Marcos Pires

Doxorubicin, a well-established chemotherapeutic agent, is known to accumulate in the cell nucleus. By using ICP-MS, we show that the conjugation of two small organometallic rhenium complexes to this structural motif results in a significant redirection of the conjugates from the nucleus to the mitochondria. Despite this relocation, the two bioconjugates display excellent toxicity toward HeLa cells. In addition, we carried out a preliminarily investigation of aspects of cytotoxicity and present evidence that the conjugates disrupt the mitochondrial membrane potential, are strong inhibitors of human Topoisomerase II, and induce apoptosis. Such derivatives may enhance the therapeutic index of the aggressive parent drug and overcome drug resistance by influencing nuclear and mitochondrial homeostasis.

The road less travelled: Doxorubicin is a strong chemotherapeutic agent known for its exclusively nuclear accumulation. Two organometallic derivatives of Doxorubicin were found to display a novel and unexpected subcellular distribution, with significant uptake into mitochondria. First in vitro studies corroborate an influence on mitochondrial homeostasis. Such derivatives could be relevant in the ongoing quest for chemotherapeutic agents that can be better tolerated.

The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance—both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.

Cysteine residues and bioconjugation: Chemical protein modification is used to construct proteins with enhanced and/or altered properties useful for a vast range of applications. Modification at only a single site ensures homogeneity, and the relatively uncommon and uniquely reactive natural residue cysteine allows for selective reactions with a diverse range of coupling partners.

The current inhibitor-based approach to therapeutics has inherent limitations owing to its occupancy-based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off-target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event-driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small-molecule-based proteolysis-targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.

Now we're just falling apart: The use of small-molecule proteolysis-targeting chimeras (PROTACs) to induce protein degradation offers an alternative therapeutic strategy to the traditional inhibitor-based approach. Recent progress in developing small-molecule PROTACs to mediate the proteasomal degradation of targeted proteins is presented, and the opportunities and challenges of this technology for drug discovery are discussed.

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

Marking the mycomembrane: Bioorthogonal chemical reporters are reported that enable sensitive, selective, and simultaneous in situ fluorescence detection of the two major mycolic acid-containing components of the mycobacterial outer membrane, which is a crucial target for tuberculosis drug development.

Home brew: Biotechnological modification of the yeast Sacharomyces cerevisiae enabled the production of opioids from glucose. This challenging extension of yeast metabolism, the result of ten years of work, involves the artificial expression of 23 genes originating from yeast, plants, and rats. This breakthrough was achieved by applying the design principles of synthetic biology, systems biology, and protein engineering.

Resistance to β-lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal-dependent activation of a water molecule. Serine β-lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo-β-lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.

Arming to fight the resistance: Metallo-β-lactamases (MBLs) are hydrolytic enzymes that cleave β-lactam rings. There is a growing need for inhibitors of MBLs that can be given as codrugs with β-lactam antibiotics. Aspergillomarasmine A (see structure) has been identified as a potential codrug. Its total synthesis confirms its stereochemical configuration and offers a route for the synthesis of derivatives.

Antimicrobial resistance poses serious public health concerns and antibiotic misuse/abuse further complicates the situation; thus, it remains a considerable challenge to optimize/improve the usage of currently available drugs. We report a general strategy to construct a bacterial strain-selective delivery system for antibiotics based on responsive polymeric vesicles. In response to enzymes including penicillin G amidase (PGA) and β-lactamase (Bla), which are closely associated with drug-resistant bacterial strains, antibiotic-loaded polymeric vesicles undergo self-immolative structural rearrangement and morphological transitions, leading to sustained release of antibiotics. Enhanced stability, reduced side effects, and bacterial strain-selective drug release were achieved. Considering that Bla is the main cause of bacterial resistance to β-lactam antibiotic drugs, as a further validation, we demonstrate methicillin-resistant S. aureus (MRSA)-triggered release of antibiotics from Bla-degradable polymeric vesicles, in vitro inhibition of MRSA growth, and enhanced wound healing in an in vivo murine model.

Smart delivery: Stimuli-responsive polymeric vesicles deliver antibiotics in response to specific enzymes including penicillin G amidase (PGA) and β-lactamase (Bla), which are relevant to drug-resistant bacterial strains. Antibiotic-loaded vesicles undergo self-immolative structural rearrangement and morphological transitions, leading to sustained antibiotic release.

Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD-CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD-CD1 was derived by an in vitro selection approach where a random-sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacteria species. RFD-CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain-specific recognition of bacterial pathogens.

A DNA molecule that is picky: A method has been established for developing catalytic DNA probes that recognize a targeted infectious strain of a specific bacterium without cross-reactivities to non-pathogenic strains of the same species.

An ability to control the assembly of peptide nanotubes (PNTs) would provide biomaterials for applications in nanotechnology and synthetic biology. Recently, we presented a modular design for PNTs using α-helical barrels with tunable internal cavities as building blocks. These first-generation designs thicken beyond single PNTs. Herein we describe strategies for controlling this lateral association, and also for the longitudinal assembly. We show that PNT thickening is pH sensitive, and can be reversed under acidic conditions. Based on this, repulsive charge interactions are engineered into the building blocks leading to the assembly of single PNTs at neutral pH. The building blocks are modified further to produce covalently linked PNTs via native chemical ligation, rendering ca. 100 nm-long nanotubes. Finally, we show that small molecules can be sequestered within the interior lumens of single PNTs.

Thick to thin: The assembly in coiled–coil peptide nanotubes (PNTs) can be controlled. Arrays of hexameric coiled–coil PNTs can be reversibly disassembled by acidification. Accordingly, repulsive-charge interactions engineered into the coiled–coil units result in the formation of single PNTs at neutral pH. Non-covalent or covalent linkage by native chemical ligation can be used to vary the stability of, and small-molecule encapsulation by, the resulting PNTs.

Michael addition reactions between biological thiols and endocyclic olefinic maleimides are extensively used for site-specific bioconjugation. The resulting thio-succinimidyl linkages, however, lack stability because of their susceptibility to thiol exchange. Reported herein is that in contrast to their endocyclic counterparts, exocyclic olefinic maleimides form highly stable thio-Michael adducts which resist thiol exchange at physiological conditions. A high-yielding approach for synthesizing a variety of exocyclic olefinic maleimides, by 4-nitrophenol-catalyzed solvent-free Wittig reactions, is reported. Mechanistic studies reveal that the catalyst facilitates the formation of the Wittig ylide intermediate through sequential proton donation and abstraction. Overall, this report details an improved thiol bioconjugation approach, a facile method for synthesizing exocyclic olefinic maleimides, and demonstrates that phenolic compounds can catalyze ylide formation.

In stable bioconjugation: A variety of exocyclic olefinic maleimides have been prepared from endocyclic olefinic maleimides under solvent-free conditions by 4-nitrophenol-mediated catalysis. These scaffolds were found to react selectively with thiols at physiological conditions to form linkages that resist thiol-exchange-mediated breakdown, thus demonstrating their potential for stable thiol bioconjugation.