Rachita Dash

J Med Chem. 2023 Aug 10;66(15):10226-10237. doi: 10.1021/acs.jmedchem.3c00565. Epub 2023 Jul 21.

ABSTRACT

Drug resistant bacterial infections have emerged as one of the greatest threats to public health. The discovery and development of new antimicrobials and anti-infective strategies are urgently needed to address this challenge. Vancomycin is one of the most important antibiotics for the treatment of Gram-positive infections. Here, we introduce the vancomycin-arginine conjugate (V-R) as a highly effective antimicrobial against actively growing mycobacteria and difficult-to-treat mycobacterial biofilm populations. Further improvement in efficacy through combination treatment of V-R to inhibit peptidoglycan synthesis and ethambutol to inhibit arabinogalactan synthesis underscores the ability to identify compound synergies to more effectively target the Achilles heel of the cell-wall assembly. Moreover, we introduce mechanistic activity data and a molecular model derived from a d-Ala-d-Ala-bound vancomycin structure that we hypothesize underlies the molecular basis for the antibacterial improvement attributed to the arginine modification that is specific to peptidoglycan chemistry employed by mycobacteria and distinct from Gram-positive pathogens.

PMID:37477249 | PMC:PMC10783851 | DOI:10.1021/acs.jmedchem.3c00565

Angew Chem Int Ed Engl. 2023 Jul 20:e202307210. doi: 10.1002/anie.202307210. Online ahead of print.

ABSTRACT

Macrocyclic peptides have become increasingly important in the pharmaceutical industry. We present a detailed computational investigation of the reaction mechanism of the recently developed "CyClick" chemistry to selectively form imidazolidinone cyclic peptides from linear peptide aldehydes, without using catalysts or directing groups (Angew. Chem. Int. Ed. 2019,58, 19073 - 19080). We conducted computational mechanistic to investigate the effects of intramolecular hydrogen bonds (IMHBs) in promoting a kinetically facile zwitterionic mechanism in "CyClick" of pentapeptide aldehyde AFGPA. Our DFT calculations highlighted the importance of IMHB in pre-organization of the resting state, stabilization of the zwitterion intermediate, and the control of the product stereoselectivity. Furthermore, we have also identified that the low ring strain energy promotes the "CyClick" of hexapeptide aldehyde AAGPFA to form a thermodynamically more stable 15+5 imidazolidinone cyclic peptide product. In contrast, large ring strain energy suppresses "CyClick" reactivity of tetra peptide aldehyde AFPA from forming the 9+5 imidazolidinone cyclic peptide product.

PMID:37475575 | DOI:10.1002/anie.202307210

J Am Chem Soc. 2023 Jul 26;145(29):16069-16080. doi: 10.1021/jacs.3c04444. Epub 2023 Jul 14.

ABSTRACT

Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification, and this dynamic labeling behavior can be "switched off" via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as a bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning 2 orders of magnitude (k₂, 1-100 M^-1 s^-1). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels-Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that "locking in" the tag through Diels-Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for "locking in" stability.

PMID:37450839 | PMC:PMC10530612 | DOI:10.1021/jacs.3c04444

Angew Chem Int Ed Engl. 2023 Sep 4;62(36):e202307157. doi: 10.1002/anie.202307157. Epub 2023 Jul 31.

ABSTRACT

Receptor tyrosine kinases (RTKs) are generally activated through their dimerization and/or oligomerization induced by their cognate ligands, and one such RTK hepatocyte growth factor (HGF) receptor, known as MET, plays an important role in tissue regeneration. Here we show the development of ubiquitin (Ub)-based protein ligand multimers, referred to as U-bodies, which act as surrogate agonists for MET and are derived from MET-binding macrocyclic peptides. Monomeric Ub constructs (U-body) were first generated by genetic implantation of a macrocyclic peptide pharmacophore into a structural loop of Ub (lasso-grafting) and subsequent optimization of its flanking spacer sequences via mRNA display. Such U-body constructs exhibit potent binding affinity to MET, thermal stability, and proteolytic stability. The U-body constructs also partially/fully inhibited or enhanced HGF-induced MET-phosphorylation. Their multimerization to dimeric, tetrameric, and octameric U-bodies linked by an appropriate peptide linker yielded potent MET activation activity and downstream cell proliferation-promoting activity. This work suggests that lasso-grafting of macrocycles to Ub is an effective approach to devising protein-based artificial RTK agonists and it can be useful in the development of a new class of biologics for various therapeutic applications.

PMID:37450419 | DOI:10.1002/anie.202307157

ACS Omega. 2023 Jun 20;8(26):23477-23483. doi: 10.1021/acsomega.3c00576. eCollection 2023 Jul 4.

ABSTRACT

DNA-encoded libraries (DELs) are a powerful platform in drug discovery. Peptides have unique properties that make them attractive pharmaceutical candidates. N-methylation of the peptide backbone can confer beneficial properties such as increased proteolytic stability and membrane permeability. Herein, we evaluate different DEL reaction systems and report a DNA-compatible protocol for forming N-methylated amide bonds. The DNA-compatible, bis(trichloromethyl)carbonate-mediated amide coupling is efficient for the formation of N-methyl peptide bonds, which promises to increase the opportunity to identify passively cell-permeable macrocyclic peptide hits by DNA-encoded technology.

PMID:37426286 | PMC:PMC10323948 | DOI:10.1021/acsomega.3c00576

Curr Microbiol. 2023 Jul 4;80(8):267. doi: 10.1007/s00284-023-03360-7.

ABSTRACT

The modulation of host's immune response plays an important role in the intracellular survival of Mycobacterium tuberculosis. The intracellular pathogen counteracts environmental stresses with help of the expression of several genes. The M. tuberculosis genome encodes several immune-modulatory proteins including PE (proline-glutamic acid)/PPE (proline-proline-glutamic acid) superfamily proteins. It is unclear how the unique PE/PPE proteins superfamily contributes to survival under different stress and pathophysiology conditions. Previously, we showed that PPE63 (Rv3539) has C-terminal esterase extension and was localized as a membrane attached and in extracellular compartment. Therefore, the probability of these proteins interacting with the host to modulate the host immune response cannot be ruled out. The physiological role of PPE63 was characterized by expressing the PPE63 in the M. smegmatis, a non-pathogenic strain intrinsically deficient of PPE63. The recombinant M. smegmatis expressing PPE63 altered the colony morphology, lipid composition, and integrity of the cell wall. It provided resistance to multiple hostile environmental stress conditions and several antibiotics. MS_Rv3539 demonstrated higher infection and intracellular survival in comparison to the MS_Vec in the PMA-differentiated THP-1 cells. The decreased intracellular level of ROS, NO, and expression of iNOS was observed in THP-1 cells upon infection with MS_Rv3539 in comparison to MS_Vec. Further, the decrease in expression of pro-inflammatory cytokines like IL-6, TNF-α, and IL-1β and enhanced anti-inflammatory cytokines like IL-10, pointed toward its role in immune modulation. Overall this study suggested the role of Rv3539 in enhanced intracellular survival of M. smegmatis via cell wall modulation and altered immune response of host.

PMID:37401981 | DOI:10.1007/s00284-023-03360-7

Free Radic Biol Med. 2023 Jun 29;206:134-142. doi: 10.1016/j.freeradbiomed.2023.06.030. Online ahead of print.

ABSTRACT

Reactive Oxygen Species (ROS) in the form of H₂O₂ can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H₂O₂ were often studied making use of exogenously added H₂O₂, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H₂O₂ production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H₂O₂ formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H₂O₂. However, a method to directly quantify the amount of H₂O₂ produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H₂O₂. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H₂O₂ production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of H₂O₂ production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H₂O₂. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H₂O₂ production to be able to discriminate the effects of H₂O₂ production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.

PMID:37392950 | DOI:10.1016/j.freeradbiomed.2023.06.030

Yakugaku Zasshi. 2023;143(7):551-557. doi: 10.1248/yakushi.23-00088.

ABSTRACT

Naturally occurring cyclopeptides are potential middle-molecule drug candidates beyond Lipinski's rule of five. This paper focuses on the structural determination and structure-activity relationship (SAR) study of two cyclopeptides: asperterrestide A and decatransin. The proposed asperterrestide A was synthesized by solution-phase peptide elongation, followed by macrolactamization. NMR analysis and molecular modeling studies revealed the stereochemistry at the two α-positions of amino acid residues as opposite to each other. This was further confirmed by the total synthesis of the revised asperterrestide A. SAR study of synthetic products revealed that the β-hydroxy group in the nonproteinogenic amino acid residue was not essential for its cytotoxicity. In addition, N-alkyl-enriched peptide fragments of decatransin were synthesized in solution-phase without diketopiperadine formation. The putative candidates of decatransin was synthesized by convergent peptide coupling, followed by macrocyclization under modified Mitsunobu conditions. The structure of the natural decatransin, including its absolute configuration, was determined through a comparison of spectral data and the cytotoxicity exhibited by the synthetic products.

PMID:37394451 | DOI:10.1248/yakushi.23-00088

J Am Chem Soc. 2023 Jul 3. doi: 10.1021/jacs.3c04833. Online ahead of print.

ABSTRACT

Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein-protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.

PMID:37395736 | DOI:10.1021/jacs.3c04833

Nat Chem Biol. 2023 Jun 29. doi: 10.1038/s41589-023-01373-8. Online ahead of print.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.

PMID:37386135 | DOI:10.1038/s41589-023-01373-8