Inhibitory prodrug mechanism for cysteine cathepsin-targeted self-controlled drug release

Tumour-associated macrophages (TAMs) support tumour development and have emerged as important regulators of therapeutic response to cytostatic agents. To target TAMs, we have developed a novel drug delivery approach which induces drug release as it inhibits cysteine cathepsin activity. This inhibitory prodrug (IPD) approach establishes a self-regulated system where drug release stops after all cysteine cathepsins are inhibited. This could improve the therapeutic window for drugs with severe side effects. We demonstrate and characterise this self-regulation concept with a fluorogenic IPD model. Next, we applied this IPD strategy to deliver cytotoxic drugs, as doxorubicin and monomethyl auristatin E, which are efficiently released and dose-dependently eliminate RAW264.7 macrophages. Lastly, by exploiting the increased cathepsin activity in TAM-like M2-polarised primary macrophages, we show that IPD-Dox selectively eliminates M2 over M1 macrophages. This demonstrates the potential of our IPD strategy for selective drug delivery and modulation of the tumour microenvironment.</p

DataSheet1_Insights in the host response towards biomaterial-based scaffolds for cancer therapy.docx

Immunotherapeutic strategies have shown promising results in the treatment of cancer. However, not all patients respond, and treatments can have severe side-effects. Adoptive cell therapy (ACT) has shown remarkable therapeutic efficacy across different leukaemia and lymphoma types. But the treatment of solid tumours remains a challenge due to limited persistence and tumour infiltration. We believe that biomaterial-based scaffolds are promising new tools and may address several of the challenges associated with cancer vaccination and ACT. In particular, biomaterial-based scaffold implants allow for controlled delivery of activating signals and/or functional T cells at specific sites. One of the main challenges for their application forms the host response against these scaffolds, which includes unwanted myeloid cell infiltration and the formation of a fibrotic capsule around the scaffold, thereby limiting cell traffic. In this review we provide an overview of several of the biomaterial-based scaffolds designed for cancer therapy to date. We will discuss the host responses observed and we will highlight design parameters that influence this response and their potential impact on therapeutic outcome.</p

O-GlcNAc Peptide Epoxyketones Are Recognized by Mammalian Proteasomes

O-GlcNAc Peptide Epoxyketones Are Recognized by Mammalian Proteasome

pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed “AND gate” multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines

The Use of a Mannitol-Derived Fused Oxacycle as a Combinatorial Scaffold

An efficient and high-yielding solid-phase synthesis of a small library of compounds containing a cis-fused pyranofuran structural motive is descibed. With use of the cheap and readily available d-(+)-mannitol, a highly functionalized sugar template was synthesized and immobilized on a solid support via an olefinic linker. Modification of this two-point molecular scaffold and subsequent ring-closing metathesis/cleavage gave access to a series of functionalized conformationally constrained fused oxacycles

The Use of a Mannitol-Derived Fused Oxacycle as a Combinatorial Scaffold

An efficient and high-yielding solid-phase synthesis of a small library of compounds containing a cis-fused pyranofuran structural motive is descibed. With use of the cheap and readily available d-(+)-mannitol, a highly functionalized sugar template was synthesized and immobilized on a solid support via an olefinic linker. Modification of this two-point molecular scaffold and subsequent ring-closing metathesis/cleavage gave access to a series of functionalized conformationally constrained fused oxacycles

A Rab20-dependent membrane trafficking pathway controls M. tuberculosis replication by regulating phagosome spaciousness and integrity

The intracellular pathogen Mycobacterium tuberculosis (Mtb) lives within phagosomes and also disrupts these organelles to access the cytosol. The host pathways and mechanisms that contribute to maintaining Mtb phagosome integrity have not been investigated. Here, we examined the spatiotemporal dynamics of Mtb-containing phagosomes and identified an interferon-gamma-stimulated and Rab20-dependent membrane trafficking pathway in macrophages that maintains Mtb in spacious proteolytic phagolysosomes. This pathway functions to promote endosomal membrane influx in infected macrophages, and is required to preserve Mtb phagosome integrity and control Mtb replication. Rab20 is specifically and significantly upregulated in the sputum of human patients with active tuberculosis. Altogether, we uncover an immune-regulated cellular pathway of defense that promotes maintenance of Mtb within intact membrane-bound compartments for efficient elimination

Application of a highly selective cathepsin S two-step activity-based probe in multicolor bio-orthogonal correlative light-electron microscopy.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology

Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity <i>in vivo</i> could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration <i>in vivo</i>. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time

Improved Quenched Fluorescent Probe for Imaging of Cysteine Cathepsin Activity

The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs