Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 µM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Discovery of the small molecular inhibitors against sclerostin loop3 as potential anti-osteoporosis agents by structural based virtual screening and molecular design.

Sclerostin is a secreted glycoprotein that expresses predominantly in osteocytes and inhibits bone formation by antagonizing the Wnt/ß-catenin signaling pathway, and the loop3 region of sclerostin has recently discovered as a novel therapeutic target for bone anabolic treatment without increasing cardiovascular risk. Herein, we used a structural based virtual screening to search for small molecular inhibitors selectively targeting sclerostin loop3. A novel natural product hit ZINC4228235 (THFA) was identified as the sclerostin loop3-selective inhibitor with a Kd value of 42.43 nM against sclerostin loop3. The simplification and derivation of THFA using molecular modeling-guided modification allowed the discovery of an effective and loop3-selective small molecular inhibitor, compound (4-(3-acetamidoprop-1-yn-1-yl)benzoyl)glycine (AACA), with improved binding affinity (Kd = 15.4 nM) compared to the hit THFA. Further in-vitro experiment revealed that compound AACA could attenuate the suppressive effect of transfected sclerostin on Wnt signaling and bone formation. These results make AACA as a potential candidate for development of anti-osteoporosis agents without increasing cardiovascular risk.

Innovative, combinatorial and high-throughput approaches to degrader synthesis.

Targeted protein degraders such as PROTACs and molecular glues are a rapidly emerging therapeutic modality within industry and academia. Degraders possess unique mechanisms of action that lead to the removal of specific proteins by co-opting the cell's natural degradation mechanisms via induced proximity. Their optimisation thus far has often been largely empirical, requiring the synthesis and screening of a large number of analogues. In addition, the synthesis and development of degraders is often challenging, leading to lengthy optimisation campaigns to deliver candidate-quality compounds. This review highlights how the synthesis of degraders has evolved in recent years, in particular focusing on means of applying high-throughput chemistry and screening approaches to expedite these timelines, which we anticipate to be valuable in shaping the future of degrader optimisation campaigns.

Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential.

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

Discovery of 4-phenyl-1H-indazole derivatives as novel small-molecule inhibitors targeting the PD-1/PD-L1 interaction.

The inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway with small molecules is a promising approach for cancer immunotherapy. Herein, novel small molecules compounds bearing various scaffolds including thiophene, thiazole, tetrahydroquinoline, benzimidazole and indazole were designed, synthesized and evaluated for their inhibitory activity against the PD-1/PD-L1 interaction. Among them, compound Z13 exhibited the most potent activity with IC50 of 189.6 nM in the homogeneous time-resolved fluorescence (HTRF) binding assay. Surface plasmon resonance (SPR) assay demonstrated that Z13 bound to PD-L1 with high affinity (KD values of 231 nM and 311 nM for hPD-L1 and mPD-L1, respectively). In the HepG2/Jurkat T co-culture cell model, Z13 decreased the viability rate of HepG2 cells in a concentration-dependent manner. In addition, Z13 showed significant in vivo antitumor efficacy (TGI = 52.6 % at 40 mg/kg) without obvious toxicity in the B16-F10 melanoma model. Furthermore, flow cytometry analysis demonstrated that Z13 inhibited tumor growth in vivo by activating the tumor immune microenvironment. These findings indicate that Z13 is a promising PD-1/PD-L1 inhibitor deserving further investigation.

Quantum chemistry calculation-aided discovery of potent small-molecule mimics of glutathione peroxidases for the treatment of cisplatin-induced hearing loss.

Hearing loss (HL) is a health burden that seriously affects the quality of life of cancer patients receiving platinum-based chemotherapy, and few FDA-approved treatment specifically targets this condition. The main mechanisms that contribute to cisplatin-induced hearing loss are oxidative stress and subsequent cell death, including ferroptosis revealed by us as a new mechanism recently. In this study, we employed the frontier molecular orbital (FMO) theory approach as a convenient prediction method for the glutathione peroxidase (GPx)-like activity of isoselenazolones and discovered new isoselenazolones with great GPx-like activity. Notably, compound 19 exhibited significant protective effects against cisplatin-induced hair cell (HC) damage in vitro and in vivo and effectively reverses cisplatin-induced hearing loss through oral administration. Further investigations revealed that this compound effectively alleviated hair cell oxidative stress, apoptosis and ferroptosis. This research highlights the potential of GPx mimics as a therapeutic strategy against cisplatin-induced hearing loss. The application of quantum chemistry (QC) calculations in the study of GPx mimics sheds light on the development of new, innovative treatments for hearing loss.

Discovery of two non-UDP-mimic inhibitors of O-GlcNAc transferase by screening a DNA-encoded library.

Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority of available OGT inhibitors are uridine-based or uridine-like compounds that mimic the main interactions of glycosyl donor UDP-GlcNAc with the enzyme. Until recently, screening of DNA-encoded libraries for discovering hits against protein targets was dedicated to a few laboratories around the world, but has become accessible to wider public with the recent launch of the DELopen platform. Here we report the results and follow-up of a DNA-encoded library screening by using the DELopen platform. This led to the discovery of two new hits with structural features not resembling UDP. Small focused libraries bearing those two scaffolds were made, leading to low micromolar inhibition of OGT and elucidation of their structure-activity relationship.

Identification of a novel histone H2A mono-ubiquitination-inhibiting cell-active small molecule.

Histone H2A mono-ubiquitination plays important roles in epigenetic gene expression and is also involved in tumorigenesis. Small molecules controlling H2A ubiquitination are of interest as potential chemical tools and anticancer drugs. To identify novel small molecule inhibitors of H2A ubiquitination, we synthesized and evaluated several compounds designed based on PRT4165 (1), which is a reported histone ubiquitin ligase RING1A inhibitor. We found that compound 11b strongly inhibited the viability and reduced histone H2A mono-ubiquitination in human osteosarcoma U2OS cells. Therefore, compound 11b is a promising lead compound for the development of H2A histone ubiquitination-inhibiting small molecules.

Expanding the ligand spaces for E3 ligases for the design of protein degraders.

Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.

Recent Advances of Aminopeptidases-Responsive Small-Molecular Probes for Bioimaging.

Aminopeptidases, enzymes with critical roles in human body, are emerging as vital biomarkers for metabolic processes and diseases. Aberrant aminopeptidase levels are often associated with diseases, particularly cancer. Small-molecule probes, such as fluorescent, fluorescent/photoacoustics, bioluminescent, and chemiluminescent probes, are essential tools in the study of aminopeptidases-related diseases. The fluorescent probes provide real-time insights into protein activities, offering high sensitivity in specific locations, and precise spatiotemporal results. Additionally, photoacoustic probes offer signals that are able to penetrate deeper tissues. Bioluminescent and chemiluminescent probes can enhance in vivo imaging abilities by reducing the background. This comprehensive review is focused on small-molecule probes that respond to four key aminopeptidases: aminopeptidase N, leucine aminopeptidase, Pyroglutamate aminopeptidase 1, and Prolyl Aminopeptidase, and their utilization in imaging tumors and afflicted regions. In this review, the design strategy of small-molecule probes, the variety of designs from previous studies, and the opportunities of future bioimaging applications are discussed, serving as a roadmap for future research, sparking innovations in aminopeptidase-responsive probe development, and enhancing our understanding of these enzymes in disease diagnostics and treatment.

Current Small Molecule-Based Medicinal Chemistry Approaches for Neurodegeneration Therapeutics.

Neurodegenerative diseases (NDDs) like Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic lateral sclerosis (ALS) possess multifactorial aetiologies. In recent years, our understanding of the biochemical and molecular pathways across NDDs has increased, however, new advances in small molecule-based therapeutic strategies targeting NDDs are obscure and scarce. Moreover, NDDs have been studied for more than five decades, however, there is a paucity of drugs that can treat NDDs. Further, the highly lipoidal blood-brain barrier (BBB) limits the uptake of many therapeutic molecules into the brain and is a complicating factor in the development of new agents to treat neurodegeneration. Considering the highly complex nature of NDDs, the association of multiple risk factors, and the challenges to overcome the BBB junction, medicinal chemists have developed small organic molecule-based novel approaches to target NDDs over the last few decades, such as designing lipophilic molecules and applying prodrug strategies. Attempts have been made to utilize a multitarget approach to modulate different biochemical molecular pathways involved in NDDs, in addition to, medicinal chemists making better decisions in identifying optimized drug candidates for the central nervous system (CNS) by using web-based computational tools. To increase the clinical success of these drug candidates, an in vitro assay modeling the BBB has been utilized by medicinal chemists in the pre-clinical phase as a further screening measure of small organic molecules. Herein, we examine some of the intriguing strategies taken by medicinal chemists to design small organic molecules to combat NDDs, with the intention of increasing our awareness of neurodegenerative therapeutics.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Synthesis of Diacylhydrazine Derivatives Based on Tetrazole-Focused DNA-Encoded Library.

Utilizing already existing DNA-encoded libraries (DELs) for the generation of a distinct DEL represents an expedited strategy for expanding the chemical space. Herein, we leverage the unique photoreactivity of tetrazoles to synthesize diacylhydrazines on DNA. Widely available carboxylic acids serving as building blocks were employed under the mild photomediated reaction conditions, affording diverse DNA-conjugated diacylhydrazines. This methodology also demonstrates robustness in DEL-compatible synthesis and facilitates the preparation of oligonucleotide-based chemical probes.

Peptide barcodes meet drug discovery.

Small-molecule libraries encoded by peptide tags may accelerate the search for therapeutics.

Abiotic peptides as carriers of information for the encoding of small-molecule library synthesis.

Encoding small-molecule information in DNA has been leveraged to accelerate the discovery of ligands for therapeutic targets such as proteins. However, oligonucleotide-based encoding is hampered by inherent limitations of information stability and density. In this study, we establish abiotic peptides for next-generation information storage and apply them for the encoding of diverse small-molecule synthesis. The chemical stability of the peptide-based tag allows the use of palladium-mediated reactions to efficiently synthesize peptide-encoded libraries (PELs) with broad chemical diversity and high purity. We demonstrate the successful de novo discovery of small-molecule protein ligands from PELs by affinity selection against carbonic anhydrase IX and the oncogenic protein targets BRD4(1) and MDM2. Collectively, this work establishes abiotic peptides as carriers of information for the encoding of small-molecule synthesis, leveraged herein for the discovery of protein ligands.

Phe19 modification of HDM2-p53 PPI inhibitors to alleviate CYP3A4 metabolism/mechanism-based inhibition liability.

The discovery of potent, bioavailable small molecule inhibitors of p53-HDM2 PPI led us to investigate subsequent modifications to address a CYP3A4 time-dependent inhibition liability. On the basis of the crystal structure of HDM2 in complex with 2, further functionalization of the solvent exposed area of the molecule that binds to Phe19 pocket were investigated as a strategy to modulate the molecule liphophilicity. Introduction of 2-oxo-nicotinic amide at Phe19 proved a viable strategy in obtaining inhibitors exempt from CYP3A4 time-dependent inhibition liability.

Harnessing an emissive guanine surrogate to design small-molecule fluorescent chemosensors of O6-methylguanine-DNA-methyltransferase (MGMT).

The fluorescence properties of an emissive guanine surrogate, thienoguanine (thGN, 2-aminothieno[3,4-d]pyrimidin-4(3H)-one), were exploited to design two real-time chemosensors of O6-methylguanine-DNA-methyltransferase (MGMT), a key DNA repair enzyme involved in the resistance to DNA-alkylating anti-cancer drugs though direct reversal of O6-alkylated guanine adducts.

First studies on tumor associated carbonic anhydrases IX and XII monoclonal antibodies conjugated to small molecule inhibitors.

We report for the first time Antibody-Drug-Conjugates (ADCs) containing human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) directed Monoclonal Antibodies (MAbs) linked to low molecular weight inhibitors of the same enzymes by means of hydrophilic peptide spacers. In agreement with the incorporated CA directed MAb fragments, in vitro inhibition data of the obtained ADCs showed sub-nanomolar KI values for the tumour associated CAs IX and XII which were up to 10-fold more potent when compared to the corresponding unconjugated MAbs. In addition, the introduction of the CA inhibitor (CAI) benzenesulfonamide allowed the ADCs to potently inhibit the housekeeping tumoral off-target human CA II isoform. Such results are supporting the definition of an unprecedented reported class of ADCs able to hit simultaneously multiple hCAs physiologically cooperative in maintaining altered cellular metabolic pathways, and therefore ideal for the treatment of chronic diseases such as cancers and inflammation diseases.

Natural Small Molecules Enabled Efficient Immunotherapy through Supramolecular Self-Assembly in P53-Mutated Colorectal Cancer.

Nanomedicine, constructed from therapeutics, presents an advantage in drug delivery for cancer therapies. However, nanocarrier-based treatment systems have problems such as interbatch variability, multicomponent complexity, poor drug delivery, and carrier-related toxicity. To solve these issues, the natural molecule honokiol (HK), an anticancer agent in a phase I clinical trial (CTR20170822), was used to form a self-assembly nanoparticle (SA) through hydrogen bonding and hydrophobicity. The preparation of SA needs no molecular precursors or excipients in aqueous solution, and 100% drug-loaded SA exhibited superior tumor-targeting ability due to the enhanced permeability and retention (EPR) effect. Moreover, SA significantly enhanced the antitumor immunity relative to free HK, and the mechanism has notable selectivity to the p53 pathway. Furthermore, SA exhibited excellent physiological stability and inappreciable toxicity. Taken together, this supramolecular self-assembly strategy provides a safe and "molecular economy" model for rational design of clinical therapies and is expected to promote targeted therapy of HK, especially in colorectal cancer patients with obvious p53 status.