Novel Concept for Super-Soft Topical Drugs: Deactivation by an Enzyme-Induced Switch into an Inactive Conformation.

We present a novel concept for the design of supersoft topical drugs. Enzymatic cleavage of the carbonate ester of the potent pan Janus kinase (JAK) inhibitor 2 releases hydroxypyridine 3. Due to hydroxypyridine-pyridone tautomerism, 3 undergoes a rapid conformational change preventing the compound to assume the bioactive conformation required for binding to JAK kinases. We demonstrate that the hydrolysis in human blood and the subsequent shape change lead to the deactivation of 2.

Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.

Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of Zwitterionic Derivatives with a Salt Bridge for the Improvement of Oral Exposure.

G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC-Derived Tensorial Constraints*.

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility.

Application of Transition-Metal Catalysis, Biocatalysis, and Flow Chemistry as State-of-the-Art Technologies in the Synthesis of LCZ696.

LCZ696 is a novel treatment for patients suffering from heart failure that combines the two active pharmaceutical ingredients sacubitril and valsartan in a single chemical compound. While valsartan is an established drug substance, a new manufacturing process suitable for large-scale commercial production had to be developed for sacubitril. The use of chemocatalysis, biocatalysis, and flow chemistry as state-of-the-art technologies allowed to efficiently build up the structure of sacubitril and achieve the defined performance targets.

Bioinspired co-crystals of Imatinib providing enhanced kinetic solubility.

Realizing the full potential of co-crystals enhanced kinetic solubility demands a comprehensive understanding of the mechanisms of dissolution, phase conversion, nucleation and crystal growth, and of the complex interplay between the active pharmaceutical ingredient (API), the coformer and co-existing forms in aqueous media. One blueprint provided by nature to keep poorly water-soluble bases in solution is the complexation with phenolic acids. Consequently, we followed a bioinspired strategy for the engineering of co-crystals of a poorly water-soluble molecule - Imatinib - with a phenolic acid, syringic acid (SYA). The dynamics of dissolution and solution-mediated phase transformations were monitored by Nuclear Magnetic Resonance (NMR) spectroscopy, providing mechanistic insights into the 60 fold-increased long lasting concentrations achieved by the syringate co-crystals as compared to Imatinib base and Imatinib mesylate. This lasting effect was linked to SYA's ability to delay the formation and nucleation of Imatinib hydrate - the thermodynamically stable form in aqueous media - through a metastable association of SYA with Imatinib in solution. Results from permeability studies evidenced that SYA did not impact Imatinib's permeability across membranes while suggesting improved bioavailability through higher kinetic solubility at the biological barriers. These results reflect that some degree of hydrophobicity of the coformer might be key to extend the kinetic solubility of co-crystals with hydrophobic APIs. Understanding how kinetic supersaturation can be shaped by the selection of an interactive coformer may help achieving the needed performance of new forms of poorly water-soluble, slowly dissolving APIs.

Design and Development of a Cyclic Decapeptide Scaffold with Suitable Properties for Bioavailability and Oral Exposure.

Permeability and oral bioavailability of macrocyclic peptides still represent difficult challenges in drug discovery. Despite the recognized potential of macrocyclic peptides as therapeutics, their use is still restricted to extracellular targets and intravenous administration. Indeed, macrocyclic peptides generally suffer from limited proteolytic stability, high clearance, and poor membrane permeability, and this leads to the absence of systemic exposure after oral administration. To overcome these limitations, we started to investigate the development of a general cyclic decapeptide scaffold that possesses ideal features for cell permeability and oral exposure. On the basis of a rigid hairpin structure, the scaffold design aimed to decrease the overall polarity of the compound, thereby limiting the energetic cost of NH desolvation and the entropy penalty during cell penetration. The results of this study also demonstrate the importance of rigidity for the ß-turn design regarding clearance. To stabilize the scaffold in the desired ß-hairpin conformation, the introduction of d-proline at the i+1 turn position proved to be beneficial for both permeability and clearance. As a result, cyclopeptide decamers with unprecedented high values for oral bioavailability and exposure are reported herein. NMR spectroscopy conformation and dynamic analysis confirmed, for selected examples, the rigidity of the scaffold and the presence of transannular hydrogen bonds in polar and apolar environments. Furthermore, we showed, for one compound, that its transition from a polar environment to an apolar one was accompanied by an increased molecular motion, revealing an entropy contribution to membrane permeation.

Small molecule-facilitated degradation of ANO1 protein: a new targeting approach for anticancer therapeutics.

ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers.

C-26 vs. C-27 hydroxylation of insect steroid hormones: regioselectivity of a microsomal cytochrome P450 from a hormone-resistant cell line.

Hydroxylation of steroids at one of the side chain terminal methyl groups, commonly linked to C-26, represents an important regulatory step established in many phyla. Discrimination between the two sites, C-26 and C-27, requires knowing the stereochemistry of the products. 26-Hydroxylation of the insect steroid hormone 20-hydroxyecdysone by a microsomal cytochrome P450 was previously found to be responsible for hormonal resistance in a Chironomus cell line mainly producing the (25S)-epimer of 20,26-dihydroxyecdysone. Here, we studied the 25-desoxy analog of 20-hydroxyecdysone, ponasterone A, to elucidate the stereochemistry of the expected 26-hydroxy product, inokosterone, which occurs as C-25 epimers in nature. We identified the predominant metabolite as the C-25 R epimer of inokosterone on comparison by RP-HPLC with the (25R)- and (25S)-epimers the stereochemistry of which was confirmed by X-ray crystallography. (25R)-inokosterone was further oxidized to the 26-aldehyde identified by mass spectroscopy, borohydride reduction and metabolic transformation to 26-carboxylic acid. The (25S)-epimers of inokosterone and its aldehyde were minor products. With 20-hydroxyecdysone as substrate, we newly identified the (25R)-epimer of 20,26-dihydroxyecdysone as a minor product. In conclusion, the present stereochemical studies revealed high regioselectivity of the Chironomus enzyme to hydroxylate both steroids at the same methyl group, denoted C-27.