Marcos Pires

The bioorthogonality of tetrazole photoclick chemistry has been reassessed. Upon photolysis of a tetrazole, the highly reactive nitrile imine formed undergoes rapid nucleophilic reaction with a variety of nucleophiles present in a biological system, along with the expected cycloaddition with alkenes. The alternative use of the tetrazole photoclick reaction was thus explored: tetrazoles were incorporated into Bodipy and Acedan dyes, providing novel photo-crosslinkers with one- and two-photon fluorescence Turn-ON properties that may be developed into protein-detecting biosensors. Further introduction of these photo-activatable, fluorogenic moieties into staurosporine resulted in the corresponding probes capable of photoinduced, no-wash imaging of endogenous kinase activities in live mammalian cells.

“Tie” and “see”: By reassessing the bioorthogonality of tetrazole photoclick chemistry, an alternative use of tetrazoles in chemical biology is proposed, as general photo-crosslinkers with potential fluorescence Turn-ON properties. This method could be used to develop affinity-based probes for live-cell imaging of endogenous kinase activities. WH=warhead.

Through the simultaneous use of three orthogonal dynamic covalent reactions, namely disulfide, boronate, and acyl hydrazone formation, we conceived a facile and versatile protocol to spatially organize tailored chromophores, which absorb in the blue, red, and yellow regions, on a preprogrammed α-helix peptide. This approach allowed the assembly of the dyes in the desired ratio and spacing, as dictated by both the relative positioning and distribution of the recognition units on the peptide scaffold. Steady-state UV/Vis absorption and emission studies suggest an energy transfer from the yellow and red donors to the blue acceptor. A molecular dynamics simulation supports the experimental findings that the helical structure is maintained after the assembly and the three dyes are confined in defined conformational spaces.

Completely self-absorbed: Tailored chromophores that absorb in the blue, red, and yellow regions can be spatially organized in a desired ratio on a preprogrammed α-helix peptide by the simultaneous use of three orthogonal dynamic covalent reactions (see figure). Energy transfer occurs from the yellow- and red- to the blue-absorbing chromophore, and the helical peptide structure is maintained after the self-assembly process.

Although peptide-based therapeutics are finding increasing application in the clinic, extensive structural modification is typically required to prevent their rapid degradation by proteases in the blood. We have evaluated the ability of erythrocytes to serve as reservoirs, protective shields (against proteases), and light-triggered launch pads for peptides. We designed lipidated peptides that are anchored to the surface of red blood cells, which furnishes a protease-resistant environment. A photocleavable moiety is inserted between the lipid anchor and the peptide backbone, thereby enabling light-triggered peptide release from erythrocytes. We have shown that a cell-permeable peptide, a hormone (melanocyte stimulating hormone), and a blood-clotting agent can be anchored to erythrocytes, protected from proteases, and photolytically released to create the desired biological effect.

Gimme shelter: Attachment to the plasma membrane of erythrocytes can be used to protect therapeutic peptides from serum proteases. A photocleavable moiety is inserted between the lipid anchor and the peptide backbone, thereby enabling light-triggered release.

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.

Stitching a protein together: A synthesis approach is reported using selenazolidine and deselenization to access a protein with non-strategically placed cysteine residues. The challenging human phosphohistidine phosphatase 1 (PHPT1) protein, a 125-residue enzyme with three cysteine residues near the C-terminus, was used as a model system.

The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N^α-deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca²⁺ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.

Returning to the fold: LDLa is small protein with complex three disulfide bridges and several aspartic acids that are sensitive to aspartimide formation. This study detailed the solution to circumvent formation of asparatimide and successful acquisition of three disulfide bridges. As a result, synthesis of LDLa was successfully achieved with excellent quality.

The use of adjuvants that rescue antibiotics against multidrug-resistant (MDR) pathogens is a promising combination strategy for overcoming bacterial resistance. While the combination of β-lactam antibiotics and β-lactamase inhibitors has been successful in restoring antibacterial efficacy in MDR bacteria, the use of adjuvants to restore fluoroquinolone efficacy in MDR Gram-negative pathogens has been challenging. We describe tobramycin–ciprofloxacin hybrid adjuvants that rescue the activity of fluoroquinolone antibiotics against MDR and extremely drug-resistant Pseudomonas aeruginosa isolates in vitro and enhance fluoroquinolone efficacy in vivo. Structure–activity studies reveal that the presence of both tobramycin and ciprofloxacin, which are separated by a C12 tether, is critical for the function of the adjuvant. Mechanistic studies indicate that the antibacterial modes of ciprofloxacin are retained while the role of tobramycin is limited to destabilization of the outer membrane in the hybrid.

Rescue me: Adjuvants that rescue the activity of fluoroquinolone antibiotics against multidrug-resistant and extremely drug-resistant Pseudomonas aeruginosa can be generated by linking tobramycin to ciprofloxacin. The adjuvant combines the antibacterial modes of ciprofloxacin with the membrane-destabilizing effects of aminoglycosides, thereby resulting in enhanced cell penetration of fluoroquinolones and other antibiotics into P. aeruginosa.

Vibrio is a model organism for the study of quorum sensing (QS) signaling and is used to identify QS-interfering drugs. Naturally occurring fimbrolides are important tool compounds known to affect QS in various organisms; however, their cellular targets have so far remained elusive. Here we identify the irreversible fimbrolide targets in the proteome of living V. harveyi and V. campbellii via quantitative mass spectrometry utilizing customized probes. Among the major hits are two protein targets with essential roles in Vibrio QS and bioluminescence. LuxS, responsible for autoinducer 2 biosynthesis, and LuxE, a subunit of the luciferase complex, were both covalently modified at their active-site cysteines leading to inhibition of activity. The identification of LuxE unifies previous reports suggesting inhibition of bioluminescence downstream of the signaling cascade and thus contributes to a better mechanistic understanding of these QS tool compounds.

Studies in­ Vibrio: Fimbrolides represent natural products that interfere with quorum sensing in various organisms. Despite their importance in biological studies their cellular mechanisms have remained unknown. Chemical proteomics have been utilized to identify proteins involved in autoinducer biosynthesis (LuxS) and luciferase activity (LuxE) as molecular targets.

Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

Induced protein degradation is an emerging field that has the potential to overcome many challenges faced by traditional inhibitor-based drug design. A modular approach to PROTAC design is presented that enables targeted degradation of the therapeutically relevant BCR-ABL oncogenic protein.

Oligonucleotide-based hepatocyte growth factor (HGF) mimetics are described. A DNA aptamer to Met, a cognate receptor for HGF, was shown to induce Met activation when used in dimer form. The most potent aptamer dimer, ss-0, which was composed solely of 100-mer single-stranded DNA, exhibited nanomolar potency. Aptamer ss-0 reproduced HGF-induced cellular behaviors, including migration and proliferation. The present work sheds light on oligonucleotides as a novel chemical entity for the design of growth factor mimetics.

Bridging the gap: A 100-mer ssDNA was developed as a potent hepatocyte growth factor (HGF) mimetic. This ssDNA was designed to induce receptor dimerization at the cell surface and subsequent signal transduction in the same way as the natural growth factor. This new class of synthetic ligands reproduced growth factor induced cellular behaviors, including cell migration and proliferation.

Cell-surface sialic acids are essential in mediating a variety of physiological and pathological processes. Sialic acid chemistry and biology remain challenging to investigate, demanding new tools for probing sialylation in living systems. The metabolic glycan labeling (MGL) strategy has emerged as an invaluable chemical biology tool that enables metabolic installation of useful functionalities into cell-surface sialoglycans by “hijacking” the sialic acid biosynthetic pathway. Here we review the principles of MGL and its applications in study and manipulation of sialic acid function, with an emphasis on recent advances.

Sialoglycans and click chemistry: The biosynthetic pathway of sialoglycans can be “hijacked” to remodel cell-surface sialic acids. Sialic acid analogues containing unnatural functional groups can be metabolically incorporated into cellular sialoglycans, thus allowing labeling, visualization, and functional modulation.

Membrane-bound proteins are important pharmaceutical drug targets, yet few strategies exist for the identification of small-molecule-targeted membrane proteins in live-cell systems. By exploiting metabolic glycan engineering of cell membrane proteins, we have developed an in situ glycan-mediated ligand-controlled click (“GLiCo-Click”) chemistry methodology that enables the attachment of small-molecule chemical probes to their receptor protein through glycans on live cells. In addition to enabling receptor enrichment from cell lysates, this strategy can be used to demonstrate target receptor engagement and enables the molecular characterization of receptors.

GLiCo Click: We demonstrate a chemical biology strategy for efficient membrane protein crosslinking with a chemical probe that enables receptor visualization with fluorescence microscopy, receptor isolation, and glycan site mapping through nanoscale liquid chromatography coupled tandem mass spectrometry.

The development of bioorthogonal reactions has classically focused on bond-forming ligation reactions. In this report, we seek to expand the functional repertoire of such transformations by introducing a new bond-cleaving reaction between N-oxide and boron reagents. The reaction features a large dynamic range of reactivity, showcasing second-order rate constants as high as 2.3×10³ M⁻¹ s⁻¹ using diboron reaction partners. Diboron reagents display minimal cell toxicity at millimolar concentrations, penetrate cell membranes, and effectively reduce N-oxides inside mammalian cells. This new bioorthogonal process based on miniscule components is thus well-suited for activating molecules within cells under chemical control. Furthermore, we demonstrate that the metabolic diversity of nature enables the use of naturally occurring functional groups that display inherent biocompatibility alongside abiotic components for organism-specific applications.

The bond-cleaving reaction between N-oxide and diboron reagents features second-order rate constants as high as 2.3×10³ M⁻¹ s⁻¹. Diboron reagents display minimal cell toxicity at millimolar concentrations, penetrate cell membranes, and reduce N-oxides inside mammalian cells. This new bioorthogonal reaction is thus well-suited for chemically activating molecules within cells.

An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.

An irreversible butelase-mediated ligation using a thiodepsipeptide substrate is described, with a ligation yield of >95 % and a minimal excess of substrate and low catalytic amounts of butelase 1. This method was used to introduce a functional tag to ubiquitin and green fluorescent protein with high yields.

Caseinolytic protease P (ClpP) is an important regulator of Staphylococcus aureus pathogenesis. A high-throughput screening for inhibitors of ClpP peptidase activity led to the identification of the first non-covalent binder for this enzyme class. Co-crystallization of the small molecule with S. aureus ClpP revealed a novel binding mode: Because of the rotation of the conserved residue proline 125, ClpP is locked in a defined conformational state, which results in distortion of the catalytic triad and inhibition of the peptidase activity. Based on these structural insights, the molecule was optimized by rational design and virtual screening, resulting in derivatives exceeding the potency of previous ClpP inhibitors. Strikingly, the conformational lock is overturned by binding of ClpX, an associated chaperone that enables proteolysis by substrate unfolding in the ClpXP complex. Thus, regulation of inhibitor binding by associated chaperones is an unexpected mechanism important for ClpP drug development.

ClpX calls the tune: The first reversible inhibitor for Staphylococcus aureus ClpP was identified, and its mode of action was visualized at the molecular level. Structure–activity relationship studies led to improved compounds that inhibit the protease in the nanomolar range. Binding of ClpX overrides the inhibitor-induced conformational lock of ClpP and leads to the formation of an active proteolytic complex.

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl (anti-DNP) antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease-relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.

Killer nanobody: We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity.