Evaluation of small-molecule modulators of the circadian clock: promising therapeutic approach to cancer.

The circadian clock is an important regulator of human homeostasis. Circadian rhythms are closely related to cell fate because they are necessary for regulating the cell cycle, cellular proliferation, and apoptosis. Clock dysfunction can result in the development of diseases such as cancer. Although certain tumors have been shown to have a malfunctioning clock, which may affect prognosis or treatment, this has been postulated but not proven in many types of cancer. Recently, important information has emerged about the basic characteristics that underpin the overt circadian rhythm and its influence on physiological outputs. This information implies that the circadian rhythm may be managed by using particular small molecules. Small-molecule clock modulators target clock components or different physiological pathways that influence the clock. Identifying new small-molecule modulators will improve our understanding of critical regulatory nodes in the circadian network and cancer. Pharmacological manipulation of the clock may be valuable for treating cancer. The discoveries of small-molecule clock modulators and their possible application in cancer treatment are examined in this review.

Autophagy modulation in cancer therapy: Challenges coexist with opportunities.

Autophagy, a crucial intracellular degradation process facilitated by lysosomes, plays a pivotal role in maintaining cellular homeostasis. The elucidation of autophagy key genes and signaling pathways has significantly advanced our understanding of this process and has led to the exploration of autophagy as a promising therapeutic approach. This review comprehensively assesses the latest developments in small molecule modulators targeting autophagy. Moreover, the review delves into the most recent strategies for drug discovery, specifically focusing on selective agents that exploit autophagosomes and lysosomes for targeted protein degradation. Additionally, this article highlights the prevailing challenges and outlines potential future advancements in the field. By amalgamating the cutting-edge knowledge in the field, we aim to offer valuable insights and references for the anti-cancer drug development of autophagy-targeted therapies, thus contributing to the advancement of novel therapeutic interventions.

Synthesis, characterization, and anti-cancer potential of novel p53-mediated Mdm2 and Pirh2 modulators: an integrated In silico and In vitro approach.

Introduction: Leukemia is a global health concern that requires alternative treatments due to the limitations of the FDA-approved drugs. Our focus is on p53, a crucial tumor suppressor that regulates cell division. It appears possible to stabilize p53 without causing damage to DNA by investigating dual-acting inhibitors that target both ligases. The paper aims to identify small molecule modulators of Mdm2 and Pirh2 by using 3D structural models of p53 residues and to further carry out the synthesis and evaluation of hit candidates for anti-cancer potency by in vitro and in silico studies. Methods: We synthesized structural analogues of MMs02943764 and MMs03738126 using a 4,5-(substituted) 1,2,4-triazole-3-thiols with 2-chloro N-phenylacetamide in acetone with derivatives of PAA and PCA were followed. Cytotoxicity assays, including MTT, Trypan Blue Exclusion, and MTS assays, were performed on cancer cell lines. Anti-proliferation activity was evaluated using K562 cells. Cell cycle analysis and protein expression studies of p53, Mdm2, and Pirh2 were conducted using flow cytometry. Results: As for results obtained from our previous studies MMs02943764, and MMs03738126 were selected among the best-fit hit molecules whose structural analogues were further subjected to molecular docking and dynamic simulation. Synthesized compounds exhibited potent anti-proliferative effects, with PAC showing significant cytotoxicity against leukemia cells. PAC induced cell cycle arrest and modulated p53, Mdm2, and Pirh2 protein expressions in K562 cells. Molecular docking revealed strong binding affinity of PAC to p53 protein, further confirmed by molecular dynamics simulation. Discussion: The study presents novel anticancer compounds targeting the p53 ubiquitination pathway, exemplified by PAC. Future perspectives involve further optimization and preclinical studies to validate PAC's potential as an effective anticancer therapy.

Small molecule inhibitors targeting m6A regulators.

As the most common form of epigenetic regulation by RNA, N6 methyladenosine (m6A) modification is closely involved in physiological processes, such as growth and development, stem cell renewal and differentiation, and DNA damage response. Meanwhile, its aberrant expression in cancer tissues promotes the development of malignant tumors, as well as plays important roles in proliferation, metastasis, drug resistance, immunity and prognosis. This close association between m6A and cancers has garnered substantial attention in recent years. An increasing number of small molecules have emerged as potential agents to target m6A regulators for cancer treatment. These molecules target the epigenetic level, enabling precise intervention in RNA modifications and efficiently disrupting the survival mechanisms of tumor cells, thus paving the way for novel approaches in cancer treatment. However, there is currently a lack of a comprehensive review on small molecules targeting m6A regulators for anti-tumor. Here, we have comprehensively summarized the classification and functions of m6A regulators, elucidating their interactions with the proliferation, metastasis, drug resistance, and immune responses in common cancers. Furthermore, we have provided a comprehensive overview on the development, mode of action, pharmacology and structure-activity relationships of small molecules targeting m6A regulators. Our aim is to offer insights for subsequent drug design and optimization, while also providing an outlook on future prospects for small molecule development targeting m6A.

Liver receptor homolog-1: structures, related diseases, and drug discovery.

Liver receptor homolog-1 (LRH-1), a member of the nuclear receptor superfamily, is a ligand-regulated transcription factor that plays crucial roles in metabolism, development, and immunity. Despite being classified as an 'orphan' receptor due to the ongoing debate surrounding its endogenous ligands, recent researches have demonstrated that LRH-1 can be modulated by various synthetic ligands. This highlights the potential of LRH-1 as an attractive drug target for the treatment of inflammation, metabolic disorders, and cancer. In this review, we provide an overview of the structural basis, functional activities, associated diseases, and advancements in therapeutic ligand research targeting LRH-1.

Discovery of PLD4 modulators by high-throughput screening and kinetic analysis.

Phospholipase D3 (PLD3) and D4 (PLD4) are endolysosomal exonucleases of ssDNA and ssRNA that regulate innate immunity. Polymorphisms of these enzymes are correlated with numerous human diseases, including Alzheimer's, rheumatoid arthritis, and systemic sclerosis. Pharmacological modulation of these immunoregulatory proteins may yield novel immunotherapies and adjuvants. A previous study reported a high-throughput screen (N = 17,952) that discovered a PLD3-selective activator and inhibitor, as well as a nonselective inhibitor, but failed to identify selective modulators of PLD4. However, modulators selective for PLD4 are therapeutically pertinent, since recent reports have shown that regulating this protein has direct implications in cancer and autoimmune diseases. Furthermore, the high expression of PLD4 in dendritic and myeloid cells, in comparison to the broader expression of PLD3, presents the opportunity for a cell-targeted immunotherapy. Here, we describe screening of an expended diversity library (N = 45,760) with an improved platform and report the discovery of one inhibitor and three activators selective for PLD4. Furthermore, kinetic modeling and structural analysis suggest mechanistic differences in the modulation of these hits. These findings further establish the utility of this screening platform and provide a set of chemical scaffolds to guide future small-molecule development for this novel immunoregulator target.

Successes and challenges in the development of BD1-selective BET inhibitors: a patent review.

INTRODUCTION: Bromodomain and ExtraTerminal (BET) domain proteins are transcriptional cofactors that, recognizing acetylated lysines of histone and non-histone proteins, can modulate gene expression. The BET family consists of four members, each of which contains two bromodomains (BD1 and BD2) able to recognize the acetylated mark. Pan-BET inhibitors (BETi) have shown a promising anticancer potential in many clinical trials; however, their further development has been in part hampered by the side effects due to their lack of selectivity. Mounting evidence suggests that BD1 is primarily involved in cancer and that its selective inhibition can phenocopy the anticancer effects of pan-BETi with increased tolerability. Therefore, the development of BD1 selective inhibitors is highly pursed in both academia and industry. AREAS COVERED: This review aims at giving an overview of the patent literature of BD1-selective BETi between 2014 and 2023. WIPO, USPTO, EPO, and SciFinder® databases were used for the search of patents. EXPERT OPINION: The development of BD1-selective BETi, despite challenging, is highly desirable as it could have a great impact on the development of new safer anticancer therapeutics. Several strategies could be applied to discover potent and selective compounds with limited side effects.

Small-molecule modulators of tumor immune microenvironment.

In recent years, tumor immunotherapy, aimed at increasing the activity of immune cells and reducing immunosuppressive effects, has attracted wide attention. Among them, immune checkpoint blocking (ICB) is the most commonly explored therapeutic approach. All approved immune checkpoint inhibitors (ICIs) are clinically effective monoclonal antibodies (mAbs). Compared with biological agents, small-molecule drugs have many unique advantages in tumor immunotherapy. Therefore, they also play an important role. Immunosuppressive signals such as PD-L1, IDO1, and TGF-ß, etc. overexpressed in tumor cells form the tumor immunosuppressive microenvironment. In addition, the efficacy of multi-pathway combined immunotherapy has also been reported and verified. Here, we mainly reviewed the mechanism of tumor immunotherapy, analyzed the research status of small-molecule modulators, and discussed drug candidates' structure-activity relationship (SAR). It provides more opportunities for further research to design more immune small-molecule modulators with novel structures.

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Challenges Coexist with Opportunities: Spatial Heterogeneity Expression of PD-L1 in Cancer Therapy.

Cancer immunotherapy using anti-programmed death-ligand 1 (PD-L1) antibodies has been used in various clinical applications and achieved certain results. However, such limitations as autoimmunity, tumor hyperprogression, and overall low patient response rate impede its further clinical application. Mounting evidence has revealed that PD-L1 is not only present in tumor cell membrane but also in cytoplasm, exosome, or even nucleus. Among these, the dynamic and spatial heterogeneous expression of PD-L1 in tumors is mainly responsible for the unsatisfactory efficacy of PD-L1 antibodies. Hence, numerous studies focus on inhibiting or degrading PD-L1 to improve immune response, while a comprehensive understanding of the molecular mechanisms underlying spatial heterogeneity of PD-L1 can fundamentally transform the current status of PD-L1 antibodies in clinical development. Herein, the concept of spatial heterogeneous expression of PD-L1 is creatively introduced, encompassing the structure and biological functions of various kinds of PD-L1 (including mPD-L1, cPD-L1, nPD-L1, and exoPD-L1). Then an in-depth analysis of the regulatory mechanisms and potential therapeutic targets of PD-L1 is provided, seeking to offer a solid basis for future investigation. Moreover, the current status of agents is summarized, especially small molecular modulators development directed at these new targets, offering a novel perspective on potential PD-L1 therapeutics strategies.

Unveiling the Therapeutic Potential of Squalene Synthase: Deciphering Its Biochemical Mechanism, Disease Implications, and Intriguing Ties to Ferroptosis.

Squalene synthase (SQS) has emerged as a promising therapeutic target for various diseases, including cancers, owing to its pivotal role in the mevalonate pathway and the antioxidant properties of squalene. Primarily, SQS orchestrates the head-to-head condensation reaction, catalyzing the fusion of two farnesyl pyrophosphate molecules, leading to the formation of squalene, which has been depicted as a highly effective oxygen-scavenging agent in in vitro studies. Recent studies have depicted this isoprenoid as a protective layer against ferroptosis due to its potential regulation of lipid peroxidation, as well as its protection against oxidative damage. Therefore, beyond its fundamental function, recent investigations have unveiled additional roles for SQS as a regulator of lipid peroxidation and programmed cell death pathways, such as ferroptosis-a type of cell death characterized by elevated levels of lipid peroxide, one of the forms of reactive oxygen species (ROS), and intracellular iron concentration. Notably, thorough explorations have shed light on the distinctive features that set SQS apart from other members within the isoprenoid synthase superfamily. Its unique biochemical structure, intricately intertwined with its reaction mechanism, has garnered significant attention. Moreover, considerable evidence substantiates the significance of SQS in various disease contexts, and its intriguing association with ferroptosis and lipid peroxidation. The objective of this report is to analyze the existing literature comprehensively, corroborating these findings, and provide an up-to-date perspective on the current understanding of SQS as a prospective therapeutic target, as well as its intricate relationship with ferroptosis. This review aims to consolidate the knowledge surrounding SQS, thereby contributing to the broader comprehension of its potential implications in disease management and therapeutic interventions.

Recent Advances in Studying Toll-like Receptors with the Use of Computational Methods.

Toll-like receptors (TLRs) are transmembrane proteins that recognize various molecular patterns and activate signaling that triggers the immune response. In this review, our goal is to summarize how, in recent years, various computational solutions have contributed to a better understanding of TLRs, regarding both their function and mechanism of action. We update the recent information about small-molecule modulators and expanded the topic toward next-generation vaccine design, as well as studies of the dynamic nature of TLRs. Also, we underline problems that remain unsolved.

Dual function of protein phosphatase 5 (PPP5C): An emerging therapeutic target for drug discovery.

Phosphorylation of proteins is reversibly controlled by the kinases and phosphatases in many posttranslational regulation patterns. Protein phosphatase 5 (PPP5C) is a serine/threonine protein phosphatase showing dual function by simultaneously exerting dephosphorylation and co-chaperone functions. Due to this special role, PPP5C was found to participate in many signal transductions related to various diseases. Abnormal expression of PPP5C results in cancers, obesity, and Alzheimer's disease, making it a potential drug target. However, the design of small molecules targeting PPP5C is struggling due to its special monomeric enzyme form and low basal activity by a self-inhibition mechanism. Through realizing the PPP5C's dual function as phosphatase and co-chaperone, more and more small molecules were found to regulate PPP5C with a different mechanism. This review aims to provide insights into PPP5C's dual function from structure to function, which could provide efficient design strategies for small molecules targeting PPP5C as therapeutic candidates.

Cellular mitophagy: Mechanism, roles in diseases and small molecule pharmacological regulation.

Cellular mitophagy means that cells selectively wrap and degrade damaged mitochondria through an autophagy mechanism, thus maintaining mitochondria and intracellular homeostasis. In recent years, mitophagy has received increasing attention as a research hotspot related to the pathogenesis of clinical diseases, such as neurodegenerative diseases, cardiovascular diseases, cancer, metabolic diseases, and so on. It has been found that the regulation of mitophagy may become a new direction for the treatment of some diseases. In addition, numerous small molecule modulators of mitophagy have also been reported, which provides new opportunities to comprehend the procedure and potential of therapeutic development. Taken together, in this review, we summarize current understanding of the mechanism of mitophagy, discuss the roles of mitophagy and its relationship with diseases, introduce the existing small-molecule pharmacological modulators of mitophagy and further highlight the significance of their development.

Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging.

Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a valuable tool for studying the NR-ligand interactions and their downstream effects. The translocation response of NRs can be studied irrespective of the nature of the interacting ligand (agonist, antagonist, or a small molecule modulator). These nuclear translocation studies offer an advantage over promoter-reporter-based transcription assays where transcription response is observed only with the activating hormones or agonistic ligands. Globally, milk serves as a major dietary source. However, suspected presence of endocrine/metabolism-disrupting chemicals like bisphenols, parabens, organochlorine pesticides, carbamates, non-steroidal anti-inflammatory drugs, chloramphenicol, brominated flame retardants, etc. has been reported. Considering that these chemicals may impart serious developmental and metabolism-related health concerns, it is essential to develop assays suitable for the detection of xenobiotics present at differing levels in milk. Since milk samples cannot be used directly on cultured cells or for microscopy, a combination of screening strategies has been developed herein based on the revelation that i) lipophilic NR ligands can be successfully retrieved in milk-fat; ii) milk-fat treatment of cells is compatible with live-cell imaging studies; and finally, iii) treatment of cells with xenobiotics-spiked and normal milk derived fat provides a visual and quantifiable response of NR translocation in living cells. Utilizing a milk-fat extraction method and Green Fluorescent Protein (GFP) tagged NRs expressed in cultured mammalian cells, followed by an assessment of NR response proved to be an effective approach for screening xenobiotics present in milk samples.HighlightsDiverse endocrine and metabolism-disrupting chemicals are suspected to contaminate milk.Nuclear receptors serve as 'xenosensors' for assessing the presence of xenobiotics in milk.Nuclear import of steroid receptors with (ant)agonist can be examined in live cells.Lipophilic xenobiotics are extracted and observed enriched in milk-fat fraction.A comprehensive cell-based protocol aids in the detection of xenobiotics in milk.

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid-liquid phase separation of tau.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.

CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy.

The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.

Effective Perturbations by Small-Molecule Modulators on Voltage-Dependent Hysteresis of Transmembrane Ionic Currents.

The non-linear voltage-dependent hysteresis (Hys(V)) of voltage-gated ionic currents can be robustly activated by the isosceles-triangular ramp voltage (Vramp) through digital-to-analog conversion. Perturbations on this Hys(V) behavior play a role in regulating membrane excitability in different excitable cells. A variety of small molecules may influence the strength of Hys(V) in different types of ionic currents elicited by long-lasting triangular Vramp. Pirfenidone, an anti-fibrotic drug, decreased the magnitude of Ih's Hys(V) activated by triangular Vramp, while dexmedetomidine, an agonist of α2-adrenoceptors, effectively suppressed Ih as well as diminished the Hys(V) strength of Ih. Oxaliplatin, a platinum-based anti-neoplastic drug, was noted to enhance the Ih's Hys(V) strength, which is thought to be linked to the occurrence of neuropathic pain, while honokiol, a hydroxylated biphenyl compound, decreased Ih's Hys(V). Cell exposure to lutein, a xanthophyll carotenoid, resulted in a reduction of Ih's Hys(V) magnitude. Moreover, with cell exposure to UCL-2077, SM-102, isoplumbagin, or plumbagin, the Hys(V) strength of erg-mediated K+ current activated by triangular Vramp was effectively diminished, whereas the presence of either remdesivir or QO-58 respectively decreased or increased Hys(V) magnitude of M-type K+ current. Zingerone, a methoxyphenol, was found to attenuate Hys(V) (with low- and high-threshold loops) of L-type Ca2+ current induced by long-lasting triangular Vramp. The Hys(V) properties of persistent Na+ current (INa(P)) evoked by triangular Vramp were characterized by a figure-of-eight (i.e., ∞) configuration with two distinct loops (i.e., low- and high-threshold loops). The presence of either tefluthrin, a pyrethroid insecticide, or t-butyl hydroperoxide, an oxidant, enhanced the Hys(V) strength of INa(P). However, further addition of dapagliflozin can reverse their augmenting effects in the Hys(V) magnitude of the current. Furthermore, the addition of esaxerenone, mirogabalin, or dapagliflozin was effective in inhibiting the strength of INa(P). Taken together, the observed perturbations by these small-molecule modulators on Hys(V) strength in different types of ionic currents evoked during triangular Vramp are expected to influence the functional activities (e.g., electrical behaviors) of different excitable cells in vitro or in vivo.

Small-molecule modulators of protein-RNA interactions.

Protein-RNA interactions (PRIs) play crucial roles in diverse cellular pathways, from transcription to liquid-liquid phase separation, and its dysregulation is associated with a wide range of human disorders. Therefore, there is great emphasis on discovering small-molecule modulators that target unexplored PRIs by developing robust PRI assays. In particular, targeting PRIs could offer innovative solutions to expand the druggable genome, as only a small portion of protein-coding genes are currently targeted by drugs. This review describes the therapeutic potential of targeting PRIs using small molecules, biochemical and cell-based experimental tools for observing PRIs, and several PRI modulators. We also highlight emerging technologies and the challenges in developing PRI modulators.