Delivery of Anticancer Drugs Using Microbubble-Assisted Ultrasound in a 3D Spheroid Model.

Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.

Therapeutic Potential of MF-TTZ-MMAE, a Site-Specifically Conjugated Antibody-Drug Conjugate, for the Treatment of HER2-Overexpressing Breast Cancer.

With three clinically approved antibody-drug conjugates targeting HER2, this target is clearly identified to be of interest in oncology. Moreover, the advent of new bioconjugation technologies producing site-specific homogenous conjugates led to the opportunity of developing new medicines linking antibodies and payloads. Here, a new relevant HER2-targeting ADC was obtained by the conjugation of monomethyl auristatin E onto trastuzumab using McSAF Inside bioconjugation technology. The antibody-drug conjugate formed presented an average drug-to-antibody ratio of 4 with a high homogeneity and an excellent stability especially when incubated with human serum albumin or in human plasma. Moreover, it demonstrated a strong efficacy in an HER2 xenograft tumor model in mice, superior to the clinically approved antibody-drug conjugate ado-trastuzumab emtansine, with a complete tumor regression observed both macroscopically and microscopically demonstrating its therapeutic potential.

Innovative Bioconjugation Technology for Antibody-Drug Conjugates: Proof of Concept in a CD30-Positive Lymphoma Mouse Model.

To overcome stability and heterogeneity issues of antibody-drug conjugates (ADCs) produced with existing bioconjugation technologies incorporating a maleimide motif, we developed McSAF Inside, a new technology based on a trifunctionalized di(bromomethyl)pyridine scaffold. Our solution allows the conjugation of a linker-payload to previously reduced interchain cysteines of a native antibody, resulting in disulfide rebridging. This leads to highly stable and homogeneous ADCs with control over the drug-to-antibody ratio (DAR) and the linker-payload position. Using our technology, we synthesized an ADC, MF-BTX-MMAE, built from anti-CD30 antibody cAC10 (brentuximab), and compared it to Adcetris, the first line treatment against CD30-positive lymphoma, in a CD30-positive lymphoma model. MF-BTX-MMAE displayed improved DAR homogeneity, with a solid batch-to-batch reproducibility, as well as enhanced stability in thermal stress conditions or in the presence of a free thiol-containing protein, such as human serum albumin (HSA). MF-BTX-MMAE showed antigen-binding, in vitro cytotoxicity, in vivo efficacy, and tolerability similar to Adcetris. Therefore, in accordance with current regulatory expectations for the development of new ADCs, McSAF Inside technology gives access to relevant ADCs with improved characteristics and stability.

Design, synthesis, and antiproliferative effect of 2,9-bis[4-(pyridinylalkylaminomethyl)phenyl]-1,10-phenanthroline derivatives on human leukemic cells by targeting G-quadruplex.

Current multiagent chemotherapy regimens have improved the cure rate in acute leukemia patients, but they are highly toxic and poorly efficient in relapsed patients. To improve the treatment approaches, new specific molecules are needed. The G-quadruplexes (G4s), which are noncanonical nucleic acid structures found in specific guanine-rich DNA or RNA, are involved in many cellular events, including control of gene expression. G4s are considered as targets for the development of anticancer agents. Heterocyclic molecules are well known to target and stabilize G4 structures. Thus, a new series of 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives (1a-i) was designed, synthesized, and evaluated against five human myeloid leukemia cell lines (K562, KU812, MV4-11, HL60, and U937). Their ability to stabilize various oncogene promoter G4 structures (c-MYC, BCL-2, and K-RAS) as well as the telomeric G4 was also determined through the fluorescence resonance energy transfer melting assay and native mass spectrometry. In addition, the more bioactive ligands 1g-i were tested for telomerase activity in HuT78 and MV4-11 protein extracts.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation.

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure-activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis.

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.

Site-Specific Conjugation of Auristatins onto Engineered scFv Using Second Generation Maleimide to Target HER2-positive Breast Cancer in Vitro.

Antibody-drug conjugates (ADC) are spearheading vectorized chemotherapy against cancer, with 4 FDA-approved ADCs and 79 in clinical trials. However, most ADCs are produced using a stochastic bioconjugation method, target hematological cancers, and are derived from a full immunoglobulin-G (IgG). These factors limit their efficacy, especially against solid tumors which remain difficult to treat. Here we report the site-specific conjugation of a single auristatin derivative onto an engineered anti-HER2 single chain fragment variable (scFv) of the trastuzumab antibody, generating new scFv-drug conjugates (SDC). Two cysteines were judiciously incorporated at the beginning of the scFv hexahistidine tag, in order to allow controlled bioconjugation of a heterobifunctional linker including a second generation maleimide (SGM), either cleavable (for monomethyl auristatin E) or noncleavable (for monomethyl auristatin F). Our data indicated that both SDCs conserved their affinity to HER2 in comparison to the native scFv, and were efficiently able to kill in vitro HER2-positive SK-BR-3 cells at subnanomolar concentrations (EC50 of 0.68 nM and 0.32 nM). No effect was observed on HER2-negative MCF-7 cells. Ours results showed efficient targeting of site-specific SDCs against HER2-positive breast cancer cells. This work represents a first important step in the design of more effective small conjugates, paving the way for future in vivo translation to evaluate their full potential.

Impact of Site-Specific Conjugation of ScFv to Multifunctional Nanomedicines Using Second Generation Maleimide.

Biocompatible multifunctional nanomedicines (NMs) are known to be an attractive platform for targeted anticancer theranosis. However, these nanomedicines are of interest only if they efficiently target diseased cells and accumulate in tumors. Here we report the synthesis of a new generation of immunotargeted nanomedicines composed of a superparamagnetic iron oxide nanoparticle (SPION) core, polyethylene glycol coating and the anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. We developed two novel bioengineered scFv carrying two cysteines located (i) at the end (4D5.1-cys2) or (ii) at the beginning (4D5.2-cys2) of its hexahistidine tag. The scFv bioconjugation was controlled via heterobifunctional linkers including a second generation maleimide (SGM). Our data indicated that the insertion of cysteines at the beginning of the hexahistidine tag was allowed to obtain nearly 2-fold conjugation efficiency (13 scFv/NP) compared to NMs using classical maleimide. As a result, the NMs-4D5.2 built using the optimal 4D5-cys2 and linkers equipped with SGM showed the enhanced recognition of HER2 in an ELISA format and on the surface of SK-BR-3 breast cancer cells in vitro. Their stability in serum was also significantly improved compared to the NMs-4D5. Our results showed the fundamental importance of the controlled ligand conjugation in the perspective of rational design of NMs with tailored physicochemical and biological properties.

Impact of cathepsin B-sensitive triggers and hydrophilic linkers on in vitro efficacy of novel site-specific antibody-drug conjugates.

Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG12 spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.

Increment in Drug Loading on an Antibody-Drug Conjugate Increases Its Binding to the Human Neonatal Fc Receptor in Vitro.

Antibody-drug conjugates, such as brentuximab vedotin (BTXv), are an innovative category of monoclonal antibodies. BTXv is bioconjugated via the chemical reduction of cysteine residues involved in disulfide bonds. Species of BTXv containing zero, two, four, six, or eight vedotin molecules per antibody coexist in the stock solution. We investigated the influence of drug loading on the binding of the antibody to FcRn, a major determinant of antibody pharmacokinetics in humans. We developed a hydrophobic interaction chromatography (HIC) method for separating the different species present in the stock solution of BTXv, and we purified and characterized the collected species before use. We assessed the binding of these different species to FcRn in a cellular assay based on flow cytometry and surface plasmon resonance. HIC separated the different species of BTXv and allowed their collection at adequate levels of purity. Physicochemical characterization showed that species with higher levels of drug loading tended to form more aggregates. FcRn binding assays showed that the most conjugated species, particularly those with saturated loading, interacted more strongly than unconjugated BTXv with the FcRn.

Rhodium-catalyzed 1,4-addition of arylboronic acids to 3-benzylidene-1H-pyrrolo[2,3-b]pyridin-2(3H)-one derivatives.

7-Azaindoles are versatile building blocks, especially in medicinal chemistry, where they serve as bioisosteres of indoles or purines. Herein, we present a novel rhodium-catalyzed asymmetric 1,4-addition of arylboronic acids to 3-benzylidene-1H-pyrrolo[2,3-b]pyridin-2(3H)-ones, as these substrates are exocyclic methylene lactamyl Michael acceptors. Ten new original derivatives of 1H-pyrrolo[2,3-b]pyridin-2(3H)-one have been obtained.

Dual intra- and extracellular release of monomethyl auristatin E from a neutrophil elastase-sensitive antibody-drug conjugate.

Antibody-drug conjugates (ADCs) are targeted therapies, mainly used in oncology, consisting in a three-component molecular architecture combining a highly potent drug conjugated via a linker onto a monoclonal antibody (mAb), designed for the selective delivery of the drug to the tumor site. The linker is a key component, defining the ADC stability and mechanism of action, and particularly the drug release strategy. In this study, we have developed and synthesized a cleavable linker, which possesses an Asn-Pro-Val (NPV) sequence sensitive to the human neutrophil elastase (HNE), overexpressed in the tumor microenvironment. This linker permitted the site-specific conjugation of the cell-permeable drug, monomethyl auristatin E (MMAE), onto trastuzumab, using a disulfide re-bridging technology. The resulting ADC was then evaluated in vitro. This conjugate demonstrated retained antigen (Ag) binding affinity and exhibited high subnanomolar potency against Ag-positive tumor cells after internalization, suggesting an intracellular mechanism of linker cleavage. While no internalization and cytotoxic activity of this ADC was observed on Ag-negative cells in classical conditions, the supplementation of exogenous HNE permitted to restore a nanomolar activity on these cells, suggesting an extracellular mechanism of drug release in these conditions. This in vitro proof of concept tends to prove that the NPV sequence could allow a dual intra- and extracellular mechanism of drug release. This work represents a first step in the design of original ADCs with a new dual intra- and extracellular drug delivery system and opens the way to further experimentations to evaluate their full potential in vivo.

Antibody-Drug Conjugates as an Emerging Therapy in Oncodermatology.

Antibody-drug conjugates (ADCs) are an emerging class of therapeutics, with twelve FDA- and EMA-approved drugs for hematological and solid cancers. Such drugs consist in a monoclonal antibody linked to a cytotoxic agent, allowing a specific cytotoxicity to tumor cells. In recent years, tremendous progress has been observed in therapeutic approaches for advanced skin cancer patients. In this regard, targeted therapies (e.g., kinase inhibitors) or immune checkpoint-blocking antibodies outperformed conventional chemotherapy, with proven benefit to survival. Nevertheless, primary and acquired resistances as well as adverse events remain limitations of these therapies. Therefore, ADCs appear as an emerging therapeutic option in oncodermatology. After providing an overview of ADC design and development, the goal of this article is to review the potential ADC indications in the field of oncodermatology.

Anticalin N- or C-Terminal on a Monoclonal Antibody Affects Both Production and In Vitro Functionality.

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

Design, synthesis and biological evaluation of new thalidomide analogues as TNF-α and IL-6 production inhibitors.

Several thalidomide analogues were synthesized and compared to thalidomide and its more active analogue, lenalidomide, for their ability to inhibit the production of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α and interleukin (IL)-6 by LPS-activated peripheral blood mononuclear cells (PBMCs). Among these compounds, two analogues containing sulfonyl group displayed interesting downregulation of TNF-α and IL-6 production.

Inhibitors Targeting STAT5 Signaling in Myeloid Leukemias: New Tetrahydroquinoline Derivatives with Improved Antileukemic Potential.

Signal transducers and activators of transcription 5A and 5B (STAT5A and STAT5B) are two closely related STAT family members that are crucial downstream effectors of tyrosine kinase oncoproteins such as FLT3-ITD in acute myeloid leukemia (AML) and BCR-ABL in chronic myeloid leukemia (CML). We recently developed and reported the synthesis of a first molecule called 17 f that selectively inhibits STAT5 signaling in myeloid leukemia cells and overcomes their resistance to chemotherapeutic agents. To improve the antileukemic effect of 17 f, we synthesized ten analogs of this molecule and analyzed their impact on cell growth, survival, chemoresistance and STAT5 signaling. Two compounds, 7 a and 7 a', were identified as having similar or higher antileukemic effects in various AML and CML cell lines. Both molecules were found to be more effective than 17 f at inhibiting STAT5 activity/expression and suppressing the chemoresistance of CML.

Uncyclized xanthommatin is a key ommochrome intermediate in invertebrate coloration.

Ommochromes are widespread pigments that mediate multiple functions in invertebrates. The two main families of ommochromes are ommatins and ommins, which both originate from the kynurenine pathway but differ in their backbone, thereby in their coloration and function. Despite its broad significance, how the structural diversity of ommochromes arises in vivo has remained an open question since their first description. In this study, we combined organic synthesis, analytical chemistry and organelle purification to address this issue. From a set of synthesized ommatins, we derived a fragmentation pattern that helped elucidating the structure of new ommochromes. We identified uncyclized xanthommatin as the elusive biological intermediate that links the kynurenine pathway to the ommatin pathway within ommochromasomes, the ommochrome-producing organelles. Due to its unique structure, we propose that uncyclized xanthommatin functions as a key branching metabolite in the biosynthesis and structural diversification of ommatins and ommins, from insects to cephalopods.