Acceleration of Protein Degradation by 20S Proteasome-Binding Peptides Generated by In Vitro Artificial Evolution.

Although the 20S core particle (CP) of the proteasome is an important component of the 26S holoenzyme, the stand-alone 20S CP acts directly on intrinsically disordered and oxidized/damaged proteins to degrade them in a ubiquitin-independent manner. It has been postulated that some structural features of substrate proteins are recognized by the 20S CP to promote substrate uptake, but the mechanism of substrate recognition has not been fully elucidated. In this study, we screened peptides that bind to the 20S CP from a random eight-residue pool of amino acid sequences using complementary DNA display an in vitro molecular evolution technique. The identified 20S CP-binding amino acid sequence was chemically synthesized and its effects on the 20S CP were investigated. The 20S CP-binding peptide stimulated the proteolytic activity of the inactive form of 20S CP. The peptide bound directly to one of the α-subunits, opening a gate for substrate entry on the α-ring. Furthermore, the attachment of this peptide sequence to α-synuclein enhanced its degradation by the 20S CP in vitro. In addition to these results, docking simulations indicated that this peptide binds to the top surface of the α-ring. These peptides could function as a key to control the opening of the α-ring gate.

Development of Gilteritinib-Based Chimeric Small Molecules that Potently Induce Degradation of FLT3-ITD Protein.

Internal tandem duplication (ITD) in the gene encoding FMS-like tyrosine kinase 3 (FLT3) (FLT3-ITD) is the most frequently observed mutation in acute myeloid leukemia (AML). Currently approved FLT3 kinase inhibitors have high efficacy, but drug resistance caused by reactivation of FLT3 kinase activity is often clinically observed. In this study, we developed novel FLT3 degraders by introducing gilteritinib, an FDA-approved FLT3 inhibitor, into targeted protein degradation technology. The most active compound, CRBN(FLT3)-8, potently degraded FLT3-ITD via the ubiquitin-proteasome system and inhibited the proliferation of FLT3-ITD mutant AML cells more effectively than gilteritinib. These findings provide a new lead compound for degradation-based drugs targeting FLT3-ITD-positive cancers.

Development of a potent small-molecule degrader against oncogenic BRAFV600E protein that evades paradoxical MAPK activation.

BRAF mutations are frequently observed in melanoma and hairy-cell leukemia. Currently approved rapidly accelerated fibrosarcoma (RAF) kinase inhibitors targeting oncogenic BRAF V600 mutations have shown remarkable efficacy in the clinic, but their therapeutic benefits are occasionally hampered by acquired resistance due to RAF dimerization-dependent reactivation of the downstream MAPK pathway, which is known as paradoxical activation. There is also a concern that paradoxical activation of the MAPK pathway may trigger secondary cancer progression. In this study, we developed chimeric compounds, proteolysis targeting chimeras (PROTACs), that target BRAFV600E protein for degradation. CRBN(BRAF)-24, the most effective chimera, potently degraded BRAFV600E in a ubiquitin-proteasome system (UPS)-dependent manner and inhibited the proliferation of BRAFV600E -driven cancer cells. In BRAF wild-type cells, CRBN(BRAF)-24 induced neither BRAFWT degradation nor paradoxical activation of the MAPK pathway. Biochemical analysis revealed that CRBN(BRAF)-24 showed more potent and sustained suppression of MAPK signaling than a BRAFV600E inhibitor, PLX-8394, in BRAFV600E -driven cancer cells. Targeted degradation of BRAFV600E by CRBN(BRAF)-24 could be a promising strategy to evade paradoxical activation of the RAF-MAPK pathway.

Discovery of a Potent, Selective, and Orally Available MTHFD2 Inhibitor (DS18561882) with in Vivo Antitumor Activity.

We report the discovery of a potent and isozyme-selective MTHFD2 inhibitor, DS18561882 (2). Through investigation of the substituents on our tricyclic coumarin scaffold (1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one), MTHFD2 inhibitory activity was shown to be elevated by incorporating an amine moiety at the 8-position and a methyl group at the 7-position of the initial lead 1. X-ray structure analysis revealed that a key interaction for enhanced potency was salt bridge formation between the amine moiety and the diphosphate linker of an NAD+ cofactor. Furthermore, ortho-substituted sulfonamide in place of benzoic acid of 1 significantly improved cell permeability and cell-based growth inhibition against a human breast cancer cell line. The thus-optimized DS18561882 showed the strongest cell-based activity (GI50 = 140 nM) in the class, a good oral pharmacokinetic profile, and thereby tumor growth inhibition in a mouse xenograft model upon oral administration.

Structure-Based Design and Synthesis of an Isozyme-Selective MTHFD2 Inhibitor with a Tricyclic Coumarin Scaffold.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays a key role in one-carbon (1C) metabolism in human mitochondria, and its high expression correlates with poor survival of patients with various types of cancer. An isozyme-selective MTHFD2 inhibitor is highly attractive for potential use in cancer treatment. Herein, we disclose a novel isozyme-selective MTHFD2 inhibitor DS44960156, with a tricyclic coumarin scaffold, which was initially discovered via high-throughput screening (HTS) and improved using structure-based drug design (SBDD). DS44960156 would offer a good starting point for further optimization based on the following features: (1) unprecedented selectivity (>18-fold) for MTHFD2 over MTHFD1, (2) a molecular weight of less than 400, and (3) good ligand efficiency (LE).

Discovery of DF-461, a Potent Squalene Synthase Inhibitor.

We report the development of a new trifluoromethyltriazolobenzoxazepine series of squalene synthase inhibitors. Structure-activity studies and pharmacokinetics optimization on this series led to the identification of compound 23 (DF-461), which exhibited potent squalene synthase inhibitory activity, high hepatic selectivity, excellent rat hepatic cholesterol synthesis inhibitory activity, and plasma lipid lowering efficacy in nonrodent repeated dose studies.

Discovery of novel tricyclic compounds as squalene synthase inhibitors.

In the present article, we have reported the design, synthesis, and identification of highly potent benzhydrol derivatives as squalene synthase inhibitors (compound 1). Unfortunately, the in vivo efficacies of the compounds were not enough for acquiring the clinical candidate. We continued our investigation to obtain a more in vivo efficacious template than the benzhydrol template. In our effort, we focused on a benzoxazepine ring and designed a new tricyclic scaffold by the incorporation of heterocycle into it. Prepared pyrrolobenzoxazepine derivatives showed further efficient in vitro and in vivo activities.

Design, synthesis and biological evaluations of novel 7-[3-(1-aminocycloalkyl)pyrrolidin-1-yl]-6-desfluoro-8-methoxyquinolones with potent antibacterial activity against multi-drug resistant Gram-positive bacteria.

A series of novel 6-desfluoro [des-F(6)] and 6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxyquinolones bearing 3-(1-aminocycloalkyl)pyrrolidin-1-yl substituents at the C-7 position (1-6) was synthesized to obtain potent drugs for nosocomial infections caused by Gram-positive pathogens. The des-F(6) compounds 4-6 exhibited at least four times more potent activity against representative Gram-positive bacteria than ciprofloxacin or moxifloxacin. Among the derivatives, 7-[(3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl] derivative 4, which showed favorable profiles in preliminary toxicological and non-clinical pharmacokinetic studies, exhibited potent antibacterial activity against clinically isolated Gram-positive pathogens that had become resistant to one or more antibiotics.

Systematic research of peptide spacers controlling drug release from macromolecular prodrug system, carboxymethyldextran polyalcohol-peptide-drug conjugates.

The primary purpose of this study was to comprehensively delineate specificity of the peptide spacer sequence to tumor-expressed proteases for the design of macromolecular carrier-peptide spacer-drug conjugate system. 225 conjugates of carboxymethyldextran polyalcohol (CM-Dex-PA) as water-soluble carrier and a dansyl derivative (N-(4-aminobutyl)-5-(dimethylamino)-1-naphthalenesulfonamide, DNS) as the model drug linked with different tetrapeptide spacers (Gly-Gly-P(2)-P(1), P(2), P(1): Ala, Asn, Gly, Cit, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) were combinatorially synthesized. First, the drug release assay of all of the fluorogenic model conjugates was performed in murine Meth A solid tumor homogenates. The drug release rate was higher with conjugates having hydrophobic amino acids at P(2). It was also found that conjugates with Asn release the drug rapidly and, in contrast, those with Pro does not. Second, we selected three peptide spacers (Gly-Gly-Phe-Gly, Gly-Gly-Ile-Gly, Gly-Gly-Pro-Leu), which release only DNS at different rates, and applied them to doxorubicin (DXR) conjugates. These three DXR conjugates were used for investigating relationships with drug release, pharmacokinetics, and antitumor activity against Meth A bearing mice of these conjugates. The release of DXR from the conjugates corresponded well with that of DNS conjugates in tumor homogenates. CM-Dex-PA-Gly-Gly-Phe-Gly-DXR and CM-Dex-PA-Gly-Gly-Ile-Gly-DXR indicated strong antitumor activity, with the comparable pharmacokinetic profile of released DXR in tumor. Taken with the fact that the drug release rate in tumor homogenates was approximately 10-fold different between these two DXR conjugates, it is likely that cellular uptake of the conjugate would be rate-limiting, rather than the drug release process under the in vivo situation. However, much weaker antitumor activity was observed with CM-Dex-PA-Gly-Gly-Pro-Leu-DXR, of which the drug release was extremely slow.

Synthesis and structure-activity relationships of 5-amino-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methylquinolonecarboxylic acid antibacterials having fluorinated 7-[(3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl] substituents.

A series of novel 5-amino-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methylquinolones bearing fluorinated (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl substituents at the C-7 position (2-4) was synthesized to obtain potent drugs for infections caused by Gram-positive pathogens, which include resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE). These fluorinated compounds 2-4 exhibited potent antibacterial activity comparable with that of a compound bearing a non-fluorinated (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidine moiety at the C-7 position (1) and had at least 4 times more potent activity against representative Gram-positive bacteria than ciprofloxacin (CPFX), gatifloxacin (GFLX), or moxifloxacin (MFLX). Among them, the 7-[(3S,4R)-4-(1-aminocyclopropan-1-yl)-3-fluoropyrrolidin-1-yl] derivative 3 (=DQ-113), which showed favorable profiles in preliminary toxicological and nonclinical pharmcokinetic studies, exhibited potent antibacterial activity against clinically isolated resistant Gram-positive pathogens.

Synthesis and mechanism of action of novel pyrimidinyl pyrazole derivatives possessing antiproliferative activity.

Pyrimidinyl pyrazole derivatives 1-4, prepared as a new scaffold of an anti-tumor agent, showed antiproliferative activity against human lung cancer cell lines and inhibited tubulin polymerization. Furthermore, it was found that compound 2 bound at the colchicine site on tubulin, but the tubulin binding pattern was different from that of colchicine. Here, we describe the synthesis of the derivatives and the differences of the action mechanism on tubulin polymerization inhibition between compound 2 and colchicine.