Generation and structure-activity relationships of novel imidazo-thienopyridine based TLR7 agonists: application as payloads for immunostimulatory antibody drug-conjugates.

Pairing immunostimulatory small molecules with the targeting capability of an antibody has emerged as a novel therapeutic modality with the potential to treat a variety of solid tumors. A series of compounds based on an imidazo-thienopyridine scaffold were synthesized and tested for their ability to agonize the innate immune sensors toll-like receptor 7 and 8 (TLR7/8). Structure-activity relationship (SAR) studies revealed that certain simple amino-substituents could enable TLR7 agonism at low nanomolar concentrations. Drug-linkers containing either payload 1 or 20h were conjugated to the HER2-targeting antibody trastuzumab at the interchain disulfide cysteine residues using a cleavable valine-citrulline dipeptide linker and stochastic thiol-maleimide chemistry. In vitro, these immune-stimulating antibody drug-conjugates (ADCs) were found to induce cytokine release in a murine splenocyte assay when co-cultured with the HER2-high NCI-N87 cancer cell line. In vivo, tumor regression was observed with a single dose in an NCI-N87 gastric carcinoma xenograft model in BALB/c nude mice.

Structure-Activity Relationships of Bis-Intercalating Peptides and Their Application as Antibody-Drug Conjugate Payloads.

Synthetic analogs based on the DNA bis-intercalating natural product peptides sandramycin and quinaldopeptin were investigated as antibody drug conjugate (ADC) payloads. Synthesis, biophysical characterization, and in vitro potency of 34 new analogs are described. Conjugation of an initial drug-linker derived from a novel bis-intercalating peptide produced an ADC that was hydrophobic and prone to aggregation. Two strategies were employed to improve ADC physiochemical properties: addition of a solubilizing group in the linker and the use of an enzymatically cleavable hydrophilic mask on the payload itself. All ADCs showed potent in vitro cytotoxicity in high antigen expressing cells; however, masked ADCs were less potent than payload matched unmasked ADCs in lower antigen expressing cell lines. Two pilot in vivo studies were conducted using stochastically conjugated DAR4 anti-FRα ADCs, which showed toxicity even at low doses, and site-specific conjugated (THIOMAB) DAR2 anti-cMet ADCs that were well tolerated and highly efficacious.

Inhibition of Sebum Production with the Acetyl Coenzyme A Carboxylase Inhibitor Olumacostat Glasaretil.

Olumacostat glasaretil (OG) is a small molecule inhibitor of acetyl coenzyme A (CoA) carboxylase (ACC), the enzyme that controls the first rate-limiting step in fatty acid biosynthesis. Inhibition of ACC activity in the sebaceous glands is designed to substantially affect sebum production, because over 80% of human sebum components contain fatty acids. OG inhibits de novo lipid synthesis in primary and transformed human sebocytes. TrueMass Sebum Panel analyses showed a reduction in saturated and monounsaturated fatty acyl chains across lipid species, including di- and triacylglycerols, phospholipids, cholesteryl esters, and wax esters in OG-treated sebocytes. There was no shift to shorter acyl chain lengths observed, suggesting that the fatty acid chain elongation process was not affected. OG is a pro-drug of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid and was designed to enhance delivery in vivo. Topical application of OG but not 5-(tetradecyloxy)-2-furoic acid significantly reduced hamster ear sebaceous gland size, indicating that this pro-drug approach was critical to obtain the desired activity in vivo. High-performance liquid chromatography analyses of hamster ear extracts showed that OG treatment increased ACC levels and the ratio of acetyl-CoA to free CoA in these animals, indicating increased fatty acid oxidation. These changes are consistent with ACC inhibition. Matrix-assisted laser desorption/ionization imaging showed that OG applied onto Yorkshire pig ears accumulated in sebaceous glands relative to the surrounding dermis. Sebaceous gland ACC represents an attractive therapeutic target given its central role in formation of sebum, a key factor in acne pathogenesis.

Synthesis and SAR of 3,5-diamino-piperidine derivatives: novel antibacterial translation inhibitors as aminoglycoside mimetics.

Aminoglycoside antibiotics target an internal RNA loop within the bacterial ribosomal decoding site. Here, we describe the synthesis and SAR of novel 3,5-diamino-piperidine derivatives as aminoglycoside mimetics, and show they act as inhibitors of bacterial translation and growth.

Structure-guided discovery of novel aminoglycoside mimetics as antibacterial translation inhibitors.

We report the structure-guided discovery, synthesis, and initial characterization of 3,5-diamino-piperidinyl triazines (DAPT), a novel translation inhibitor class that targets bacterial rRNA and exhibits broad-spectrum antibacterial activity. DAPT compounds were designed as structural mimetics of aminoglycoside antibiotics which bind to the bacterial ribosomal decoding site and thereby interfere with translational fidelity. We found that DAPT compounds bind to oligonucleotide models of decoding-site RNA, inhibit translation in vitro, and induce translation misincorporation in vivo, in agreement with a mechanism of action at the ribosomal decoding site. The novel DAPT antibacterials inhibit growth of gram-positive and gram-negative bacteria, including the respiratory pathogen Pseudomonas aeruginosa, and display low toxicity to human cell lines. In a mouse protection model, an advanced DAPT compound demonstrated efficacy against an Escherichia coli infection at a 50% protective dose of 2.4 mg/kg of body weight by single-dose intravenous administration.

Novel 2,5-dideoxystreptamine derivatives targeting the ribosomal decoding site RNA.

The ribosomal decoding site is the target of aminoglycoside antibiotics that specifically recognize an internal loop RNA structure. We synthesized RNA-targeted 2,5-dideoxystreptamine-4-amides in which a sugar moiety in natural aminoglycosides is replaced by heterocycles.

Novel paromamine derivatives exploring shallow-groove recognition of ribosomal-decoding-site RNA.

Natural aminoglycoside antibiotics recognize an internal loop of bacterial ribosomal-decoding-site RNA by binding to the deep groove of the RNA structure. We have designed, synthesized, and tested RNA-targeted paromamine derivatives that exploit additional interactions on the shallow groove face of the decoding-site RNA. An in vitro transcription-translation assay of a series of 6'-derivatives showed the 6'-position to be very sensitive to substitution. This result suggests that the group at the 6'-position plays a pivotal role in RNA target recognition.

Rational design of azepane-glycoside antibiotics targeting the bacterial ribosome.

RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of Staphylococcus aureus, including aminoglycoside-resistant strains.