Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: Structural basis of their function.

The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.

Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF.

Several cancer core regulatory circuitries (CRCs) depend on the sustained generation of DNA accessibility by SWI/SNF chromatin remodelers. However, the window when SWI/SNF is acutely essential in these settings has not been identified. Here we used neuroblastoma (NB) cells to model and dissect the relationship between cell-cycle progression and SWI/SNF ATPase activity. We find that SWI/SNF inactivation impairs coordinated occupancy of non-pioneer CRC members at enhancers within 1 hour, rapidly breaking their autoregulation. By precisely timing inhibitor treatment following synchronization, we show that SWI/SNF is dispensable for survival in S and G2/M, but becomes acutely essential only during G1 phase. We furthermore developed a new approach to analyze the oscillating patterns of genome-wide DNA accessibility across the cell cycle, which revealed that SWI/SNF-dependent CRC binding sites are enriched at enhancers with peak accessibility during G1 phase, where they activate genes involved in cell-cycle progression. SWI/SNF inhibition strongly impairs G1-S transition and potentiates the ability of retinoids used clinically to induce cell-cycle exit. Similar cell-cycle effects in diverse SWI/SNF-addicted settings highlight G1-S transition as a common cause of SWI/SNF dependency. Our results illustrate that deeper knowledge of the temporal patterns of enhancer-related dependencies may aid the rational targeting of addicted cancers.

Cancer cells driven by runaway transcription factor networks frequently depend on the cellular machinery that promotes DNA accessibility. For this reason, recently developed small molecules that impair SWI/SNF (or BAF) chromatin remodeling activity have been under active evaluation as anti-cancer agents. However, exactly when SWI/SNF activity is essential in dependent cancers has remained unknown. By combining live-cell imaging and genome-wide profiling in neuroblastoma cells, Cermakova et al. discover that SWI/SNF activity is needed for survival only during G1 phase of the cell cycle. The authors reveal that in several cancer settings, dependency on SWI/SNF arises from the need to reactivate factors involved in G1-S transition. Because of this role, authors find that SWI/SNF inhibition potentiates cell-cycle exit by retinoic acid.

Design, synthesis and evaluation of novel thieno[2,3d]pyrimidine derivatives as potent and specific RIPK2 inhibitors.

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2.

Exploring positions 6 and 7 of a quinazoline-based scaffold leads to changes in selectivity and potency towards RIPK2/3 kinases.

Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research.

Inhibition of FLT3-ITD Kinase in Acute Myeloid Leukemia by New Imidazo[1,2-b]pyridazine Derivatives Identified by Scaffold Hopping.

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

Rational Design of Highly Potent SARS-CoV-2 nsp14 Methyltransferase Inhibitors.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Hydrogen-Bonding Interactions of 8-Substituted Purine Derivatives.

Hydrogen bonding between nucleobases is a crucial noncovalent interaction for life on Earth. Canonical nucleobases form base pairs according to two main geometries: Watson-Crick pairing, which enables the static functions of nucleic acids, such as the storing of genetic information; and Hoogsteen pairing, which facilitates the dynamic functions of these biomacromolecules. This precisely tuned system can be affected by oxidation or substitution of nucleobases, leading to changes in their hydrogen-bonding patterns. This paper presents an investigation into the intermolecular interactions of various 8-substituted purine derivatives with their hydrogen-bonding partners. The systems were analyzed using nuclear magnetic resonance spectroscopy and density functional theory calculations. Our results demonstrate that the stability of hydrogen-bonded complexes, or base pairs, depends primarily on the number of intermolecular H-bonds and their donor-acceptor alternation. No strong preferences for a particular geometry, either Watson-Crick or Hoogsteen, were found.

SWI/SNF Blockade Disrupts PU.1-Directed Enhancer Programs in Normal Hematopoietic Cells and Acute Myeloid Leukemia.

In acute myeloid leukemia (AML), SWI/SNF chromatin remodeling complexes sustain leukemic identity by driving high levels of MYC. Previous studies have implicated the hematopoietic transcription factor PU.1 (SPI1) as an important target of SWI/SNF inhibition, but PU.1 is widely regarded to have pioneer-like activity. As a result, many questions have remained regarding the interplay between PU.1 and SWI/SNF in AML as well as normal hematopoiesis. Here we found that PU.1 binds to most of its targets in a SWI/SNF-independent manner and recruits SWI/SNF to promote accessibility for other AML core regulatory factors, including RUNX1, LMO2, and MEIS1. SWI/SNF inhibition in AML cells reduced DNA accessibility and binding of these factors at PU.1 sites and redistributed PU.1 to promoters. Analysis of nontumor hematopoietic cells revealed that similar effects also impair PU.1-dependent B-cell and monocyte populations. Nevertheless, SWI/SNF inhibition induced profound therapeutic response in an immunocompetent AML mouse model as well as in primary human AML samples. In vivo, SWI/SNF inhibition promoted leukemic differentiation and reduced the leukemic stem cell burden in bone marrow but also induced leukopenia. These results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. SIGNIFICANCE: Disruption of PU.1-directed enhancer programs upon SWI/SNF inhibition causes differentiation of AML cells and induces leukopenia of PU.1-dependent B cells and monocytes, revealing the on- and off-tumor effects of SWI/SNF blockade.

The hydrogen bond continuum in solid isonicotinic acid.

The understanding and correct description of intermolecular hydrogen bonds are crucial in the field of multicomponent pharmaceutical solids, such as salts and cocrystals. Solid isonicotinic acid can serve as a suitable model for the development of methods that can accurately characterize these hydrogen bonds. Experimental solid-state NMR has revealed a remarkable temperature dependence and deuterium-isotope-induced changes of the chemical shifts of the atoms involved in the intermolecular hydrogen bond; these NMR data are related to changes of the average position of the hydrogen atom. These changes of NMR parameters were interpreted using periodic DFT path-integral molecular dynamics (PIMD) simulations. The small size of the unit cell of isonicotinic acid allowed for PIMD simulations with the computationally demanding hybrid DFT functional. Calculations of NMR parameters based on the hybrid-functional PIMD simulations are in excellent agreement with experiment. It is thus demonstrated that an accurate characterization of intermolecular hydrogen bonds can be achieved by a combination of NMR experiments and advanced computations.

Structure-based design and modular synthesis of novel PI4K class II inhibitors bearing a 4-aminoquinazoline scaffold.

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.

Discovery of isonucleotidic CDNs as potent STING agonists with immunomodulatory potential.

Stimulator of interferon genes (STING) is an adaptor protein of the cGAS-STING signaling pathway involved in the sensing of cytosolic DNA. It functions as a receptor for cyclic dinucleotides (CDNs) and, upon their binding, mediates cytokine expression and host immunity. Besides naturally occurring CDNs, various synthetic CDNs, such as ADU-S100, have been reported to effectively activate STING and are being evaluated in clinical trials for the treatment of cancer. Here, we describe the preparation of a unique new class of STING agonists: isonucleotidic cyclic dinucleotides and the synthesis of their prodrugs. The presented CDNs stimulate STING with comparable efficiency to ADU-S100, whereas their prodrugs demonstrate activity up to four orders of magnitude better due to the improved cellular uptake. The compounds are very potent inducers of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs). We also report the X-ray crystal structure of the lead inhibitor bound to the wild-type (WT) STING.

Non-Nucleotide RNA-Dependent RNA Polymerase Inhibitor That Blocks SARS-CoV-2 Replication.

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.

The Structure-Based Design of SARS-CoV-2 nsp14 Methyltransferase Ligands Yields Nanomolar Inhibitors.

In this study, we have focused on the structure-based design of the inhibitors of one of the two SARS-CoV-2 methyltransferases (MTases), nsp14. This MTase catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to cap the guanosine triphosphate moiety of the newly synthesized viral RNA, yielding the methylated capped RNA and S-adenosyl-l-homocysteine (SAH). As the crystal structure of SARS-CoV-2 nsp14 is unknown, we have taken advantage of its high homology to SARS-CoV nsp14 and prepared its homology model, which has allowed us to identify novel SAH derivatives modified at the adenine nucleobase as inhibitors of this important viral target. We have synthesized and tested the designed compounds in vitro and shown that these derivatives exert unprecedented inhibitory activity against this crucial enzyme. The docking studies nicely explain the contribution of an aromatic part attached by a linker to the position 7 of the 7-deaza analogues of SAH.

Synthesis and Biological Evaluation of Phosphoester and Phosphorothioate Prodrugs of STING Agonist 3',3'-c-Di(2'F,2'dAMP).

Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3',3'-c-di(2'F,2'dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2' position of 3',3'-c-di(2'F,2'dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.

Remdesivir triphosphate can efficiently inhibit the RNA-dependent RNA polymerase from various flaviviruses.

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.

Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer's Disease.

Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.

Optimization of norbornyl-based carbocyclic nucleoside analogs as cyclin-dependent kinase 2 inhibitors.

We report on the discovery of norbornyl moiety as a novel structural motif for cyclin-dependent kinase 2 (CDK2) inhibitors which was identified by screening a carbocyclic nucleoside analogue library. Three micromolar hits were expanded by the use of medicinal chemistry methods into a series of 16 novel compounds. They had prevailingly micromolar activities against CDK2 and the best compound of the series attained IC50 of 190 nM. The binding modes were explored in molecular details by modeling and docking. Quantum mechanics-based scoring was used to rationalize the affinities. In conclusion, the discovered 9-hydroxymethylnorbornyl moiety was shown by joint experimental-theoretical efforts to be able to serve as a novel substituent for CDK2 inhibitors. This finding opens door to the exploration of chemical space towards more effective derivatives targeting this important class of protein kinases.

Could 5'-N and S ProTide analogues work as prodrugs of antiviral agents?

The nucleoside/nucleotide derived antiviral agents have been the most important components of antiviral therapy used in clinics. Recently, the focus of the medicinal chemists within this exciting research field has been affected mainly by the lack of effective therapies for the Hepatitis C virus (HCV) infection and several other "neglected" diseases caused by viruses such as Zika or Dengue. 2'-Methyl modified nucleosides and their monophosphate prodrugs (ProTides) have revolutionized the therapies for HCV in the last few years and, according to the latest research efforts, have also brought a promise for treatment of diseases caused by other members of Flaviviridae family. Here, we report on the design and synthesis of 5'-N and S modified ProTides derived from 2'-methyladenosine. We studied potential applicability of these derivatives as prodrugs of this archetypal antiviral compound.

Determination of nucleobase-pairing free energies from rotamer equilibria of 2-(methylamino)pyrimidines.

A fast straightforward method for the determination of free energies of modified nucleobase pairing is proposed. The method is based on the nuclear magnetic resonance (NMR) spectroscopy monitoring of conformational changes of 2-(methylamino)-pyrimidines upon intermolecular binding.

A novel and potent brain penetrant inhibitor of extracellular vesicle release.

BACKGROUND AND PURPOSE: Extracellular vesicles (EVs) are constitutively shed from cells and released by various stimuli. Their protein and RNA cargo are modified by the stimulus, and in disease conditions can carry pathological cargo involved in disease progression. Neutral sphingomyelinase 2 (nSMase2) is a major regulator in at least one of several independent routes of EV biogenesis, and its inhibition is a promising new therapeutic approach for neurological disorders. Unfortunately, known inhibitors exhibit µM potency, poor physicochemical properties, and/or limited brain penetration. Here, we sought to identify a drug-like inhibitor of nSMase2. EXPERIMENTAL APPROACH: We conducted a human nSMase2 high throughput screen (>365,000 compounds). Selected hits were optimized focusing on potency, selectivity, metabolic stability, pharmacokinetics, and ability to inhibit EV release in vitro and in vivo. KEY RESULTS: We identified phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC), a potent (pIC50  = 6.57) and selective non-competitive inhibitor of nSMase2. PDDC was metabolically stable, with excellent oral bioavailability (%F = 88) and brain penetration (AUCbrain /AUCplasma  = 0.60). PDDC dose-dependently (pEC50  = 5.5) inhibited release of astrocyte-derived extracellular vesicles (ADEV). In an in vivo inflammatory brain injury model, PDDC robustly inhibited ADEV release and the associated peripheral immunological response. A closely related inactive PDDC analogue was ineffective. CONCLUSION AND IMPLICATIONS: PDDC is a structurally novel, potent, orally available, and brain penetrant inhibitor of nSMase2. PDDC inhibited release of ADEVs in tissue culture and in vivo. PDDC is actively being tested in animal models of neurological disease and, along with closely related analogues, is being considered for clinical translation.