Class Of Small Molecules Research Articles

Abstract ND04: BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy

Abstract Velcrin compounds are a class of small molecules that induce complex formation between PDE3A and SLFN12, killing cancer cells that express elevated levels of these two proteins by a mechanism independent of PDE3A enzymatic inhibition. Instead, PDE3A binding stimulates the RNase activity of SLFN12, resulting in cleavage of the specific SLFN12 substrate, tRNA-Leu-TAA. Cleavage of tRNA-Leu-TAA in turn causes ribosomal pausing, inhibition of protein synthesis, and cancer cell death. Unlike traditional targeted therapies that leverage dependencies created in cancer cells by genomic alterations, velcrins instead kill cancer cells by a gain-of-function mechanism dependent on the RNase activity of SLFN12. In a collaboration between the Broad Institute and Bayer Pharmaceuticals, we developed the first velcrin, BAY 2666605, to enter Phase I clinical trials. BAY 2666605 is active in cell line and patient-derived xenografts of several tumor types, specifically where elevated levels of the two biomarkers, PDE3A and SLFN12, are expressed. Biomarker-positive tumors are especially enriched among melanomas, and we have consistently observed tumor regression in biomarker-positive melanoma tumor models in vivo. BAY 2666605 furthermore shows drug-like properties, excellent brain penetration, increased stimulation of SLFN12 RNase activity, and reduced inhibition of PDE3A enzymatic activity compared with most other velcrins and approved PDE3A inhibitors. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors that co-express PDE3A and SLFN12, including melanoma, ovarian cancer, and sarcoma. Citation Format: Stefan Gradl, Sooncheol Lee, Martin Lange, Xiaoyun Wu, Silvia Goldoni, Timothy Lewis, Charlotte Kopitz, Colin Garvie, Philip Lienau, Stephanie Hoyt, Henrik Seidel, Stephan Kaulfuss, Manuel Ellermann, Luc de Waal, Adrian Tersteegen, Sven Golfier, Detlev Suelzle, Christa Hegele-Hartung, James Carr, Frederick Brookfield, Michael Bruening, Melanie Berthold, Thibaud Jourdan, Monica Schenone, Galen Gao, Joseph McGaunn, Antje Wengner, Elisa Aquilanti, Franziska Siegel, Marine Garrido, Annette Walter, Isabelle Genvresse, Andrew Cherniack, Stuart Schreiber, Knut Eis, Ashley Eheim, Matthew Meyerson, Heidi Greulich. BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND04.

Abstract 1823: Imipridones show pre-clinical efficacy in MYCN-amplified and MYCN non-amplified neuroblastoma cell lines

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood, accounting for 15% of all pediatric cancer-related deaths, with around 63% survival for high-risk patients. Treatment remains limited for advanced disease, calling for the development of novel therapies. We investigated the anti-tumor effect of three imipridones (ONC201, ONC206, and ONC212), a promising new class of small molecules, in the treatment of neuroblastoma. Imipridones are potent anticancer drugs that have previously shown efficacy for a variety of adult and pediatric malignancies. ONC201 and ONC206 are antagonists of dopamine receptor D2 (DRD2), while ONC212 is an agonist of GPR132, both of which are overexpressed in many cancers. We performed drug treatment with ONC201, ONC206, and ONC212 on established MYCN-amplified SK-N-BE(2) and MYCN non-amplified SH-SY5Y pediatric neuroblastoma cell lines in vitro. Cell viability assays were performed 72 hours post-treatment to generate dose responses curves. The IC50s for ONC201, ONC206, and ONC212 were 17.82 uM, 547 nM, and 70 nM for SK-N-BE(2), and 997 nM, 314 nM, and 6 nM for SH-SY5Y. Imipridones demonstrated greater efficacy for the non-MYCN-amplified cell line. Using protein quantification studies and downstream target analysis on drug-treated cells, we investigated the mechanisms of how these therapies caused cell death. We showed that imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). They also induce tumor cell apoptosis by modifying the mitochondrial Clp protease complex, decreasing expression of chaperone subunit ClpX and activating mitochondrial proteolysis. Histone deacetylases (HDACs) play a role in controlling MYCN function, which is amplified in more aggressive neuroblastoma; HDAC activity and MYCN are also upregulated in chemotherapy-resistant neuroblastoma. Cell viability assays with HDAC inhibitors Vorinostat and Panobinostat demonstrated single-agent efficacy in vitro. The IC50s for Vorinostat and Panobinostat were 609 nM and 5.51 nM for SK-N-BE(2), and 884 nM and 6.13 nM for SH-SY5Y. However, further investigation is needed to determine synergy and mechanisms of synergy when histone deacetylase (HDAC) inhibitors are used in novel combinations with imipridones. Overall, our data reveals promise in imipridone therapy for neuroblastoma, and future studies are proposed to explore potential novel therapeutic combinations in this difficult-to-treat pediatric cancer. Citation Format: Claire Lin, Wen-I Chang, Joshua N. Honeyman, Lanlan Zhou, Varun V. Prabhu, Joshua Allen, Wafik El-Deiry. Imipridones show pre-clinical efficacy in MYCN-amplified and MYCN non-amplified neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1823.

Chimeric Ligands of Pili and Lectin A Inhibit Tolerance, Persistence, and Virulence Factors of Pseudomonas aeruginosa over a Wide Range of Phenotypes.

Bacteria readily form resilient phenotypes to counter environmental and antibiotic stresses. Here, we demonstrate a class of small molecules that inhibit a wide range of Pseudomonas aeruginosa phenotypes and enable antibiotics to kill previously tolerant bacteria, preventing the transition of tolerant bacteria into a persistent population. We identified two proteins, type IV pili and lectin LecA, as receptors for our molecules by methods including a new label-free assay based on bacterial motility sensing the chemicals in the environment, the chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure–activity relationship studies reveal a molecule that inhibits only pili appendage and a class of chimeric ligands that inhibit both LecA and pili. Important structural elements of the ligand are identified for each protein. This selective ligand binding identifies the phenotypes each protein receptor controls. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and is correlated with killing previously tolerant bacteria. Inhibiting pili appendages impedes swarming and twitching motilities and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously controls the multiple signaling mechanisms preventing bacteria to elude antibiotic treatments.

Resolution of the Working Meeting of Experts in “Dermatology”: Possibilities of systemic therapy of atopic dermatitis with selective immunosuppressants

On February 19, 2022, a working meeting of experts on the problem of atopic dermatitis was held in Moscow under the chairmanship of the Director of the State Scientific Center for Dermatovenereology and Cosmetology of the Ministry of Health of Russia, Doctor of Medical Sciences, Professor, Corresponding Member of the Russian Academy of Sciences A.A. Kubanov. Also in attendance was the Academician of the Russian Academy of Sciences, Chief Freelance Childrens Specialist in Preventive Medicine, Head of the Research Institute of Pediatrics and Childrens Health of the Central Clinical Hospital of the Russian Academy of Sciences of the Ministry of Science, and Higher Education of the Russian Federation, Doctor of Medical Sciences, Professor, Academician of the Russian Academy of Sciences, L.S. Namazova-Baranova, and also the Deputy Director for Clinical Work ― Chief Physician of the Federal State Budgetary Institution SSC Institute of Immunology FMBA of Russia Doctor of Medical Sciences, Professor N.I. Ilyina.
 The meeting raised germane issues of providing medical care, reducing the burden of the disease, and improving the quality of life of patients with atopic dermatitis. New possibilities and principles of systemic therapy in patients with moderate and severe atopic dermatitis were discussed. We analyzed data on a new representative of the small molecule class the selective Janus kinase 1 inhibitor, upadacitinib, which was registered in the Russian Federation in June 2021. This analysis aimed to determine its place in atopic dermatitis therapy algorithms and the possibility of the impact of its use as a therapy on the quality of life of patients in relation to the burden of diseases.
 The article has been published in several journals simultaneously with permission from authors and publishers. The parallel publication is available here: Capabilities of atopic dermatitis systemic therapy with selective immunosuppressors: resolution of the experts workshop of dermatology profile. Pediatric pharmacology. 2022;19(2):209211. DOI: https://doi.org/10.15690/pf.v19i2.2424

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma.

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein‐protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co‐elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic—Diffuse B‐cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

A Toolbox for the Generation of Chemical Probes for Baculovirus IAP Repeat Containing Proteins.

E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.

Synthesis, X-ray Crystallography, Hirshfeld Surface Analysis, Spectral Analysis, and Molecular Docking Studies on (E)–2–(1–(4–Fluorophenyl)Ethylidene) Hydrazine Carbothioamide

Synthesis, X-ray crystallography, spectral analysis, and molecular docking analysis on (E)–2–(1–(4–fluorophenyl) ethylidene) hydrazine carbothioamide (EFEHC) are performed. Thiosemicarbazones are a class of small molecules used as anticancer therapeutics. The FT–IR and FT-Raman spectra of EFEHC have been recorded in 4000–400 and 4000–100 cm–1, respectively. The present communication deals with the quantum chemical calculations of energies, geometrical structure, and vibrational wavenumbers of EFEHC using the density functional (DFT/B3LYP) method with 6–31G (d, p) basis set. The conformational analysis gives the energy values based on the change in the position of atoms in the EFEHC molecule. Hirshfeld surface analysis (HSs) is used to discuss the evaluation of intermolecular interactions. Molecular docking analysis of the small molecule EFEHC with macromolecule HMG–CoA protein showed that this is a good molecule and is suitable for anti-cholesterol targets.

In Vitro Pharmacological Characterization of CRN04894: The First Reported Oral, Selective Nonpeptide Melanocortin 2 Receptor Antagonist Evaluated in Phase 1 First‐in‐Human Clinical Trials

Adrenocorticotropic hormone (ACTH) is secreted from the pituitary gland and selectively activates the melanocortin type 2 receptor (MC2R) in the adrenal glands, where it modulates steroidal hormone (e.g., cortisol) synthesis and secretion. Excess ACTH contributes to the pathophysiology of Cushing’s disease (CD), ectopic ACTH syndrome (EAS), and congenital adrenal hyperplasia (CAH). ACTH‐secreting tumors cause CD (in pituitary) and EAS (outside pituitary) and lead to hypercortisolemia and various metabolic comorbidities. CAH is caused by inactivating mutations in adrenal steroid synthesis pathway proteins leading to low levels of cortisol that are insufficient to provide negative feedback on ACTH production. Chronic ACTH elevation can lead to life‐threatening salt wasting and significant virilization. We hypothesize that blocking the ACTH‐MC2R interaction with a selective MC2R antagonist may provide a key new therapeutic option for these patients.We initiated an iterative medicinal chemistry program to identify potent and selective nonpeptide MC2R antagonists suitable for evaluation in clinical trials. Initial assessment consisted of equilibrium radioligand binding competition assays using [125I]ACTH(1‐39) to determine Ki and EC50 shift functional assays using ACTH(1‐24) to determine ΔpEC50 values. Select compounds were then characterized using a full Schild regression analysis to determine KB values and gain insight into mechanism. There was a strong correlation between the Ki and KB values for this class of small molecule.Our efforts identified CRN04894 as a highly potent MC2R antagonist (Ki = 2.1 nM, KB = 0.34 nM). The Schild regression data for CRN04894 was consistent with a competitive, orthosteric interaction with ACTH. The individual ACTH dose‐response curves with the addition of different concentrations of CRN04894 showed the same maximum response and slopes near unity, and the Schild plot had a slope not statistically different than unity. Furthermore, CRN04894 did not affect the dissociation rate of [125I]ACTH(1‐39) initiated by a saturating concentration of ACTH(1‐39), also consistent with an orthosteric interaction. Because CRN04894 appeared to bind the orthosteric site on MC2R, it was further tested in a kinetic radioligand binding competition assay using [125I]ACTH(1‐39) to determine its binding rate constants. CRN04894 rate constants are comparable to published values for other GPCR ligands with kon = 1.2 e7 M‐1 min‐1 and koff = 0.018 min‐1, corresponding to a dissociation t1/2of 39 min. The kinetic Ki (1.5 nM) calculated from the kon and koff is in accordance with the equilibrium competition binding Ki and Schild analysis KB. In addition, CRN04894 selectively blocks the ACTH‐MC2R interaction, showing no binding to MC1R, MC3R, MC4R, and MC5R at concentrations ≥ 1 µM. To our knowledge, this class of molecule represents the first reported potent nonpeptide MC2R antagonists, and CRN04894 is currently being evaluated in a first‐in‐human phase 1 clinical trial evaluating pharmacokinetics, pharmacodynamics, and tolerability in healthy subjects.

Targeting Pantothenate Kinase as an Effective Strategy for Antifungal Drug Development

Fungal infections affect over one billion people worldwide each year and are the leading cause of mortality by eukaryotic pathogens with an estimated 150 million severe life‐threatening cases and 1.7 million deaths reported annually. The rapid increase in the number of clinical cases and the emergence of multidrug resistant pathogens such as Candida auris emphasize the urgent need for new strategies to develop more effective, selective and safe antifungals. Coenzyme A (CoA) plays a central role in key cellular processes including the citric acid cycle, lipid metabolism, and ergosterol biosynthesis. The first step in the CoA biosynthesis pathway is catalyzed by a pantothenate kinase (PanK), which phosphorylates pantothenic acid (PA, or vitamin B5) to form phosphopantothenate. In fungi, this enzyme is encoded by a single copy PanK gene, which plays an essential role in cell viability. Here, we report the first crystal structure of the S. cerevisiae PanK enzyme, Cab1, at 1.8Å resolution. Cab1 was crystallized alone and in the presence of three lead high‐affinity inhibitors identified in a high‐throughput chemical screen of a library of 156,593 small molecules. Structure‐guided mutagenesis together with functional complementation assays in yeast identified key residues in Cab1 that are essential for catalysis, substrate binding and dimerization. Structure activity relationship and biological assays identified a lead inhibitor YU385599, with excellent activity against the human pathogens Candida albicans, C. glabrata, and C. parapsilosis. Together, our data validate PanK activity as a novel drug target and identify a novel class of small molecules for the treatment of fungal diseases.

A unique small molecule class of fluorophores with large Stokes shift based on the electron deficient 9-methoxypyrroloisoquinolinetrione core

A novel fluorescent core based on electron-deficient 9-methoxypyrroloisoquinolinetrione moiety with a condensed imide cycle is developed. The new class of dyes show large Stokes shift up to 6519 cm-1 (Δλ up to 130 nm), however, unlike parent 5-hydroxyisoquinolones, 9-methoxypyrroloisoquinolinetrione are not superphotoacidic which could be responsible for the large Stokes shifts. At the same time methylation of 5-hydroxyisoquinolones reduces Stokes shift from 5086 to 4689 cm-1. The increase in Stokes shift upon the formation of an imide cycle was shown to be due to unusual bond-lengths relaxation involving the imide ring.

Proton magnetic resonance spectroscopy detects cerebral metabolic derangement in a mouse model of brain coenzyme a deficiency

BackgroundPantothenate kinase (PANK) is the first and rate-controlling enzymatic step in the only pathway for cellular coenzyme A (CoA) biosynthesis. PANK-associated neurodegeneration (PKAN), formerly known as Hallervorden–Spatz disease, is a rare, life-threatening neurologic disorder that affects the CNS and arises from mutations in the human PANK2 gene. Pantazines, a class of small molecules containing the pantazine moiety, yield promising therapeutic effects in an animal model of brain CoA deficiency. A reliable technique to identify the neurometabolic effects of PANK dysfunction and to monitor therapeutic responses is needed.MethodsWe applied 1H magnetic resonance spectroscopy as a noninvasive technique to evaluate the therapeutic effects of the newly developed Pantazine BBP-671.Results1H MRS reliably quantified changes in cerebral metabolites, including glutamate/glutamine, lactate, and N-acetyl aspartate in a neuronal Pank1 and Pank2 double-knockout (SynCre+Pank1,2 dKO) mouse model of brain CoA deficiency. The neuronal SynCre+Pank1,2 dKO mice had distinct decreases in Glx/tCr, NAA/tCr, and lactate/tCr ratios compared to the wildtype matched control mice that increased in response to BBP-671 treatment.ConclusionsBBP-671 treatment completely restored glutamate/glutamine levels in the brains of the mouse model, suggesting that these metabolites are promising clinically translatable biomarkers for future therapeutic trials.

High-resolution structures of the bound effectors avadomide (CC-122) and iberdomide (CC-220) highlight advantages and limitations of the MsCI4 soaking system

Cereblon (CRBN) is the substrate receptor of the CRL4CRBN E3 ubiquitin ligase and is a central player in targeted protein degradation. It is the target of the thalidomide-derived immunomodulatory drugs (IMiDs) and is one of the most widely employed receptors for proteolysis-targeting chimeras (PROTACs), both of which induce the ubiquitination and subsequent proteasomal degradation of target proteins. Structural studies of ligand binding to CRBN are crucial to elucidate the mechanisms of action and for mediation of side effects, ultimately aiding the development of next-generation IMiDs and PROTACs. With this aim, a crystal-soaking system based on the single-domain bacterial homologue MsCI4 has previously been established and used to delineate the binding modes of several classes of small molecules, including FDA-approved drugs, at the molecular level. Here, this system was used to characterize the binding of the next-generation IMiDs avadomide (CC-122) and iberdomide (CC-220) at high resolution, highlighting the advantages and limitations of the MsCI4 system and its implications for the development of future cereblon effectors.

Abstract PD3-10: Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer

Abstract Clinical Significance: Despite initial response to therapies, patients with triple negative breast cancer (TNBC) have the highest risk of metastatic relapse within 5 years of diagnosis. There is an urgent need for better treatments for this deadly disease. To this end, understanding the molecular drivers can reveal novel therapeutic targets. Background: The TNBC oncogenic program is known to be driven by MYC. MYC expression is regulated by bromodomain proteins and several bromodomain inhibitors such as JQ1 have proven effective at inhibiting MYC expression. However, resistance can occur rapidly upon activation of alternative cell survival mechanisms, such as autophagy. TNBC is noted for its elevated basal autophagy. Interestingly, publicly available datasets show that ULK3, an initial effector of the autophagy program, is upregulated in TNBC patients while the opposite is observed for the classical autophagy initiator ULK1. Moreover, TNBC patients with low ULK3 expression levels tend to achieve longer relapse free survival (RFS) than higher expression cohort, in opposition to ULK1. Importantly, ULK3 has not been investigated to date in regulating TNBC cell intrinsic autophagy and no drugs exist that can inhibit ULK3. Methods and Results: Given the importance of MYC and ULK3 in TNBC progression, our team developed a novel class of small molecules, namely dual BRD4/ULK3 inhibitors, that are superior to JQ1 alone in limiting TNBC viability. In vitro data demonstrate that TNBC cells are more sensitive to the cytotoxic activity of the dual inhibitors compared to other breast cancer subtypes. Further investigation through western blot analysis confirms the effectiveness of these novel dual inhibitors in abrogating MYC expression overtime, and completely blocking the autophagy program (measured by downstream ULK3, ULK1, LC3B, p62 protein levels), resulting in cell death. Interestingly, the human cell line SUM159R, that is resistant to BRD4 inhibitor JQ1 and cross-resistant to standard chemotherapeutics (doxorubicin) is also highly sensitive to our dual inhibitors, implying their potential use for the treatment of refractory TNBC disease. Excitingly, and for the first time, using a genetic silencing approach (shULK3 in SUM159), we demonstrated that ULK3 is critical for TNBC autophagy and cell survival. As a proof of concept, we reproduced the same genetic silencing experiment in a different cell line, namely WM1366-LC3BmCherry-GFP, an LC3B conveniently engineered system used to visualize proper autophagosome formation and material degradation through autophagy. We verified that both genetic ablation and pharmacologic inhibition of ULK3 are strategies that lead to cancer cell death. Conclusions: In conclusion, we 1) characterized a novel class of dual BRD4/kinase compounds, capable of inhibiting BRD4 activity and autophagy, and superior to the BRD4 inhibitor, JQ1. 2) showed that ULK3 is over-expressed in TNBC. 3) demonstrated that silencing ULK3 blocks autophagy in TNBC cell lines and results in significantly lower cell viability. 4) found that our dual inhibitors present a double bind for TNBC cells that results in increased cytotoxicity and potentially will be useful for the treatment of resistant TNBC. Clinically, we also posit that our dual inhibitors could be easily administered as a single agent, avoiding the potential complication of pharmacokinetics/pharmacodynamics associated with administering multiple therapies. Citation Format: Marilena Tauro, Tao Li, Conor C Lynch. Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-10.

K+ takes the crown: Selective activation of non-selective crown ether channels

K+ channel proteins form spectacularly K+-selective ion channels (1). Can anything compare with these chemical wonders that pass more than 10,000 K+ ions for every Na+ ion they let leak through? Are there any other viable chemical architectures for K+ channels? Crown ethers are a class of small molecules chemically dissimilar to proteinaceous K+ channels, but some can selectively bind K+, and molecules containing crown ethers can form ion channels (2). Crown ether channels have been proposed to be K+ selective.

Synthesis and inverse virtual screening of new bi-cyclic structures towards cancer-relevant cellular targets

We report here synthetic approaches to access new classes of small molecules based on three heterocyclic scaffolds, i.e. 3,7-dihydropyrimido[4,5-d]pyridazine-4,8-dione, 1,8-naphthyridin-4(1H)-one and 4H-pyrido[1,2-a]pyrimidin-4-one. The bi-cyclic structure 3,7-dihydropyrimido[4,5-d]pyridazine-4,8-dione is a new heterocycle, described here for the first time. In silico methodologies of inverse virtual screening have been used to preliminary analyse the molecules, in order to explore their potential as hits for chemical biology investigations. Our computational study has been conducted with 43 synthetically accessible small molecules towards 31 cellular proteins involved in cancer pathogenesis. Binding energies were quantified using molecular docking calculations, allowing to define the relative affinities of the ligands for the cellular targets. Through this methodology, 16 proteins displayed effective interactions with distinct small molecules within the matrix. In addition, 23 ligands have demonstrated high affinity for at least one cellular protein, using as reference the co-crystallised ligand in the X-ray structure. The evaluation of ADME and drug score for selected hits also highlights that these new molecular series can serve as sources of lead candidates for further structure optimisation and biological studies.

Ruthenium 360 and mitoxantrone inhibit mitochondrial calcium uniporter channel to prevent liver steatosis induced by high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) affects over 25% of the general population and lacks an effective treatment. Recent evidence implicates disrupted mitochondrial calcium homeostasis in the pathogenesis of hepatic steatosis. In this study, mitochondrial calcium uniporter (MCU) was inhibited through classical genetic approaches, viral vectors or small molecule inhibitors in vivo to study its role in hepatic steatosis induced by high-fat diet (HFD). In vitro, MCU was overexpressed or inhibited to change mitochondrial calcium homeostasis, endoplasmic reticulum-mitochondrial linker was adopted to increase mitochondria-associated membranes (MAMs) and MICU1-EF hand mutant was used to decrease the sensitivity of mitochondrial calcium uptake 1 (MICU1) to calcium and block MCU channel. Here, we found that inhibition of liver MCU by AAV virus and classical genetic approaches can prevent HFD-induced liver steatosis. MCU regulates mitochondrial calcium homeostasis and affects lipid accumulation in liver cells. In addition, a HFD in mice enlarged the MAM. The high-calcium environment produced by MAM invalidated the function of MICU1 and led to persistent open of MCU channels. Therefore, it caused mitochondrial calcium overload and liver fat deposition. Inhibition of MAM and MCU alleviated HFD-induced hepatic steatosis. MCU inhibitors (Ru360 and mitoxantrone) can block MCU channels and reduce mitochondrial calcium levels. Intraperitoneal injection of MCU inhibitors (0.01-μM·kg-1 bodyweight) can alleviate HFD-induced hepatic steatosis. These findings provide molecular insights into the way HFD disrupts mitochondrial calcium homeostasis and identify MCU as a promising drug target for the treatment of hepatic steatosis.

Mavacamten depresses human atrial contractility in the same EC50% range as human ventricle

In obstructive HCM patients, recent phase III placebo-controlled randomized EXPLORERHCM clinical trial demonstrated the efficacy and safety of Mavacamten in reducing left ventricular (LV) outflow tract gradient and ameliorating exercise capacity (Olivotto et al. 2021). Both effects are attributable to a local reduction of LV septal contractility. Although very promising, this class of small molecules can globally depress cardiac muscle contractility with potential detrimental consequences on the overall cardiac output.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

The mitochondrial anti-apoptotic dependencies of hematologic malignancies: from disease biology to advances in precision medicine.

Mitochondria are critical organelles in the regulation of intrinsic apoptosis. As a general feature of blood cancers, different antiapoptotic members of the BCL-2 protein family localize at the outer mitochondrial membrane to sequester variable amounts of proapoptotic activators, and hence protect cancer cells from death induction. However, the impact of distinct anti-apoptotic members on apoptosis prevention, a concept termed anti-apoptotic dependence, differs remarkably across disease entities. Over the last two decades, several genetic and functional methodologies have been established to uncover the anti-apoptotic dependencies of the majority of blood cancers, inspiring the development of a new class of small molecules called BH3 mimetics. In this review, we highlight the rationale of targeting mitochondrial apoptosis in hematology, and provide a comprehensive map of the anti-apoptotic dependencies that are currently guiding novel therapeutic strategies. Cell-extrinsic and -intrinsic mechanisms conferring resistance to BH3 mimetics are also examined, with insights on potential strategies to overcome them. Finally, we discuss how the field of mitochondrial apoptosis might be complemented with other dimensions of precision medicine for more successful treatment of ‘highly complex’ hematologic malignancies.

Identification of a novel class of cyclic penta-peptides against hepatitis C virus as p7 channel blockers

The hepatitis C virus (HCV) p7 viroporin protein is essential for viral assembly and release, suggesting its unrealised potential as a target for HCV interventions. Several classes of small molecules that can inhibit p7 through allosteric mechanisms have shown low efficacy. Here, we used a high throughput virtual screen to design a panel of eight novel cyclic penta-peptides (CPs) that target the p7 channel with high binding affinity. Further examination of the effects of these CPs in viral production assays indicated that CP7 exhibits the highest potency against HCV among them. Moreover, the IC50 efficacy of CP7 in tests of strain Jc1-S282T suggested that this cyclopeptide could also effectively inhibit a drug-resistant HCV strain. A combination of nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations revealed that CP7 blocking activity relies on direct binding to the p7 channel lumen at the N-terminal bottleneck region. These findings thus present a promising anti-HCV cyclic penta-peptide targeting p7 viroporin, while also describing an alternative strategy for designing a new class of p7 channel blockers for strains resistant to direct-acting antiviral agents (DAA).