Crystal Packing Reveals a Potential Autoinhibited KRAS Dimer Interface and a Strategy for Small-Molecule Inhibition of RAS Signaling.

KRAS GTPases harbor oncogenic mutations in more than 25% of human tumors. KRAS is considered to be largely undruggable due to the lack of a suitable small-molecule binding site. Here, we report a unique crystal structure of His-tagged KRASG12D that reveals a remarkable conformational change. The Switch I loop of one His-KRASG12D structure extends into the Switch I/II pocket of another His-KRASG12D in an adjacent unit cell to create an elaborate interface that is reminiscent of high-affinity protein-protein complexes. We explore the contributions of amino acids at this interface using alanine-scanning studies with alchemical free energy perturbation calculations based on explicit-solvent molecular dynamics simulations. Several interface amino acids were found to be hot spots as they contributed more than 1.5 kcal/mol to the protein-protein interaction. Computational analysis of the complex revealed the presence of two large binding pockets that possess physicochemical features typically found in pockets considered druggable. Small-molecule binding to these pockets may stabilize this autoinhibited structure of KRAS if it exists in cells to provide a new strategy to inhibit RAS signaling.

Exploring Covalent Bond Formation at Tyr-82 for Inhibition of Ral GTPase Activation.

Ral RAS GTPases are directly activated by KRAS through a trimeric complex with a guanine exchange factor. Ral is considered undruggable and lacks an accessible cysteine for covalent drug development. Previously we had reported an aryl sulfonyl fluoride fragment that formed a covalent bond at Tyr-82 on Ral and created a deep and well-defined pocket. Here, we explore this pocket further through design and synthesis of several fragment derivatives. The fragment core is modified by introducing tetrahydronaphthalene or benzodioxane rings to enhance affinity and stability of the sulfonyl fluoride reactive group. The deep pocket in the Switch II region is also explored by modifying the aromatic ring of the fragment that is ensconced into the pocket. Compounds 19 (SOF-658) and 26 (SOF-648) formed a single robust adduct specifically at Tyr-82, inhibited Ral GTPase exchange in buffer and in mammalian cells, and blocked invasion of pancreatic ductal adenocarcinoma cancer cells. Compound 19 (SOF-658) was stable in buffer, mouse, and human microsomes suggesting that further optimization could lead to small molecules to probe Ral activity in tumor models.

Synthesis and Evaluation of Marine Natural Product-Inspired Meroterpenoids with Selective Activity toward Dormant Mycobacterium tuberculosis.

Tuberculosis is a disease caused primarily by the organism Mycobacterium tuberculosis (Mtb), which claims about 1.5 million lives every year. A challenge that impedes the elimination of this pathogen is the ability of Mtb to remain dormant after primary infection, thus creating a reservoir for the disease in the population that reactivates under more ideal conditions. A better understanding of the physiology of dormant Mtb and therapeutics able to kill these phenotypically tolerant bacilli will be critical for completely eradicating Mtb. Our groups are focusing on characterizing the activity of derivatives of the marine natural product (+)-puupehenone (1). Recently, the Rohde group reported that puupehedione (2) and 15-α-methoxypuupehenol (3) exhibit enhanced activity in an in vitro multi-stress dormancy model of Mtb. To optimize the antimycobacterial activity of these terpenoids, novel 15-α-methoxy- and 15-α-acetoxy-puupehenol esters were prepared from (+)-puupehenone (1) accessed through a (+)-sclareolide-derived ß-hydroxyl aldehyde. For added diversity, various congeners related to (1) were also prepared from a common borono-sclareolide donor, which resulted in the synthesis of epi-puupehenol and the natural products (+)-chromazonarol and (+)-yahazunol. In total, we generated a library of 24 compounds, of which 14 were found to be active against Mtb, and the most active compounds retained the enhanced activity against dormant Mtb seen in the parent compound. Several of the 15-α-methoxy- and 15-α-acetoxy-puupehenol esters possessed potent activity against actively dividing and dormant Mtb. Intriguingly, the closely related triisobutyl derivative 16 showed similar activity to 1 in actively dividing Mtb but lost about 178-fold activity against dormant Mtb. However, the monopivaloyl compound 13 showed a modest 3- to 4-fold loss in activity in both actively dividing and dormant Mtb relative to the activity of 1 revealing the importance of the free OH at C19 supporting the potential role of quinone methide formation as critical for activity in dormant Mtb. Elucidating important structure-activity relationships and the mechanism of action of this natural product-inspired chemical series may yield insights into vulnerable drug targets in dormant bacilli and new therapeutics to more effectively target dormant Mtb.

Evaluation of Derivatives of (+)-Puupehenone against Clostridioides difficile and Other Gram-Positive Bacteria.

Patients receiving healthcare are at higher risk of acquiring healthcare-associated infections, which cause a significant number of illnesses and deaths. Most pathogens responsible for these infections are highly resistant to multiple antibiotics, prompting the need for discovery of new therapeutics to combat these evolved threats. We synthesized structural derivatives of (+)-puupehenone, a marine natural product, and observed growth inhibition of several clinically relevant Gram-positive bacteria, particularly Clostridioides difficile. The most potent compounds-(+)-puupehenone, 1, 15, 19, and 20-all inhibited C. difficile in the range of 2.0-4.0 µg/mL. Additionally, when present in the range of 1-8 µg/mL, a subset of active compounds-(+)-puupehenone, 1, 6, 15, and 20-greatly reduced the ability of C. difficile to produce exotoxins, which are required for disease in infected hosts. Our findings showcase a promising class of compounds for potential drug development against Gram-positive pathogens, such as C. difficile.

Total Synthesis of Tetrahydrolipstatin, Its Derivatives, and Evaluation of Their Ability to Potentiate Multiple Antibiotic Classes against Mycobacterium Species.

Tetrahydrolipstatin (THL, 1a) has been shown to inhibit both mammalian and bacterial α/ß hydrolases. In the case of bacterial systems, THL is a known inhibitor of several Mycobacterium tuberculosis hydrolases involved in mycomembrane biosynthesis. Herein we report a highly efficient eight-step asymmetric synthesis of THL using a route that allows modification of the THL α-chain substituent to afford compounds 1a through 1e. The key transformation in the synthesis was use of a (TPP)CrCl/Co2(CO)8-catalyzed regioselective and stereospecific carbonylation on an advanced epoxide intermediate to yield a trans-ß-lactone. These compounds are modest inhibitors of Ag85A and Ag85C, two α/ß hydrolases of M. tuberculosis involved in the biosynthesis of the mycomembrane. Among these compounds, 10d showed the highest inhibitory effect on Ag85A (34 ± 22 µM) and Ag85C (66 ± 8 µM), and its X-ray structure was solved in complex with Ag85C to 2.5 Å resolution. In contrast, compound 1e exhibited the best-in-class MICs of 50 µM (25 µg/mL) and 16 µM (8.4 µg/mL) against M. smegmatis and M. tuberculosis H37Ra, respectively, using a microtiter assay plate. Combination of 1e with 13 well-established antibiotics synergistically enhanced the potency of few of these antibiotics in M. smegmatis and M. tuberculosis H37Ra. Compound 1e applied at concentrations 4-fold lower than its MIC enhanced the MIC of the synergistic antibiotic by 2-256-fold. In addition to observing synergy with first-line drugs, rifamycin and isoniazid, the MIC of vancomycin against M. tuberculosis H37Ra was 65 µg/mL; however, the MIC was lowered to 0.25 µg/mL in the presence of 2.1 µg/mL 1e demonstrating the potential of targeting mycobacterial hydrolases involved in mycomembrane and peptidoglycan biosynthesis.

Neuroprotective and Anti-neuroinflammatory Properties of Ebselen Derivatives and Their Potential to Inhibit Neurodegeneration.

Ebselen (EBS) is an organo-selenium-containing compound that has anti-inflammatory, antitumor, and antibacterial properties. EBS is being explored as a possible treatment for reperfusion injury and stroke and is under clinical trials as a mimetic of lithium for the treatment of bipolar disorder [Mota et al. Synapse 2020, 74 (7), 1-6] and noise-induced hearing loss as a result of these actives [Martini et al. J. Psychiatr. Res. 2019, 109, 107-117. Slusarczyk et al. Neural Regener. Res. 2019, 17 (7), 1255-1261. Thangamani et al. PLoS One 2015, 10 (7), e0133877. Kil et al. Lancet 2017, 390 (10098), 969-979]. However, we wanted to characterize derivatives of EBS as neuroprotective, anti-neuroinflammatory, and antioxidant compounds. Recently, we have reported on a new thermal and photoinduced copper-mediated cross-coupling between potassium selenocyanate (KSeCN) and N-substituted ortho-halobenzamides to form ebselen derivatives with increased synthetic efficiency [Thanna et al. J. Org. Chem. 2017, 82 (7), 3844-3854]. Our synthesis allows for the varying of the remote benzene ring with various substituents or replacing that ring with heterocyclic rings such as pyridine, pyrrole, thiophene, etc. In this study, we synthesized seven new heterocyclic EBS derivatives to further diversify our EBS library. These 21 compounds were then evaluated for their neuroprotective properties, with four compounds showing an equal or better neuroprotective profile than EBS. Compounds 5, 9, 23, and 27 showed 73, 86, 80, 84% cell viability, respectively, at a 10 µM concentration. These studies were performed using human neuroblastoma SH-SY5Y cells in an oxygen and glucose deprivation (OGD) model of ischemia. At the same concentration, these compounds significantly inhibited lipopolysaccharide-induced nitric oxide and tumor necrosis factor alpha release from Human microglia clone 3 microglial cells. Compounds 9 and 27 showed significantly increased cell viability (84 and 80%, respectively) for SH-SY5Y cells exposed to microglia-activated media. These compounds showed only mild GPx-like reductive activity, with compounds 2, 7, 12, and 14 (115, 96, 95, and 82%, respectively) showing a higher percent rate of oxidation of NADPH in a coupled reaction assay compared to ebselen. This research highlights several derivatives of ebselen that show improved activity as neuroprotective agents over the parent compound.

Biochemical and microbiological evaluation of N-aryl urea derivatives against mycobacteria and mycobacterial hydrolases.

A focused library of 24 N-aryl urea derivatives was prepared and evaluated against serine esterases of Mycobacterium tuberculosis (Mtb) Rv3802c and Mtb Ag85C. The members of the library were evaluated for both selectivity and mode of inhibition. Furan-based urea derivative 6c was found to be the most potent non-covalent inhibitor of Rv3802c with a K i value of 5.2 ± 0.7 µM. On the other hand, triazole-based ureas 10a and 10b selectively inhibited Ag85C irreversibly with a k inact/K i value of 2.3 ± 0.3 and 5.5 ± 0.4 × 10-3 µM-1 min-1, respectively. The library was also evaluated for minimum inhibitory concentration (MIC) against two strains of Mtb, Mycobacterium smegmatis, and Mycobacterium abscessus. Compounds 4a and 4c were active against Mtb H37Rv mc26206 with MIC values of 3.12 and 1.5 µM, respectively. Closely related 4e showed similar activity against Mtb H37Rv mc26206 but also possessed activity against Mtb H37Ra, Mycobacterium smegmatis and Mycobacterium abscessus. Compounds 4a, 4c, and 4e all contained a common 1-(cyclohexylmethyl)-3-phenylurea motif. In summary, we identified a selective non-covalent inhibitor of Rv3802c and covalently irreversible inhibitors of Ag85C as well as the 1-(cyclohexylmethyl)-3-phenylurea motif which showed activity against a variety of mycobacteria.