Design of a Lead-Like Cysteine-Targeting Covalent Library and the Identification of Hits to Cys55 of Bfl-1.

Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

A Mild Synthesis of Aryl Triflates Enabling the Late-Stage Modification of Drug Analogs and Complex Peptides.

We report the discovery of a straightforward protocol to convert phenols into the corresponding aryl triflates using 1-methyl-3-((trifluoromethyl)sulfonyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one in the presence of a fluoride source. This novel reagent can be handled without any precautions to exclude air or moisture making this method highly convenient. The reactions generally show very clean conversions within only a few minutes at room temperature. The mild conditions allow the so far unprecedented O-triflation of tyrosine in peptides bearing challenging side chains present for example in arginine and histidine including the late-stage triflation of complex bioactive peptides. We show how aryl triflates - an interesting but so far underutilized group - can be used to optimize physicochemical and in vitro properties of compound series in medicinal chemistry. We believe that this method is highly attractive for applications in peptide functionalization as well as automated and medicinal chemistry.

Advances in the design of new types of inhaled medicines.

Inhalation of small molecule drugs has proven very efficacious for the treatment of respiratory diseases due to enhanced efficacy and a favourable therapeutic index compared with other dosing routes. It enables targeted delivery to the lung with rapid onset of therapeutic action, low systemic drug exposure, and thereby reduced systemic side effects. An increasing number of pharmaceutical companies and biotechs are investing in new modalities-for this review defined as therapeutic molecules with a molecular weight >800Da and therefore beyond usual inhaled small molecule drug-like space. However, our experience with inhaled administration of PROTACs, peptides, oligonucleotides (antisense oligonucleotides, siRNAs, miRs and antagomirs), diverse protein scaffolds, antibodies and antibody fragments is still limited. Investigating the retention and metabolism of these types of molecules in lung tissue and fluid will contribute to understanding which are best suited for inhalation. Nonetheless, the first such therapeutic molecules have already reached the clinic. This review will provide information on the physiology of healthy and diseased lungs and their capacity for drug metabolism. It will outline the stability, aggregation and immunogenicity aspects of new modalities, as well as recap on formulation and delivery aspects. It concludes by summarising clinical trial outcomes with inhaled new modalities based on information available at the end of 2021.

Importance of Binding Site Hydration and Flexibility Revealed When Optimizing a Macrocyclic Inhibitor of the Keap1-Nrf2 Protein-Protein Interaction.

Upregulation of the transcription factor Nrf2 by inhibition of the interaction with its negative regulator Keap1 constitutes an opportunity for the treatment of disease caused by oxidative stress. We report a structurally unique series of nanomolar Keap1 inhibitors obtained from a natural product-derived macrocyclic lead. Initial exploration of the structure-activity relationship of the lead, followed by structure-guided optimization, resulted in a 100-fold improvement in inhibitory potency. The macrocyclic core of the nanomolar inhibitors positions three pharmacophore units for productive interactions with key residues of Keap1, including R415, R483, and Y572. Ligand optimization resulted in the displacement of a coordinated water molecule from the Keap1 binding site and a significantly altered thermodynamic profile. In addition, minor reorganizations of R415 and R483 were accompanied by major differences in affinity between ligands. This study therefore indicates the importance of accounting both for the hydration and flexibility of the Keap1 binding site when designing high-affinity ligands.

Discovery and optimization of cyclohexane-1,4-diamines as allosteric MALT1 inhibitors.

Inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a promising strategy to modulate NF-κB signaling, with the potential to treat B-cell lymphoma and autoimmune diseases. We describe the discovery and optimization of (1s,4s)-N,N'-diaryl cyclohexane-1,4-diamines, a novel series of allosteric MALT1 inhibitors, resulting in compound 8 with single digit micromolar cell potency. X-ray analysis confirms that this compound binds to an induced allosteric site in MALT1. Compound 8 is highly selective and has an excellent in vivo rat PK profile with low clearance and high oral bioavailability, making it a promising lead for further optimization.

Mechanism-Based Insights into Removing the Mutagenicity of Aromatic Amines by Small Structural Alterations.

Aromatic and heteroaromatic amines (ArNH2) are activated by cytochrome P450 monooxygenases, primarily CYP1A2, into reactive N-arylhydroxylamines that can lead to covalent adducts with DNA nucleobases. Hereby, we give hands-on mechanism-based guidelines to design mutagenicity-free ArNH2. The mechanism of N-hydroxylation of ArNH2 by CYP1A2 is investigated by density functional theory (DFT) calculations. Two putative pathways are considered, the radicaloid route that goes via the classical ferryl-oxo oxidant and an alternative anionic pathway through Fenton-like oxidation by ferriheme-bound H2O2. Results suggest that bioactivation of ArNH2 follows the anionic pathway. We demonstrate that H-bonding and/or geometric fit of ArNH2 to CYP1A2 as well as feasibility of both proton abstraction by the ferriheme-peroxo base and heterolytic cleavage of arylhydroxylamines render molecules mutagenic. Mutagenicity of ArNH2 can be removed by structural alterations that disrupt geometric and/or electrostatic fit to CYP1A2, decrease the acidity of the NH2 group, destabilize arylnitrenium ions, or disrupt their pre-covalent transition states with guanine.

Cell Permeability of Isomeric Macrocycles: Predictions and NMR Studies.

Conformation-dependent 3D descriptors have been shown to provide better predictions of the physicochemical properties of macrocycles than 2D descriptors. However, the computational identification of relevant conformations for macrocycles is nontrivial. Herein, we report that the Caco-2 cell permeability difference between a pair of diastereomeric macrocycles correlated with their solvent accessible 3D polar surface area and radius of gyration. The descriptors were calculated from the macrocycles' solution-phase conformational ensembles and independently from ensembles obtained by conformational sampling. Calculation of the two descriptors for three other stereo- and regioisomeric macrocycles also allowed the correct ranking of their cell permeability. Methods for conformational sampling may thus allow ranking of passive permeability for moderately flexible macrocycles, thereby contributing to the prioritization of macrocycles for synthesis in lead optimization.

Mining Natural Products for Macrocycles to Drug Difficult Targets.

Lead generation for difficult-to-drug targets that have large, featureless, and highly lipophilic or highly polar and/or flexible binding sites is highly challenging. Here, we describe how cores of macrocyclic natural products can serve as a high-quality in silico screening library that provides leads for difficult-to-drug targets. Two iterative rounds of docking of a carefully selected set of natural-product-derived cores led to the discovery of an uncharged macrocyclic inhibitor of the Keap1-Nrf2 protein-protein interaction, a particularly challenging target due to its highly polar binding site. The inhibitor displays cellular efficacy and is well-positioned for further optimization based on the structure of its complex with Keap1 and synthetic access. We believe that our work will spur interest in using macrocyclic cores for in silico-based lead generation and also inspire the design of future macrocycle screening collections.

N-Trifluoromethyl Amines and Azoles: An Underexplored Functional Group in the Medicinal Chemist's Toolbox.

Introducing trifluoromethyl groups is a common strategy to improve the properties of biologically active compounds. However, N-trifluoromethyl moieties on amines and azoles are very rarely used. To evaluate their suitability in drug design, we synthesized a series of N-trifluoromethyl amines and azoles, determined their stability in aqueous media, and investigated their properties. We show that N-trifluoromethyl amines are prone to hydrolysis, whereas N-trifluoromethyl azoles have excellent aqueous stability. Compared to their N-methyl analogues, N-trifluoromethyl azoles have a higher lipophilicity and can show increased metabolic stability and Caco-2 permeability. Furthermore, N-trifluoromethyl azoles can serve as bioisosteres of N-iso-propyl and N-tert-butyl azoles. Consequently, we suggest that N-trifluoromethyl azoles are valuable substructures to be considered in medicinal chemistry.

Toward the Design of Molecular Chameleons: Flexible Shielding of an Amide Bond Enhances Macrocycle Cell Permeability.

A series of macrocycles inspired by natural products were synthesized to investigate how side-chains may shield amide bonds and influence cell permeability. NMR spectroscopy and X-ray crystallography revealed that the phenyl group of phenylalanine, but not the side-chains of homologous or aliphatic amino acids, shields the adjacent amide bond through an intramolecular NH-π interaction. This resulted in increased cell permeability, suggesting that NH-π interactions may be used in the design of molecular chameleons.

Concise total synthesis of (+)-asperazine A and (+)-pestalazine B.

The highly convergent total synthesis of dimeric diketopiperazine alkaloids (+)-asperazine A and (+)-pestalazine B is described. A critical aspect of our expedient route was the development of a directed regio- and diastereoselective C3-N1' coupling of complex tetracyclic diketopiperazine components. This late-stage heterodimerization reaction was made possible by design of tetracyclic diketopiperazines that allow C3-carbocation coupling of the electrophilic component to the N1' locus of the nucleophilic fragment. The application of this new coupling reaction to the first total synthesis of (+)-asperazine A led to our revision of the sign and magnitude of the optical rotation for the reported structure.

Site-Specific Isotope-Labeling of Inosine Phosphoramidites and NMR Analysis of an Inosine-Containing RNA Duplex.

Structural features and internal dynamics of inosine-containing RNAs are poorly understood. NMR studies of such RNAs require 13 C,15 N-labeling, which cannot be achieved using in vitro transcription as inosine and guanosine are not distinguished by RNA polymerase. Herein, we report the synthesis of an inosine phosphoramidite with selective 13 C8 and 15 N7-isotope incorporation in the base and uniform 13 C-labeling of the ribose. Chemical synthesis of an RNA duplex containing four consecutive IU base pairs with this optimized isotope-labeling scheme greatly simplifies NMR spectra and resolves signal overlap. The absence of detectable NMR signals of imino protons and unusual inter-residue NOE correlations in this RNA indicate deviations from standard A-form geometry, consistent with reduced stability of this duplex seen in UV melting studies compared to its nonedited RNA counterparts. These studies indicate that the introduction of IU base pairs distorts and destabilizes RNA helices significantly compared to the also noncanonical GU base-pairs. Our optimized isotope-labeling scheme enables high-resolution NMR studies of inosine-edited RNAs.

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

Synthesis of a DNA promoter segment containing all four epigenetic nucleosides: 5-methyl-, 5-hydroxymethyl-, 5-formyl-, and 5-carboxy-2'-deoxycytidine.

A 5-formyl-2'-deoxycytidine (fdC) phosphoramidite building block that enables the synthesis of fdC-containing DNA with excellent purity and yield has been developed. In combination with phosphoramidites for 5-methyl-dC, 5-hydroxymethyl-dC, and carboxy-dC, it was possible to prepare a segment of the OCT-4 promoter that contains all four epigenetic bases. Because of the enormous interest in these new epigenetic bases, the ability to insert all four of them into DNA should be of great value for the scientific community.

Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine.

Three new cytosine derived DNA modifications, 5-hydroxymethyl-2'-deoxycytidine (hmdC), 5-formyl-2'-deoxycytidine (fdC) and 5-carboxy-2'-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C-C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2'-deoxyuridine (hmdU). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C-C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C-C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C-C bond cleavage.

Synthesis of 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine-triphosphates and their incorporation into oligonucleotides by polymerase chain reaction.

The synthesis of the triphosphates of 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine and the incorporation of these building blocks into long DNA fragments using the polymerase chain reaction (PCR) are reported. In this way DNA fragments containing multiple hmC, fC, and caC nucleobases are readily accessible.

Mechanism and stem-cell activity of 5-carboxycytosine decarboxylation determined by isotope tracing.

Eraserhead: Stem cells seem to erase epigenetic information by decarboxylation of the newly discovered epigenetic base 5-carboxycytosine (caC; see picture). This reaction is likely to involve a nucleophilic attack of the C5-C6 double bond.