Prodrug Activation Research Articles

Palladium-Mediated Bioorthogonal System for Prodrug Activation of N-Benzylbenzamide-Containing Tubulin Polymerization Inhibitors for the Treatment of Solid Tumors.

Bioorthogonal cleavage reactions have been developed as an intriguing strategy to enhance the safety of chemotherapeutics. Aiming to reduce the toxicity and improve the targeted release properties of the colchicine binding site inhibitors (CBSIs) based on previous work, a series of biologically inert prodrugs were further designed and synthesized through a bioorthogonal prodrug strategy. The therapeutic effects of prodrugs could be "turned-on" once combined with palladium resins. Particularly, prodrug 2b was 68.3-fold less cytotoxic compared to the parent compound, while its cytotoxicity was recovered in situ in the presence of palladium resins. Mechanism studies confirmed that 2b inhibited cell growth in the same manner as CBSIs. More importantly, in vivo efficacy studies demonstrated the efficient activation of 2b by palladium resins, resulting in significant inhibition of tumor growth (63.2%). These results suggest that prodrug 2b with improved safety and targeted release property catalyzed by a Pd-mediated bioorthogonal cleavage reaction deserves further investigation.

DBPR116, a Prodrug of BPRMU191, in Combination with Naltrexone as a Safer Opioid Analgesic Than Morphine via Peripheral Administration.

The development of opioid analgesics with reduced adverse effects is an unmet need. In a previous study, we discovered a unique combination of BPRMU191 and morphinan antagonists that produced potent antinociception with reduced adverse effects after central administration (intrathecal or intracerebroventricular). BPRMU191/naltrexone exhibits notable in vitro and in vivo pharmacological properties. However, the poor blood-brain barrier penetrative ability of BPRMU191 restricts its clinical application. In this study, we utilized a prodrug strategy to deliver sufficient brain concentrations of BPRMU191 and selected compound 2 (DBPR116) with the best physicochemical and pharmacological properties among other in vivo active prodrugs. The in vivo pharmacological studies of compound 2/naltrexone, including thermally stimulated pain, cancer pain, constipation, sedation, psychological dependence, heart rate, and respiratory frequency measurements, demonstrated that it was a safer opioid analgesic than morphine in pain control.

Advancing Cancer Immunotherapy through Engineering New PD-L1 Degraders: A Comprehensive Study from Small Molecules to PD-L1-Specific Peptide-Drug Conjugates.

Despite the considerable achievements of antibodies targeting PD-1/PD-L1 in cancer immunotherapy, limitations in antitumor immune response and pharmacokinetics hinder their clinical adoption. Small molecules toward PD-L1 degradation signifies an innovative avenue to modulate PD-1/PD-L1 axis. Herein, we unveil a comprehensive engineering involving the development of new PD-L1 degraders based on the berberine (BBR) and palmatine (PMT) bioactive frameworks and explore their translational potential for cancer immunotherapy using a peptide-drug conjugate strategy. Chemical modifications at the O-9 position of PMT dramatically enhance the PD-L1 degradation capacity. Further conjugation of PMT degraders with an anti-PD-L1 peptide featuring disulfide linkers enables efficient GSH-specific prodrug activation, yielding synergistic immunotherapeutic benefits through both external PD-L1 blockade and internal PD-L1 degradation mechanisms. This work elucidates the compelling charm of the discovery and application of PD-L1 degraders, offering solutions to the challenges in advancing cancer immunotherapy in widespread clinics.

Prodrug Nanomedicine for Synovium Targeted Therapy of Inflammatory Arthritis: Insights from Animal Model and Human Synovial Joint Fluid.

Many patients cannot tolerate low-dose weekly methotrexate (MTX) therapy for inflammatory arthritis treatment due to life-threatening toxicity. Although biologics offer a target-specific therapy, it raises the risk of serious infections and even cancer due to immune system suppression. We introduce an anti-inflammatory arthritis MTX ester prodrug using a long-circulating biocompatible polymeric macromolecule: folic acid (FA) functionalized hyperbranched polyglycerol (HPG). In vitro the drug MTX is incrementally released through pH and enzymatic degradation over 2 weeks. The role of matrix metalloproteinases (MMPs) in site-specific prodrug activation was verified using synovial fluid (SF) of 26 rheumatology patients and 4 healthy controls. Elevated levels of specific MMPs-markers of joint inflammation-positively correlated with enhanced prodrug release explained by acid-catalyzed hydrolysis of esters by proteases. Intravenously administered 111In-radiolabeled prodrug confirmed by SPECT/CT imaging that it accumulated preferentially in inflamed joints while reducing off-target side-effects in a mouse model of rheumatoid arthritis (RA). Added FA as a targeting vector prolonged prodrug action; prodrug with 4x less MTX applied every 2 weeks was as effective as weekly MTX therapy. The preclinical results suggest a prodrug-based strategy for the treatment of inflammatory joint diseases, with potential for other chronic inflammatory diseases and cancer.

Prodrug activation by 4,4’-bipyridine-mediated aromatic nitro reduction

Unleashing prodrugs through nitro-reduction is a promising strategy in cancer treatment. In this study, we present a unique bioorthogonal reaction for aromatic nitro reduction, mediated by 4,4‘-bipyridine. The reaction is a rare example of organocatalyst-mediated bioorthogonal reaction. This bioorthogonal reaction demonstrates broad substrate scope and proceeds at low micromolar concentrations under biocompatible conditions. Our mechanistic study reveals that water is essential for the reaction to proceed at biorelevant substrate concentrations. We illustrate the utility of our reaction for controlled prodrug activation in mammalian cells, bacteria, and mouse models. Furthermore, a nitro-reduction-annulation cascade is developed for the synthesis of indole derivatives in living cells.

Widespread loss-of-function mutations implicating preexisting resistance to new or repurposed anti-tuberculosis drugs

BackgroundFive New or Repurposed Drugs (NRDs) were approved in the last decade for treatment of multi-drug resistant tuberculosis: bedaquiline, clofazimine, linezolid, delamanid, and pretomanid. Unfortunately, resistance to these drugs emerged faster than anticipated, potentially due to preexisting resistance in naïve strains. Previous investigations into the rapid emergence have mostly included short variants. For the first time, we utilize de novo-assembled genomes, and systematically include Structural Variations (SV) and heterogeneity to comprehensively study this rapid emergence. We show high prevalence of preexisting resistance, identify novel markers of resistance, and lay the foundation for preventing preexisting resistance in future drug development. MethodsFirst, a systematic literature review revealed 313 NRD resistance variants in 13 genes. Next, 409 globally diverse clinical isolates collected prior to the drugs’ programmatic use (308 were multidrug resistant, 106 had de novo assembled genomes) were utilized to study the 13 genes comprehensively for conventional, structural, and heterogeneous variants. FindingsWe identified 5 previously reported and 67 novel putative NRD resistance variants. These variants were 2 promoter mutations (in 8/409 isolates), 13 frameshifts (21/409), 6 SVs (9/409), 35 heterogeneous frameshifts (32/409) and 11 heterogeneous SVs (12/106). Delamanid and pretomanid resistance mutations were most prevalent (48/409), while linezolid resistance mutations were least prevalent (8/409). InterpretationPreexisting mutations implicated in resistance to at least one NRD was highly prevalent (85/409, 21 %). This was mostly caused by loss-of-function mutations in genes responsible for prodrug activation and efflux pump regulation. These preexisting mutations may have emerged through a bet-hedging strategy, or through cross-resistance with non-tuberculosis drugs such as metronidazole. Future drugs that could be resisted through loss-of-function in non-essential genes may suffer from preexisting resistance. The methods used here for comprehensive preexisting resistance assessment (especially SVs and heterogeneity) may mitigate this risk during early-stage drug development.

Paclitaxel prodrug nanoparticles boost antitumor efficacy via hitchhiking of human serum albumin

Improving drug delivery efficacy is the key point for enhancing the therapeutic index of medicines. Herein, we report fatty chain conjugated paclitaxel (PTX) prodrugs with a disulfide bond as linker. The formed prodrugs can self-assemble into stable nanoparticles in aqueous solutions, and rapidly transform into long-circulating nanocomplexes via the non-covalent binding to serum albumin in blood, enabling efficient drug delivery and robust antitumor effect. PTX prodrug (PC) with single-chain possess the improved self-assembly and interaction with albumins. The formed PC@albumin nanocomplexes reinforce the responsiveness of prodrug activation, and exhibit the enhanced tumor suppression ability. This strategy of hitchhiking albumin to deliver therapeutic agents holds great promise for enhanced chemotherapy.

Senolytic Prodrugs: A Promising Approach to Enhancing Senescence-targeting Intervention.

Cellular senescence has emerged as a potential therapeutic target for aging and a wide range of age-related disorders. Despite the encouraging therapeutic impact of senolytic agents on improving lifespan and the outcomes of pharmacological intervention, the senolytic induced side effects pose barriers to clinical application. There is a pressing need for selective ablation of senescent cells (SnCs). The design of senolytic prodrugs has been demonstrated as a promising approach to addressing these issues. These prodrugs are generally designed via modification of senolytics with a cleavable galactose moiety to respond to the senescent biomarker - senescence-associated β-galactosidase (SA-β-gal) to restore their therapeutic effects. In this Concept, we summarize the developments by categorizing these prodrugs into two classes: 1) galactose-modified senolytic prodrugs, in which sensing unit galactose is either directly conjugated to the drug or via a self-immolative linker and 2) bioorthogonal activation of senolytic prodrugs. In the bioorthogonal prodrug design, galactose is incorporated into dihydrotetrazine to sense SA-β-gal for click activation. Notably, in addition to repurposed chemotherapeutics and small molecule inhibitors, PROTACs and photodynamic therapy have been introduced as new senolytics in the prodrug design. It is expected that the senolytic prodrugs would facilitate translating small-molecule senolytics into clinical use.

Synthesis and release studies on amylose-based ester prodrugs of fenamic acid NSAIDs.

Aim: To achieve colon-targeted release of mefenamic acid from its ester-linked amylose prodrugs.Materials & methods: The prodrug was characterized by 1H NMR and IR spectroscopy. Drug activation and release profile was studied in enzyme enriched simulated physiological media via UV-vis spectroscopy and was validated with HPLC analysis. ELISA assay was employed for evaluating the % inhibition of COX-1 and COX-2 inhibition at different concentrations of the prodrug preincubated with ester and/ or amylose hydrolyzing enzymes. SEM studies further validated the performance of the prodrug under simulated physiological conditions.Results: Pancreatin was essential for the prodrug activation in SIM to make the ester bonds in prodrug vulnerable to hydrolysis by esterase. This evidence was confirmed by drug release studies, HPLC analysis, ELISA assay and SEM investigation where the ester conjugated prodrug showed marked stability in physiological media only to get activated in the presence of amylose degrading enzyme.Conclusion: Ester linked amylose-mefenamic acid conjugate showed both enzyme responsive activation and release in SIM.

Transforming Aryl‐Tetrazines into Bioorthogonal Scissors for Systematic Cleavage of trans‐Cyclooctenes

Bioorthogonal bond‐cleavage reactions have emerged as a powerful tool for precise spatiotemporal control of (bio)molecular function in the biological context. Among these chemistries, the tetrazine‐triggered elimination of cleavable trans‐cyclooctenes (click‐to‐release) stands out due to high reaction rates, versatility, and selectivity. Despite an increasing understanding of the underlying mechanisms, application of this reaction remains limited by the cumulative performance trade‐offs (i.e., click kinetics, release kinetics, release yield) of existing tools. Efficient release has been restricted to tetrazine scaffolds with comparatively low click reactivity, while highly reactive aryl‐tetrazines give only minimal release. By introducing hydroxyl groups onto phenyl‐ and pyridyl‐tetrazine scaffolds, we have developed a new class of ‘bioorthogonal scissors’ with unique chemical performance. We demonstrate that hydroxyaryl‐tetrazines achieve near‐quantitative release upon accelerated click reaction with cleavable trans‐cyclooctenes, as exemplified by click‐triggered activation of a caged prodrug, intramitochondrial cleavage of a fluorogenic probe (turn‐on) in live cells, and rapid intracellular bioorthogonal disassembly (turn‐off) of a ligand‐dye conjugate.

Transforming Aryl-Tetrazines into Bioorthogonal Scissors for Systematic Cleavage of trans-Cyclooctenes.

Bioorthogonal bond-cleavage reactions have emerged as a powerful tool for precise spatiotemporal control of (bio)molecular function in the biological context. Among these chemistries, the tetrazine-triggered elimination of cleavabletrans-cyclooctenes (click-to-release) stands out due to high reaction rates, versatility, and selectivity. Despite an increasing understanding of the underlying mechanisms, application of this reaction remains limited by the cumulative performance trade-offs (i.e., click kinetics, release kinetics, release yield) of existing tools. Efficient release has been restricted to tetrazine scaffolds with comparatively low click reactivity, while highly reactive aryl-tetrazines give only minimal release. By introducing hydroxyl groups onto phenyl- and pyridyl-tetrazine scaffolds, we have developed a new class of 'bioorthogonal scissors' with unique chemical performance. We demonstrate that hydroxyaryl-tetrazines achieve near-quantitative release upon accelerated click reaction with cleavabletrans-cyclooctenes, as exemplified by click-triggered activation of a caged prodrug, intramitochondrial cleavage of a fluorogenic probe (turn-on) in live cells, and rapid intracellular bioorthogonal disassembly (turn-off) of a ligand-dye conjugate.

Phenotypic-Based Discovery and Exploration of a Resorufin Scaffold with Activity against Mycobacterium tuberculosis.

Tuberculosis remains a leading cause of death by infectious disease. The long treatment regimen and the spread of drug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) necessitates the development of new treatment options. In a phenotypic screen, nitrofuran-resorufin conjugate 1 was identified as a potent sub-micromolar inhibitor of whole cell Mtb. Complete loss of activity was observed for this compound in Mtb mutants affected in enzyme cofactor F420 biosynthesis (fbiC), suggesting that 1 undergoes prodrug activation in a manner similar to anti-tuberculosis prodrug pretomanid. Exploration of the structure-activity relationship led to the discovery of novel resorufin analogues that do not rely on the deazaflavin-dependent nitroreductase (Ddn) bioactivation pathway for their antimycobacterial activity. These analogues are of interest as they work through an alternative, currently unknown mechanism that may expand our chemical arsenal towards the treatment of this devastating disease.

Prodrug activation in malaria parasites mediated by an imported erythrocyte esterase, acylpeptide hydrolase (APEH).

The continued emergence of antimalarial drug resistance highlights the need to develop new antimalarial therapies. Unfortunately, new drug development is often hampered by poor drug-like properties of lead compounds. Prodrugging temporarily masks undesirable compound features, improving bioavailability and target penetration. We have found that lipophilic diester prodrugs of phosphonic acid antibiotics, such as fosmidomycin, exhibit significantly higher antimalarial potency than their parent compounds (1). However, the activating enzymes for these prodrugs were unknown. Here, we show that an erythrocyte enzyme, acylpeptide hydrolase (APEH) is the major activating enzyme of multiple lipophilic ester prodrugs. Surprisingly, this enzyme is taken up by the malaria parasite, Plasmodium falciparum, where it localizes to the parasite cytoplasm and retains enzymatic activity. Using a novel fluorogenic ester library, we characterize the structure activity relationship of APEH, and compare it to that of P. falciparum esterases. We show that parasite-internalized APEH plays an important role in the activation of substrates with branching at the alpha carbon, in keeping with its exopeptidase activity. Our findings highlight a novel mechanism for antimicrobial prodrug activation, relying on a host-derived enzyme to yield activation at a microbial target. Mutations in prodrug activating enzymes are a common mechanism for antimicrobial drug resistance (2-4). Leveraging an internalized host enzyme would circumvent this, enabling the design of prodrugs with higher barriers to drug resistance.

Reductants supplement boost the antitumor efficacy of nanomedicine

The elevated redox state has become a distinctive characteristic in the tumor environment for designing novel nanomedicines. However, the excessive oxidation stress brought out several plights, such as insufficient reduction-responsive drug release, dense extracellular matrix, and immunosuppressive tumor microenvironment. Here, we explored a potential strategy of replenishing exogenous reductants to boost the efficacy of nanomedicines. Disulfide bond-bridged cabazitaxel prodrug nanoassemblies were developed with high drug loading and reduction responsivity. As relying on endogenous reductants only was not sufficient enough to trigger the activation of prodrug, exogenous reductants were further supplemented to accelerate the activation efficiency. In addition, we found exogenous reductants would increase the tumor penetration of prodrug nanoassemblies, reverse the immunosuppressive system caused by high oxidation stress, and mediate a remarkable antitumor efficacy combined with Anti-PD1. This work elucidated the functions of reductants in cancer treatment, and highlighted their prominent role in combination with nanomedicines.

Enhancing the therapeutic window for Spectinamide anti-tuberculosis Agents: Synthesis, Evaluation, and activation of phosphate prodrug 3408

Spectinamides are a novel class of narrow-spectrum antitubercular agents with the potential to treat drug-resistant tuberculosis infections. Spectinamide 1810 has shown a good safety record following subcutaneous injection in mice or infusion in rats but exhibits transient acute toxicity following bolus administration in either species. To improve the therapeutic index of 1810, an injectable prodrug strategy was explored. The injectable phosphate prodrug 3408 has a superior maximum tolerated dose compared to 1810 or Gentamicin. Following intravenous administration in rodents, prodrug 3408 was quickly converted to 1810. The resulting 1810 exposure and pharmacokinetic profile after 3408 administration was identical to equivalent molar amounts of 1810 given directly by intravenous administration. 3408 and the parent 1810 exhibited similar overall efficacy in a BALB/c acute tuberculosis efficacy model. Delivery of 1810 in phosphate prodrug form, therefore, holds the potential to improve further the therapeutic index of an already promising tuberculosis antibiotic.

Acid-Responsive Polymer Micelles for Targeted Delivery and Bioorthogonal Activation of Prodrug through Ru Catalyst in Tumor Cells.

Bioorthogonal reactions present a promising strategy for minimizing off-target toxicity in cancer chemotherapy, yet a dependable nanoplatform is urgently required. Here, we have fabricated an acid-responsive polymer micelle for the specific delivery and activation of the prodrug within tumor cells through Ru catalyst-mediated bioorthogonal reactions. The decomposition of micelles, triggered by the cleavage of the hydrazone bond in the acidic lysosomal environment, facilitated the concurrent release of Alloc-DOX and the Ru catalyst within the cells. Subsequently, the uncaging process of Alloc-DOX was demonstrated to be induced by the high levels of glutathione within tumor cells. Notably, the limited glutathione inside normal cells prevented the conversion of Alloc-DOX into active DOX, thereby minimizing the toxicity toward normal cells. In tumor-bearing mice, this nanoplatform exhibited enhanced efficacy in tumor suppression while minimizing off-target toxicity. Our study provides an innovative approach for in situ drug activation that combines safety and effectiveness in cancer chemotherapy.

Fibroblast Activation Protein Is Expressed by Altered Osteoprogenitors and Associated to Disease Burden in Fibrous Dysplasia.

Fibrous dysplasia (FD) is a mosaic skeletal disorder involving the development of benign, expansile fibro-osseous lesions during childhood that cause deformity, fractures, pain, and disability. There are no well-established treatments for FD. Fibroblast activation protein (FAPα) is a serine protease expressed in pathological fibrotic tissues that has promising clinical applications as a biomarker and local pro-drug activator in several pathological conditions. In this study, we explored the expression of FAP in FD tissue and cells through published genetic expression datasets and measured circulating FAPα in plasma samples from patients with FD and healthy donors. We found that FAP genetic expression was increased in FD tissue and cells, and present at higher concentrations in plasma from patients with FD compared to healthy donors. Moreover, FAPα levels were correlated with skeletal disease burden in patients with FD. These findings support further investigation of FAPα as a potential imaging and/or biomarker of FD, as well as a pro-drug activator specific to FD tissue.

Lignin-Based Nanoparticles for Combination of Tumor Oxidative Stress Amplification and Reactive Oxygen Species Responsive Drug Release.

In this study, maleic anhydride-modified lignin (LG-M), a ROS-cleavable thioketal (TK) bond, and polyethylene glycol (PEG) were used to synthesize a lignin-based copolymer (LG-M(TK)-PEG). Doxorubicin (DOX) was attached to the ROS-cleavable bond in the LG-M(TK)-PEG for the preparation of the ROS-activatable DOX prodrug (LG-M(TK-DOX)-PEG). Nanoparticles (NPs) with a size of 125.7 ± 3.1 nm were prepared by using LG-M(TK-DOX)-PEG, and they exhibited enhanced uptake by cancer cells compared to free DOX. Notably, the presence of lignin in the nanoparticles could boost ROS production in breast cancer 4T1 cells while showing little effect on L929 normal cells. This selective effect facilitated the specific activation of the DOX prodrug in the tumor microenvironment, resulting in the superior tumor inhibitory effects and enhanced biosafety relative to free DOX. This work demonstrates the potential of the LG-M(TK-DOX)-PEG NPs as an efficient drug delivery system for cancer treatment.

Gold(III)-Induced Amide Bond Cleavage In Vivo: A Dual Release Strategy via π-Acid Mediated Allyl Substitution.

Selective cleavage of amide bonds holds prominent significance by facilitating precise manipulation of biomolecules, with implications spanning from basic research to therapeutic interventions. However, achieving selective cleavage of amide bonds via mild synthetic chemistry routes poses a critical challenge. Here, we report a novel amide bond-cleavage reaction triggered by Na[AuCl4] in mild aqueous conditions, where a crucial cyclization step leads to the formation of a 5-membered ring intermediate that rapidly hydrolyses to release the free amine in high yields. Notably, the reaction exhibits remarkable site-specificity to cleave peptide bonds at the C-terminus of allyl-glycine. The strategic introduction of a leaving group at the allyl position facilitated a dual-release approach through π-acid catalyzed substitution. This reaction was employed for the targeted release of the cytotoxic drug monomethyl auristatin E in combination with an antibody-drug conjugate in cancer cells. Finally, Au-mediated prodrug activation was shown in a colorectal zebrafish xenograft model, leading to a significant increase in apoptosis and tumor shrinkage. Our findings reveal a novel metal-based cleavable reaction expanding the utility of Au complexes beyond catalysis to encompass bond-cleavage reactions for cancer therapy.

Bio-inspired biorthogonal compartmental microparticles for tumor chemotherapy and photothermal therapy

Microcarrier is a promising drug delivery system demonstrating significant value in treating cancers. One of the main goals is to devise microcarriers with ingenious structures and functions to achieve better therapeutic efficacy in tumors. Here, inspired by the nucleus-cytoplasm structure of cells and the material exchange reaction between them, we develop a type of biorthogonal compartmental microparticles (BCMs) from microfluidics that can separately load and sequentially release cyclooctene-modified doxorubicin prodrug (TCO-DOX) and tetrazine-modified indocyanine green (Tz-ICG) for tumor therapy. The Tz-ICG works not only as an activator for TCO-DOX but also as a photothermal agent, allowing for the combination of bioorthogonal chemotherapy and photothermal therapy (PTT). Besides, the modification of DOX with cyclooctene significantly decreases the systemic toxicity of DOX. As a result, the developed BCMs demonstrate efficient in vitro tumor cell eradication and exhibit notable tumor growth inhibition with favorable safety. These findings illustrate that the formulated BCMs establish a platform for bioorthogonal prodrug activation and localized delivery, holding significant potential for cancer therapy and related applications.Graphical