Photodynamic therapy of cancer using graphene nanomaterials.

INTRODUCTION: High incidence and fatality rates of cancer remain a global challenge. The success of conventional treatment modalities is being questioned on account of adverse effects. Photodynamic therapy (PDT) is a potential alternative. It utilizes a combination of photosensitizer (PS), light and oxygen to target the tissues locally, thereby minimizing the damage to neighboring healthy tissues. Conventional PSs suffer from poor selectivity, high hydrophobicity and sub-optimal yield of active radicals. Graphene nanomaterials (GNs) exhibit interesting particulate and photophysical properties in the context of their use in PDT. AREA COVERED: We focus on describing the mechanistic aspects of PDT-mediated elimination of cancer cells and the subsequent development of adaptive immunity. After covering up-to-date literature on the significant enhancement of PDT capability with GNs, we have discussed the probability of combining PDT with chemo-, immuno-, and photothermal therapy to make the treatment more effective. EXPERT OPINION: GNs can be synthesized in various size ranges, and their biocompatibility can be improved through surface functionalization and doping. These can be used as PS to generate ROS or conjugated with other PS molecules for treating deep-seated tumors. With increasing evidence on biosafety, such materials offer hope as antitumor therapeutics.

Sortases: structure, mechanism, and implications for protein engineering.

Sortase enzymes are critical cysteine transpeptidases on the surface of bacteria that attach proteins to the cell wall and are involved in the construction of bacterial pili. Due to their ability to recognize specific substrates and covalently ligate a range of reaction partners, sortases are widely used in protein engineering applications via sortase-mediated ligation (SML) strategies. In this review, we discuss recent structural studies elucidating key aspects of sortase specificity and the catalytic mechanism. We also highlight select recent applications of SML, including examples where fundamental studies of sortase structure and function have informed the continued development of these enzymes as tools for protein engineering.

Macrocycles and macrocyclization in anticancer drug discovery: Important pieces of the puzzle.

Increasing disease-related proteins have been identified as novel therapeutic targets. Macrocycles are emerging as potential solutions, bridging the gap between conventional small molecules and biomacromolecules in drug discovery. Inspired by successful macrocyclic drugs of natural origins, macrocycles are attracting more attention for enhanced binding affinity and target selectivity. Due to the conformation constraint and structure preorganization, macrocycles can reach bioactive conformations more easily than parent acyclic compounds. Also, rational macrocyclization combined with sequent structural modification will help improve oral bioavailability and combat drug resistance. This review introduces various strategies to enhance membrane permeability in macrocyclization and subsequent modification, such as N-methylation, intramolecular hydrogen bonding modulation, isomerization, and reversible bicyclization. Several case studies highlight macrocyclic inhibitors targeting kinases, HDAC, and protein-protein interactions. Finally, some macrocyclic agents targeting tumor microenvironments are illustrated.

An Expedition on Synthetic Methodology of FDA-approved Anticancer Drugs (2018-2021).

New drugs being established in the market every year produce specified structures for selective biological targeting. With medicinal insights into molecular recognition, these begot molecules open new rooms for designing potential new drug molecules. In this review, we report the compilation and analysis of a total of 56 drugs including 33 organic small molecules (Mobocertinib, Infigratinib, Sotorasib, Trilaciclib, Umbralisib, Tepotinib, Relugolix, Pralsetinib, Decitabine, Ripretinib, Selpercatinib, Capmatinib, Pemigatinib, Tucatinib, Selumetinib, Tazemetostat, Avapritinib, Zanubrutinib, Entrectinib, Pexidartinib, Darolutamide, Selinexor, Alpelisib, Erdafitinib, Gilteritinib, Larotrectinib, Glasdegib, Lorlatinib, Talazoparib, Dacomitinib, Duvelisib, Ivosidenib, Apalutamide), 6 metal complexes (Edotreotide Gallium Ga-68, fluoroestradiol F-18, Cu 64 dotatate, Gallium 68 PSMA-11, Piflufolastat F-18, 177Lu (lutetium)), 16 macromolecules as monoclonal antibody conjugates (Brentuximabvedotin, Amivantamab-vmjw, Loncastuximabtesirine, Dostarlimab, Margetuximab, Naxitamab, Belantamabmafodotin, Tafasitamab, Inebilizumab, SacituzumabGovitecan, Isatuximab, Trastuzumab, Enfortumabvedotin, Polatuzumab, Cemiplimab, Mogamulizumab) and 1 peptide enzyme (Erwiniachrysanthemi-derived asparaginase) approved by the U.S. FDA between 2018 to 2021. These drugs act as anticancer agents against various cancer types, especially non-small cell lung, lymphoma, breast, prostate, multiple myeloma, neuroendocrine tumor, cervical, bladder, cholangiocarcinoma, myeloid leukemia, gastrointestinal, neuroblastoma, thyroid, epithelioid and cutaneous squamous cell carcinoma. The review comprises the key structural features, approval times, target selectivity, mechanisms of action, therapeutic indication, formulations, and possible synthetic approaches of these approved drugs. These crucial details will benefit the scientific community for futuristic new developments in this arena.

Target-Specific Bioorthogonal Reactions for Precise Biomedical Applications.

Bioorthogonal chemistry is a promising toolbox for dissecting biological processes in the native environment. Recently, bioorthogonal reactions have attracted considerable attention in the medical field for treating diseases, since this approach may lead to improved drug efficacy and reduced side effects via in situ drug synthesis. For precise biomedical applications, it is a prerequisite that the reactions should occur in the right locations and on the appropriate therapeutic targets. In this minireview, we highlight the design and development of targeted bioorthogonal reactions for precise medical treatment. First, we compile recent strategies for achieving target-specific bioorthogonal reactions. Further, we emphasize their application for the precise treatment of different therapeutic targets. Finally, a perspective is provided on the challenges and future directions of this emerging field for safe, efficient, and translatable disease treatment.

Target Deconvolution by Limited Proteolysis Coupled to Mass Spectrometry.

Limited proteolysis coupled to mass spectrometry (LiP-MS) is a recent proteomics technique that allows structure-based target engagement profiling on a proteome-wide level. To achieve this, native lysates are first incubated with a compound, followed by a short incubation with a nonspecific protease. Binding of a compound can change accessibility at the binding site or induce other structural changes in the target. This leads to treatment-specific proteolytic fingerprints upon limited proteolysis, which can be analyzed by standard bottom-up MS-based proteomics. Here, we describe a basic LiP-MS protocol using the natural product rapamycin as an example compound. Along with the provided LiP-MS reference data available via ProteomeXchange with identifier PXD035183, this enables the straightforward implementation of the method by scientists with a basic biochemistry and mass spectrometry background. We describe how the procedure can easily be adapted to other protein samples and small molecules.

A patent review of MAPK inhibitors (2018 - present).

INTRODUCTION: The mitogen-activated protein kinase (MAPK) family consist of p38 MAP kinases, c-Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs). They are involved in a multitude of diseases, including inflammatory, autoimmune, neurodegenerative, and metabolic diseases as well as cancer. In recent years, further developments in the field of MAPK-inhibitors have been reported, including an isoform or downstream target selective inhibition of MAPKs as well as target protein degradation approaches. AREAS COVERED: This review summarizes newly patented MAPK-inhibitors that were claimed between 2018 and early 2023. Presented are the patents as well as their corresponding publications, the storyline of development, and clinical trials involving these compounds. This article elaborates a total of 27 patents, which were identified using established search engines. EXPERT OPINION: Although industrial research on MAPK-inhibitors has been ongoing for more than 20 years, novel clinical trials of MAPK-inhibitors as potential drug candidates are still being conducted in the period under review. Recently reported inhibitors show an excellent selectivity profile and are even achieving selectivity between closely related isoforms. This progression offers the possibility to eliminate unwanted side effects and may finally lead to the approval of the first MAPK-inhibitor.

Signal Amplification in Electrochemical DNA Biosensors Using Target-Capturing DNA Origami Tiles.

Electrochemical DNA (e-DNA) biosensors are feasible tools for disease monitoring, with their ability to translate hybridization events between a desired nucleic acid target and a functionalized transducer, into recordable electrical signals. Such an approach provides a powerful method of sample analysis, with a strong potential to generate a rapid time to result in response to low analyte concentrations. Here, we report a strategy for the amplification of electrochemical signals associated with DNA hybridization, by harnessing the programmability of the DNA origami method to construct a sandwich assay to boost charge transfer resistance (RCT) associated with target detection. This allowed for an improvement in the sensor limit of detection by two orders of magnitude compared to a conventional label-free e-DNA biosensor design and linearity for target concentrations between 10 pM and 1 nM without the requirement for probe labeling or enzymatic support. Additionally, this sensor design proved capable of achieving a high degree of strand selectivity in a challenging DNA-rich environment. This approach serves as a practical method for addressing strict sensitivity requirements necessary for a low-cost point-of-care device.

cGMP Analogues with Opposing Actions on CNG Channels Selectively Modulate Rod or Cone Photoreceptor Function.

The vertebrate retina harbors rod and cone photoreceptors. Human vision critically depends on cone photoreceptor function. In the phototransduction cascade, cGMP activates distinct rod and cone isoforms of the cyclic nucleotide-gated (CNG) channel. Excessive cGMP levels initiate a pathophysiological rollercoaster, which starts with CNG channel over-activation, typically in rod photoreceptors. This triggers cell death of rods first, and then cones, and is the root cause of many blinding retinal diseases, including Retinitis pigmentosa. While targeting of CNG channels has been proposed for therapeutic purposes, thus far, it has not been possible to inhibit rod CNG channels without compromising cone function. Here, we present a novel strategy, based on cGMP analogues with opposing actions on CNG channels, which enables the selective modulation of either rod or cone photoreceptor activity. The combined treatment with the weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and the cone-selective CNG-channel activator (8-pCPT-cGMP) essentially normalized rod CNG-channel function while preserving cone functionality at physiological and pathological cGMP levels. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Moreover, this strategy may allow for improvements in visual performance in certain light environments.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition.

The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal-containing protein to the peptidoglycan precursor lipid II (LII). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl-enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

Functional ligands for improving anticancer drug therapy: current status and applications to drug delivery systems.

Conventional chemotherapy lacking target selectivity often leads to severe side effects, limiting the effectiveness of chemotherapy. Therefore, drug delivery systems ensuring both selective drug release and efficient intracellular uptake at the target sites are highly demanded in chemotherapy to improve the quality of life of patients with low toxicity. One of the effective approaches for tumor-selective drug delivery is the adoption of functional ligands that can interact with specific receptors overexpressed in malignant cancer cells. Various functional ligands including folic acid, hyaluronic acid, transferrin, peptides, and antibodies, have been extensively explored to develop tumor-selective drug delivery systems. Furthermore, cell-penetrating peptides or ligands for tight junction opening are also actively pursued to improve the intracellular trafficking of anticancer drugs. Sometimes, multiple ligands with different roles are used in combination to enhance the cellular uptake as well as target selectivity of anticancer drugs. In this review, the current status of various functional ligands applicable to improve the effectiveness of cancer chemotherapy is overviewed with a focus on their roles, characteristics, and preclinical/clinical applications.

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes.

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative ß7-ß8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1' position of the substrate motif, underlined in LPXTG, in contrast to the strict P1' Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of ß7-ß8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified ß7-ß8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Differential p53-Mediated Cellular Responses to DNA-Damaging Therapeutic Agents.

The gene TP53, which encodes the tumor suppressor protein p53, is mutated in about 50% of cancers. In response to cell stressors like DNA damage and after treatment with DNA-damaging therapeutic agents, p53 acts as a transcription factor to activate subsets of target genes which carry out cell fates such as apoptosis, cell cycle arrest, and DNA repair. Target gene selection by p53 is controlled by a complex regulatory network whose response varies across contexts including treatment type, cell type, and tissue type. The molecular basis of target selection across these contexts is not well understood. Knowledge gained from examining p53 regulatory network profiles across different DNA-damaging agents in different cell types and tissue types may inform logical ways to optimally manipulate the network to encourage p53-mediated tumor suppression and anti-tumor immunity in cancer patients. This may be achieved with combination therapies or with p53-reactivating targeted therapies. Here, we review the basics of the p53 regulatory network in the context of differential responses to DNA-damaging agents; discuss recent efforts to characterize differential p53 responses across treatment types, cell types, and tissue types; and examine the relevance of evaluating these responses in the tumor microenvironment. Finally, we address open questions including the potential relevance of alternative p53 transcriptional functions, p53 transcription-independent functions, and p53-independent functions in the response to DNA-damaging therapeutics.

Domain Analysis and Motif Matcher (DAMM): A Program to Predict Selectivity Determinants in Monosiga brevicollis PDZ Domains Using Human PDZ Data.

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.

A Structure Based Study of Selective Inhibition of Factor IXa over Factor Xa.

Blood coagulation is an essential physiological process for hemostasis; however, abnormal coagulation can lead to various potentially fatal disorders, generally known as thromboembolic disorders, which are a major cause of mortality in the modern world. Recently, the FDA has approved several anticoagulant drugs for Factor Xa (FXa) which work via the common pathway of the coagulation cascade. A main side effect of these drugs is the potential risk for bleeding in patients. Coagulation Factor IXa (FIXa) has recently emerged as the strategic target to ease these risks as it selectively regulates the intrinsic pathway. These aforementioned coagulation factors are highly similar in structure, functional architecture, and inhibitor binding mode. Therefore, it remains a challenge to design a selective inhibitor which may affect only FIXa. With the availability of a number of X-ray co-crystal structures of these two coagulation factors as protein-ligand complexes, structural alignment, molecular docking, and pharmacophore modeling were employed to derive the relevant criteria for selective inhibition of FIXa over FXa. In this study, six ligands (three potent, two selective, and one inactive) were selected for FIXa inhibition and six potent ligands (four FDA approved drugs) were considered for FXa. The pharmacophore hypotheses provide the distribution patterns for the principal interactions that take place in the binding site. None of the pharmacophoric patterns of the FXa inhibitors matched with any of the patterns of FIXa inhibitors. Based on pharmacophore analysis, a selectivity of a ligand for FIXa over FXa may be defined quantitatively as a docking score of lower than -8.0 kcal/mol in the FIXa-grids and higher than -7.5 kcal/mol in the FXa-grids.

Sequence variation in the ß7-ß8 loop of bacterial class A sortase enzymes alters substrate selectivity.

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this ß7-ß8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by ß7-ß8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.

Transforming targeted cancer therapy with PROTACs: A forward-looking perspective.

Small-molecule targeted protein degraders have in recent years made a great impact on the strategies of many industry and academic cancer research endeavours. We seek here to provide a concise perspective on the opportunities and challenges that lie ahead for bifunctional degrader molecules, so-called 'Proteolysis Targeting Chimeras (PROTACs),' in the context of cancer therapy. We highlight high-profile studies that support the potential for PROTAC approaches to broaden drug target scope, address drug resistance, enhance target selectivity and provide tissue specificity, but also assess where the modality is yet to fully deliver in these contexts. Future opportunities presented by the unique bifunctional nature of these molecules are also discussed.

Molecular Basis of P131 Cryptosporidial-IMPDH Selectivity-A Structural, Dynamical and Mechanistic Stance.

Cryptosporidiosis accounts for a surge in infant (<5 years) mortality and morbidity. To date, several drug discovery efforts have been put in place to develop effective therapeutic options against the causative parasite. Based on a recent report, P131 spares inosine monophosphate dehydrogenase (IMPDH) in a eukaryotic model (mouse IMPDH (mIMPDH)) while binding selectively to the NAD+ site in Cryptosporidium parvum (CpIMPDH). However, no structural detail exists on the underlining mechanisms of P131-CpIMPDH selective targeting till date. To this effect, we investigate the selective inhibitory dynamics of P131 in CpIMPDH relative to mIMPDH via molecular biocomputation methods. Pairwise sequence alignment revealed prominent variations at the NAD+ binding regions of both proteins that accounted for disparate P131 binding activities. The influence of these variations was further revealed by the MM/PBSA energy estimations coupled with per-residue energy decomposition which monitored the systematic binding of the compound. Furthermore, relative high-affinity interactions occurred at the CpIMPDH NAD+ site which were majorly mediated by SER22, VAL24, PRO26, SER354, GLY357, and TYR358 located on chain D. These residues are unique to the parasite IMPDH form and not in the eukaryotic protein, highlighting variations that account for preferential P131 binding. Molecular insights provided herein corroborate previous experimental reports and further underpin the basis of CpIMPDH inhibitor selectivity. Findings from this study could present attractive prospects toward the design of novel anticryptosporidials with improved selectivity and binding affinity against parasitic targets.

On the Different Mode of Action of Au(I)/Ag(I)-NHC Bis-Anthracenyl Complexes Towards Selected Target Biomolecules.

Gold and silver N-heterocyclic carbenes (NHCs) are emerging for therapeutic applications. Multiple techniques are here used to unveil the mechanistic details of the binding to different biosubstrates of bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) silver chloride [Ag(EIA)2]Cl and bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) gold chloride [Au(EIA)2]Cl. As the biosubstrates, we tested natural double-stranded DNA, synthetic RNA polynucleotides (single-poly(A), double-poly(A)poly(U) and triple-stranded poly(A)2poly(U)), DNA G-quadruplex structures (G4s), and bovine serum albumin (BSA) protein. Absorbance and fluorescence titrations, mass spectrometry together with melting and viscometry tests show significant differences in the binding features between silver and gold compounds. [Au(EIA)2]Cl covalently binds BSA. It is here evidenced that the selectivity is high: low affinity and external binding for all polynucleotides and G4s are found. Conversely, in the case of [Ag(EIA)2]Cl, the binding to BSA is weak and relies on electrostatic interactions. [Ag(EIA)2]Cl strongly/selectively interacts only with double strands by a mechanism where intercalation plays the major role, but groove binding is also operative. The absence of an interaction with triplexes indicates the major role played by the geometrical constraints to drive the binding mode.