Topical application of an ERK inhibitor alleviated the clinical symptoms, histological changes, TEWL, and decrease in expression of filaggrin in the AD-like NC/Nga murine model. The ERK inhibitor also restored the IL-4 induced reduction in the expression of filaggrin in NHEK, and inhibited chemokine production from BMDC induced by IL-4. These results indicate that the ERK pathway is involved in the pathogenesis of AD, and suggest that the ERK pathway has potential as a therapeutic target for AD in the future.     

New Insights into the Role of Insulin and Hypothalamic-Pituitary-Adrenal (HPA) Axis in the Metabolic Syndrome

Recent data suggests that (pre)diabetes onset is preceded by a period of hyperinsulinemia. Consumption of the “modern” Western diet, over-nutrition, genetic background, decreased hepatic insulin clearance, and fetal/metabolic programming may increase insulin secretion, thereby causing chronic hyperinsulinemia. Hyperinsulinemia is an important etiological factor in the development of metabolic syndrome, type 2 diabetes, cardiovascular disease, polycystic ovarian syndrome, and Alzheimer’s disease. Recent data suggests that the onset of prediabetes and diabetes are preceded by a variable period of hyperinsulinemia. Emerging data suggest that chromic hyperinsulinemia is also a driving force for increased activation of the hypothalamic-adrenal-pituitary (HPA) axis in subjects with the metabolic syndrome, leading to a state of “functional hypercortisolism”. This “functional hypercortisolism” by antagonizing insulin actions may prevent hypoglycemia. It also disturbs energy balance by shifting energy fluxes away from muscles toward abdominal fat stores. Synergistic effects of hyperinsulinemia and “functional hypercortisolism” promote abdominal visceral obesity and insulin resistance which are core pathophysiological components of the metabolic syndrome. It is hypothesized that hyperinsulinemia-induced increased activation of the HPA axis plays an important etiological role in the development of the metabolic syndrome and its consequences. Numerous studies have demonstrated reversibility of hyperinsulinemia with lifestyle, surgical, and pharmaceutical-based therapies. Longitudinal studies should be performed to investigate whether strategies that reduce hyperinsulinemia at an early stage are successfully in preventing increased activation of the HPA axis and the metabolic syndrome.     

Analysis of Results of Specific IgE in 100 Atopic Dermatitis Patients with the Use of Multiplex Examination ALEX2—Allergy Explorer

Background and aim: Progress in laboratory diagnostics of IgE-mediated allergy is the use of component-resolved diagnosis. Our study analyses the results of specific IgE to 295 allergen reagents (117 allergenic extracts and 178 molecular components) in patients suffering from atopic dermatitis (AD) with the use of ALEX2 Allergy Explorer. Method: The complete dermatological and allergological examination, including the examination of the sensitization to molecular components with ALEX2 Allergy Explorer testing, was performed. The statistical analysis of results was performed with these methods: TURF (total unduplicated reach and frequency), best reach and frequency by group size, two-sided tests, Fisher’s exact test, and chi-square test (at an expected minimum frequency of at least 5). Results: Altogether, 100 atopic dermatitis patients were examined: 48 men, 52 women, the average age 40.9 years, min. age 14 years, max. age 67 years. The high and very high level of specific IgE was reached in 75.0% of patients to 18 molecular components: from PR-10 proteins (Aln g 1, Bet v 1, Cor a1.0103, Cor a1.0401, Fag s 1), lipocalin (Can f 1), NPC2 family (Der f 2, Der p 2), uteroglobin (Fel d 1), from Alternaria alternata (Alt a 1), Beta expansin (Lol p 1, Phl p 1), molecular components from Timothy, cultivated rye (Secc pollen) and peritrophin-like protein domain Der p 23. The high and very high level of specific IgE to other lipocalins (Fel d 7, Can f 4), to arginine kinase (Bla g 9, German cockroach), and to allergen extracts Art v (mugwort), and Cyn d (Bermuda grass) reached 52.0% of patients. The severity of AD is in significant relation to the sensitization to molecular components of storage mites (Gly d 2, Lep d 2—NPC2 family), lipocalins (Can f 1, Can f 2, Can f 4, and Can f 6), arginine kinase (Asp f 6, Bla g 9, Der p 20, Pen m 2), uteroglobin (Fel d 1, Ory c 3), Mn superoxide dismutase (Mala s 11), PR-10 proteins (Fag s 1, Mal d 1, Cor a 1.0401, Cor a 1.0103), molecular components of the peritrophin-like domain (Der p 21, Der p 23), and to Secc pollen. In the subgroup of patients suffering from bronchial asthma, the significant role play molecular components from house dust mites and storage mites (Lep d 2, Der p 2, Der f 2—NPC2 family), cysteine protease (Der p 1), peritrophin-like protein domain (Der p 21, Der p 23), enolase from Alternaria alternata (Alt a 6), and Beta expansin Phl p 1. Conclusion: The results of our study demonstrate the detailed profile of sensitization to allergens reagents (allergen extract and molecular components) in patients with atopic dermatitis. We show the significance of disturbed epidermal barrier, resulting in increased penetration of allergens. We confirmed the significant relationship between the severity of AD, the occurrence of bronchial asthma and allergic rhinitis, and high levels of specific IgE to allergen reagents. Our results may be important for regime measures and immunotherapy; Der p 23 shall be considered as an essential component for the diagnosis and specific immunotherapy of house dust mite allergy.     

Molecular Mechanism of Small-Molecule Inhibitors in Blocking the PD-1/PD-L1 Pathway through PD-L1 Dimerization

Programmed cell death-1 (PD-1), which is a molecule involved in the inhibitory signal in the immune system and is important due to blocking of the interactions between PD-1 and programmed cell death ligand-1 (PD-L1), has emerged as a promising immunotherapy for treating cancer. In this work, molecular dynamics simulations were performed on complex systems consisting of the PD-L1 dimer with (S)-BMS-200, (R)-BMS-200 and (MOD)-BMS-200 (i.e., S, R and MOD systems) to systematically evaluate the inhibitory mechanism of BMS-200-related small-molecule inhibitors in detail. Among them, (MOD)-BMS-200 was modified from the original (S)-BMS-200 by replacing the hydroxyl group with a carbonyl to remove its chirality. Binding free energy analysis indicates that BMS-200-related inhibitors can promote the dimerization of PD-L1. Meanwhile, no significant differences were observed between the S and MOD systems, though the R system exhibited a slightly higher energy. Residue energy decomposition, nonbonded interaction, and contact number analyses show that the inhibitors mainly bind with the C, F and G regions of the PD-L1 dimer, while nonpolar interactions of key residues Ile54, Tyr56, Met115, Ala121 and Tyr123 on both PD-L1 monomers are the dominant binding-related stability factors. Furthermore, compared with (S)-BMS-200, (R)-BMS-200 is more likely to form hydrogen bonds with charged residues. Finally, free energy landscape and protein–protein interaction analyses show that the key residues of the PD-L1 dimer undergo remarkable conformational changes induced by (S)-BMS-200, which boosts its intimate interactions. This systematic investigation provides a comprehensive molecular insight into the ligand recognition process, which will benefit the design of new small-molecule inhibitors targeting PD-L1 for use in anticancer therapy.     

Cutting Edge of the Pathogenesis of Atopic Dermatitis: Sphingomyelin Deacylase,the Enzyme Involved in Its Ceramide Deficiency,Plays a Pivotal Role

Atopic dermatitis (AD) is characterized clinically by severe dry skin and functionally by both a cutaneous barrier disruption and an impaired water-holding capacity in the stratum corneum (SC) even in the nonlesional skin. The combination of the disrupted barrier and water-holding functions in nonlesional skin is closely linked to the disease severity of AD, which suggests that the barrier abnormality as well as the water deficiency are elicited as a result of the induced dermatitis and subsequently trigger the recurrence of dermatitis. These functional abnormalities of the SC are mainly attributable to significantly decreased levels of total ceramides and the altered ceramide profile in the SC. Clinical studies using a synthetic pseudo-ceramide (pCer) that can function as a natural ceramide have indicated the superior clinical efficacy of pCer and, more importantly, have shown that the ceramide deficiency rather than changes in the ceramide profile in the SC of AD patients plays a central role in the pathogenesis of AD. Clinical studies of infants with AD have shown that the barrier disruption due to the ceramide deficiency is not inherent and is essentially dependent on postinflammatory events in those infants. Consistently, the recovery of trans-epidermal water loss after tape-stripping occurs at a significantly slower rate only at 1 day post-tape-stripping in AD skin compared with healthy control (HC) skin. This resembles the recovery pattern observed in Niemann–Pick disease, which is caused by an acid sphingomyelinase (aSMase) deficiency. Further, comparison of ceramide levels in the SC between before and after tape-stripping revealed that whereas ceramide levels in HC skin are significantly upregulated at 4 days post-tape-stripping, their ceramide levels remain substantially unchanged at 4 days post-tape-stripping. Taken together, the sum of these findings strongly suggests that an impaired homeostasis of a ceramide-generating process may be associated with these abnormalities. We have discovered a novel enzyme, sphingomyelin (SM) deacylase, which cleaves the N-acyl linkage of SM and glucosylceramide (GCer). The activity of SM deacylase is significantly increased in AD lesional epidermis as well as in the involved and uninvolved SC of AD skin, but not in the skin of patients with contact dermatitis or chronic eczema, compared with HC skin. SM deacylase competes with aSMase and β-glucocerebrosidase (BGCase) to hydrolyze their common substrates, SM and GCer, to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine (GSP), respectively, instead of ceramide. Consistently, those reaction products (SPC and GSP) accumulate to a greater extent in the involved and uninvolved SC of AD skin compared with chronic eczema or contact dermatitis skin as well as HC skin. Successive chromatographies were used to purify SM deacylase to homogeneity with a single band of ≈43 kDa and with an enrichment of >14,000-fold. Analysis of a protein spot with SM deacylase activity separated by 2D-SDS-PAGE using MALDI-TOF MS/MS allowed its amino acid sequence to be determined and to identify it as the β-subunit of acid ceramidase (aCDase), an enzyme consisting of α- and β-subunits linked by amino-bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ≈56 and ≈13 kDa and the β-subunit at ≈43 kDa, the purified SM deacylase was detectable only by the antibody to the β-subunit at ≈43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with an apparent size of ≈40 kDa upon gel chromatography in contrast to aCDase activity with an apparent size of ≈50 kDa in untreated recombinant human aCDase. These results provide new insights into the essential role of SM deacylase as the β-subunit aCDase that causes the ceramide deficiency in AD skin.     

Role of PD-L1 in Gut Mucosa Tolerance and Chronic Inflammation

The gastrointestinal (GI) mucosa is among the most complex systems in the body. It has a diverse commensal microbiome challenged continuously by food and microbial components while delivering essential nutrients and defending against pathogens. For these reasons, regulatory cells and receptors are likely to play a central role in maintaining the gut mucosal homeostasis. Recent lessons from cancer immunotherapy point out the critical role of the B7 negative co-stimulator PD-L1 in mucosal homeostasis. In this review, we summarize the current knowledge supporting the critical role of PD-L1 in gastrointestinal mucosal tolerance and how abnormalities in its expression and signaling contribute to gut inflammation and cancers. Abnormal expression of PD-L1 and/or the PD-1/PD-L1 signaling pathways have been observed in the pathology of the GI tract. We also discuss the current gap in our knowledge with regards to PD-L1 signaling in the GI tract under homeostasis and pathology. Finally, we summarize the current understanding of how this pathway is currently targeted to develop novel therapeutic approaches.     

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys¹⁸ in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg¹⁷ was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys¹⁸ does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.