Uncialamycin-based antibody-drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect.

Antibody-drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin [Formula: see text], have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.

Design, Synthesis, and Biological Evaluation of Tubulysin Analogues, Linker-Drugs, and Antibody-Drug Conjugates, Insights into Structure-Activity Relationships, and Tubulysin-Tubulin Binding Derived from X-ray Crystallographic Analysis.

Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/ß interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations.

Streamlined Symmetrical Total Synthesis of Disorazole B1 and Design, Synthesis, and Biological Investigation of Disorazole Analogues.

Taking advantage of the C2-symmetry of the antitumor naturally occurring disorazole B1 molecule, a symmetrical total synthesis was devised with a monomeric advanced intermediate as the key building block, whose three-step conversion to the natural product allowed for an expeditious entry to this family of compounds. Application of the developed synthetic strategies and methods provided a series of designed analogues of disorazole B1, whose biological evaluation led to the identification of a number of potent antitumor agents and the first structure-activity relationships (SARs) within this class of compounds. Specifically, the substitutions of the epoxide units and lactone moieties with cyclopropyl and lactam structural motifs, respectively, were found to be tolerable for biological activities and beneficial with regard to chemical stability.

Synthesis and Biological Evaluation of Shishijimicin A-Type Linker-Drugs and Antibody-Drug Conjugates.

Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

Design, Synthesis, and Biological Investigation of Epothilone B Analogues Featuring Lactone, Lactam, and Carbocyclic Macrocycles, Epoxide, Aziridine, and 1,1-Difluorocyclopropane and Other Fluorine Residues.

Despite previous studies within the epothilone field, only one member of this compound family, ixabepilone, made it to approval for clinical use. Recent advances in organic synthesis and medicinal chemistry allow further optimization of lead epothilone analogues aiming to improve their potencies and other pharmacological properties as part of the quest for discovery and development of new anticancer drugs, including antibody-drug conjugates as potential targeted cancer therapies. Herein, we report the design, synthesis, and biological evaluation of a series of new epothilone B analogues equipped with novel structural motifs, including fluorine-containing residues, 12,13-difluorocyclopropyl moieties, mono- and dimethylated macrolactones, and 1-keto macrocyclic systems, as well as two N-substituted ixabepilone analogues in which the 12,13-epoxide and macrolactam NH moieties were replaced, the former with a substituted aziridine moiety and the latter with an NCO-alkyl residue (imide or carbamate). Biological evaluation of these analogues revealed a number of exceptionally potent epothilone B analogues, demonstrating the potency enhancing effects of the fluorine residues and the aziridinyl moiety within the structure of the epothilone molecule and providing new and useful structure-activity relationships within this class of compounds.

Evaluation of PNU-159682 antibody drug conjugates (ADCs).

PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo.

UNLABELLED: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

Measuring experimental cyclohexane-water distribution coefficients for the SAMPL5 challenge.

Small molecule distribution coefficients between immiscible nonaqueuous and aqueous phases-such as cyclohexane and water-measure the degree to which small molecules prefer one phase over another at a given pH. As distribution coefficients capture both thermodynamic effects (the free energy of transfer between phases) and chemical effects (protonation state and tautomer effects in aqueous solution), they provide an exacting test of the thermodynamic and chemical accuracy of physical models without the long correlation times inherent to the prediction of more complex properties of relevance to drug discovery, such as protein-ligand binding affinities. For the SAMPL5 challenge, we carried out a blind prediction exercise in which participants were tasked with the prediction of distribution coefficients to assess its potential as a new route for the evaluation and systematic improvement of predictive physical models. These measurements are typically performed for octanol-water, but we opted to utilize cyclohexane for the nonpolar phase. Cyclohexane was suggested to avoid issues with the high water content and persistent heterogeneous structure of water-saturated octanol phases, since it has greatly reduced water content and a homogeneous liquid structure. Using a modified shake-flask LC-MS/MS protocol, we collected cyclohexane/water distribution coefficients for a set of 53 druglike compounds at pH 7.4. These measurements were used as the basis for the SAMPL5 Distribution Coefficient Challenge, where 18 research groups predicted these measurements before the experimental values reported here were released. In this work, we describe the experimental protocol we utilized for measurement of cyclohexane-water distribution coefficients, report the measured data, propose a new bootstrap-based data analysis procedure to incorporate multiple sources of experimental error, and provide insights to help guide future iterations of this valuable exercise in predictive modeling.

Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism.

Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18-36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at 2-8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage stability appears to aid in identifying batches with higher probability for particulate formation upon extended storage at 5 °C.

Dapagliflozin attenuates fat accumulation and insulin resistance in obese mice with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), a common endocrine disorder affecting premenopausal women, is associated with various metabolic consequences such as insulin resistance, hyperlipidemia, obesity, and type 2 diabetes mellitus (T2DM). Insulin sensitizers, such as metformin and pioglitazone, though effective, often leads to significant gastrointestinal adverse effects or weight gain, limiting its suitability for women with PCOS. There is an urgent need for safe, effective and affordable agents. Dapagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, enhances glucose elimination through urine, thereby reducing body weight and improving glucose and lipid metabolism. Nevertheless, it is not currently recommended as a therapeutic option for PCOS in clinical guidelines. In this study, we systematically examined the impact of dapagliflozin on an obese PCOS mouse model, focusing on alterations in glucose metabolism, adipose tissue morphology, and plasma lipid profile. Obese PCOS was induced in mice by continuous dihydrotestosterone (DHEA) injections over 21 days and high-fat diet (HFD) feeding. PCOS mice were then orally gavaged with dapagliflozin (1 mg/kg), metformin (50 mg/kg), or vehicle daily for 8 weeks, respectively. Our results demonstrated that dapagliflozin significantly prevented body weight gain and reduced fat mass in obese PCOS mice. Meanwhile, dapagliflozin treatment improved glucose tolerance and increased insulin sensitivity compared to the control PCOS mice. Furthermore, dapagliflozin significantly improved adipocyte accumulation and morphology in white adipose tissue, resulting in a normalized plasma lipid profile in PCOS mice. In conclusion, our results suggest that dapagliflozin is an effective agent in managing glucose and lipid metabolism disorders in obese PCOS mice.

Small-molecule inhibition of glycogen synthase 1 for the treatment of Pompe disease and other glycogen storage disorders.

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.

Fat-free mass index is a feasible predictor of insulin resistance in women with polycystic ovary syndrome: Evidence from a cross-sectional study.

BACKGROUND: Insulin resistance (IR) and adipose tissue amplify the metabolic and reproductive outcomes in women with polycystic ovary syndrome (PCOS). It has been widely discussed that body composition influences metabolic health. Still, limited studies were focused on the role of the fat-free mass index (FFMI) in assessing IR in PCOS women. AIMS: We aimed to explore the associations between FFMI/fat mass index (FMI) and IR in women with PCOS and assess the role of FFMI in predicting IR in women with PCOS. METHODS: In the current cross-sectional study, women with PCOS aged between 18 and 40 years were enrolled from October 2018 to July 2022. Baseline demographic information was obtained using standardized self-administered questionnaires. Anthropometric, biochemical, and hormonal information was measured and recorded by investigators. Pearson's correlation and multivariable logistical regression were used to analyze the associations of FFMI/FMI and IR. In addition, receiver operating characteristic (ROC) curves were implied to measure the predictive role of FFMI/FMI for IR in women with PCOS. RESULTS: A total of 371 women with PCOS, reproductive age (27.58 ± 4.89) were enrolled. PCOS women with IR have higher levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), homeostatic model assessment of insulin resistance (HOMA-IR), FMI, and FFMI than that without IR. FMI (r = 0.492, p < 0.001) and FFMI (r = 0.527, p < 0.001) were positively associated with IR. After adjusting for potential confounders, FMI and FFMI were significantly associated with IR in PCOS women, and the OR was 1.385 (95%CI: 1.212-1.583) and 2.306 (95%CI: 1.675-3.174), respectively. Additionally, the FFMI (0.847, 95%CI: 0.784-0.888) has a larger area of ROC (AUC) than the FMI (0.836, 95%CI: 0.799-0.896), while there is no difference in predicting IR (95%CI: -0.18-0.41, p = 0.456). CONCLUSION: These results indicated that FFMI and FMI could significantly increase the risk of IR, both of which could be feasible predictors of IR in PCOS women.

Time-restricted feeding improves metabolic and endocrine profiles in mice with polycystic ovary syndrome.

Objectives: Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathy disorders in premenopausal women, which is characterized by hyperandrogenemia, anovulation, and polycystic ovarian morphology (PCOM). Time-restricted feeding (TRF) is a new intermittent restriction dietary pattern, which has been shown to have positive benefits on obesity and glycolipid metabolism disorders. We aimed to explore the effect of the feeding regimen (ad libitum vs. TRF) on the glycolipid metabolism and reproductive endocrine disorders in a PCOS mouse model. Methods: PCOS mouse model was induced by continuous subcutaneous administration of dihydrotestosterone for 21 days. Mice were fed a high-fat diet (HFD) for 8 weeks on an ad libitum or time- restricted diet (from 10:30 p.m. to 6:30 a.m.). Results: Compared to control mice, PCOS mice that received TRF treatment had significantly lower body weight, reduced adiposity, lower area under the curve (AUC) of glucose response in the oral glucose tolerance test (OGTT), and lower AUC in the insulin tolerance test (ITT). TRF also ameliorated lipid metabolism, as shown by a reduction in plasma lipid profiles (triglycerides and cholesterol) and the triglyceride content in the liver of PCOS mice. In terms of reproduction, the plasma androgen level, plasma estrogen (E2) level, and luteinizing hormone (LH)/follicle stimulating hormone (FSH) ratio in PCOS mice were significantly reduced after 8 weeks of TRF treatment. In addition, ovarian histology showed that TRF inhibits cyst formation and promotes corpus luteum formation. Conclusion: In conclusion, TRF improved metabolic and endocrine profiles in mice with PCOS.

Novel markers to track oxidative polysorbate degradation in pharmaceutical formulations.

Polysorbates can undergo oxidative degradation in pharmaceutical formulations resulting in both soluble and insoluble degradation products. The insoluble degradants may precipitate to form subvisible and visible particulates, which are undesirable in liquid parenteral products. To date, no oxidation byproduct has been identified as an established marker to track Polysorbate 20 oxidation. Herein, we identified the aldehyde derivative of free fatty acid esters as a byproduct of polysorbate oxidation that can be derivatized using 2,4-dinitrophenylhydrazine and tracked analytically to monitor oxidative polysorbate degradation in pharmaceutical formulations.

A high throughput solubility assay for drug discovery using microscale shake-flask and rapid UHPLC-UV-CLND quantification.

The rapid determination of key physical properties of lead compounds is essential to the drug discovery process. Solubility is one of the most important properties since good solubility is needed not only for obtaining reliable in vitro and in vivo assay results in early discovery but also to ensure sufficient concentration of the drug being in circulation to get the desired therapeutic exposure at the target of interest. In order for medicinal chemists to tune solubility of lead compounds, a rapid assay is needed to provide solubility data that is accurate and predictive so that it can be reliably used for designing the next generation of compounds with improved properties. To ensure speed and data quality, we developed a high throughput solubility assay that utilizes a single calibration UHPLC-UV-CLND method and a 24h shake-flask format for rapid quantification. A set of 46 model compounds was used to demonstrate that the method is accurate, reproducible and predictive. Here we present development of the assay, including evaluation of quantification method, filtration membranes, equilibrium times, DMSO concentrations, and buffer conditions. A comparison of thermodynamic solubility results to our high throughput 24h shake-flask solubility assay results is also discussed.

Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety.

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

A novel method for high throughput lipophilicity determination by microscale shake flask and liquid chromatography tandem mass spectrometry.

Modern small molecule drug design requires the optimization of not only the binding characteristics of the molecule but also its physicochemical properties for ADMET performance. A key physical property is lipophilicity and medicinal chemists need rapid access to high quality data in order to drive their decision making. Traditionally lipophilicity (log D) measurements are performed with a shake flask method and UV determination. This method suffers from low sensitivity and is not easily converted to a high throughput format. Over the past decade, several groups have taken different approaches to improve this assay, including replacing the shake flask method with one that utilizes reverse phase HPLC. Here we describe a new microscale shake flask method that utilizes UPLC-MS/MS to achieve increased throughput, sensitivity and accuracy. Approaches for assessing data quality are also described. This platform technology only requires micrograms of compound and is routinely used by most small molecule drug discovery project teams at Genentech.

Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins.

Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements obtained from FRET analysis. Following modification, stoichiometrically labeled cytochrome c was digested with trypsin and derivatized fragments were analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry to identify Lys25 as the predominant site of covalent modification. FRET analysis on derivatized protein demonstrated nearly complete quenching of Cascade Blue fluorescence, indicating the labeled lysine residue to reside within 30 A of the heme prosthetic group. Sodium dodecyl sulfate (SDS) denaturation resulted in an approximately 28% recovery of fluorescence, demonstrating the utility of this donor-acceptor pair for evaluating distance changes of 30-90 A. Modeling the Cascade Blue donor molecule onto Lys25 of a cytochrome c NMR structure confirmed a distance of < or =30 A from the heme acceptor, as determined by FRET analysis. Further modeling of the SDS-denatured state as an extended chain suggested a maximum separation distance of 45 A, also consistent with results derived from FRET analysis.