Cytokines are crucial regulators of the immune system that orchestrate interactions between cells and, when dysregulated, contribute to the progression of chronic inflammation, cancer, and autoimmunity. Numerous biologic-based clinical agents, mostly monoclonal antibodies, have validated cytokines as important clinical targets and are now part of the standard of care for a number of diseases. These agents, while impactful, still suffer from limitations including a lack of oral bioavailability, high cost of production, and immunogenicity. Small-molecule cytokine inhibitors are attractive alternatives that can address these limitations. Although targeting cytokine-cytokine receptor complexes with small molecules has been a challenging research endeavor, multiple small-molecule inhibitors have now been identified, with a number of them undergoing clinical evaluation. In this review, we highlight the recent advancements in the discovery and development of small-molecule inhibitors targeting soluble cytokines. The strategies for identifying these novel ligands as well as the structural and mechanistic insights into their activity represent important milestones in tackling these challenging and clinically important protein-protein interactions.

Small molecules play important roles in a variety of biological processes such as metabolism, cell signaling and enzyme regulation, and can serve as valuable biomarkers for human diseases. Moreover, they are essential to drug discovery and development, and are important targets for environmental monitoring and food safety. Due to the size incompatibility, small molecule transport is difficult to be monitored with a nanopore. A popular strategy for nanopore detection of small molecules is to introduce a molecular probe as a ligand (or recognition element) and rely on their effect on the ligand transport. One limitation for this sensing strategy is that the probe molecule needs to have a slightly smaller size than the nanopore constriction or can be easily unfolded or unzipped through the pore. Herein, by taking advantage of replacement and complexation chemical interactions, a generic approach is reported for detection of small molecules by using large biomolecules with well-defined stable 3D structures such as aptamers as recognition elements. Given the versatile use of aptamers as capture agents for a wide variety of species, the developed nanopore sensing strategy should find applications in many fields.

Organelle targeting is a useful approach in drug development for cancer therapy. Peptide amphiphiles are good candidates for targeting specific organelles because they can be engineered into a wide range of molecular structures, enabling customization for specific functional needs. We have developed a peptide amphiphile, C16-(EY)3, that can respond to tyrosine kinase activity and undergo phosphorylation inside cancer cells. C16-(EY)3 selectively induced apoptosis in cancer cells that overexpressed tyrosine kinase. The self-assembly of peptide amphiphiles on the endoplasmic reticulum (ER) membrane reduced the ER membrane fluidity and triggered ER stress. The mechanism of the cancer cell death induced by C16-(EY)3 was shown to involve phosphorylation by tyrosine kinase, ER stress induction, and the subsequent activation of caspase-4, -12, and -9, which ultimately triggered apoptosis through the activation of caspase-3 and -7. In vivo studies further validated the antitumor efficacy of C16-(EY)3, as transcutaneous administration of the peptide amphiphile inhibited tumor growth in mice. This study elucidated the mechanism of apoptosis induced by the peptide amphiphile, indicating the potential of peptide amphiphiles as organelle-targeting cancer therapeutics and providing a novel strategy for the development of selective and potent anticancer drugs.

Current small-molecule-regulated synthetic gene switches face clinical limitations such as cytotoxicity, long-term side-effects and metabolic disturbances. Here, we describe an advanced synthetic platform inducible by risk-free input medication (ASPIRIN), which is activated by acetylsalicylic acid (ASA/aspirin), a multifunctional drug with pain-relieving, anti-inflammatory, and cardiovascular benefits. To construct ASPIRIN, we repurpose plant salicylic acid receptors NPR1 and NPR4. Through domain truncations and high-throughput mutant library screening, we enhance their ASA sensitivity. Optimized NPR1 fused with a membrane-tethering myristoylation signal (Myr-NPR1) forms a complex with NPR4, which is fused with a DNA binding domain (VanR) and a transactivation domain (VP16). ASA induces dissociation of the Myr-NPR1/NPR4-VanR-VP16 complex, allowing nuclear translocation of NPR4-VanR-VP16 to activate VanR-operator-controlled gene expression. In male diabetic mice implanted with microencapsulated ASPIRIN-engineered cells, ASA regulates insulin expression, restores normoglycemia, alleviates pain and reduces biomarkers of diabetic neuropathy and inflammation. We envision this system will pave the way for aspirin-based combination gene therapies.

Compared to single-drug therapy, combination therapy involves the use of two or more drugs to reduce drug dosage, decrease drug toxicity, and improve treatment efficacy. We developed an extreme gradient boosting (XGBoost)-based drug-drug cell line prediction model (XDDC) to predict synergistic drug combinations. XDDC was based on XGBoost and used one of the largest drug combination datasets, NCI-ALMANAC. In XDDC, drug chemical structures, adverse drug reactions, and target information were selected as drug features; gene expression, methylation, mutations, copy number variations, and RNA interference data were used as cell line features; and pathway information was incorporated to link drug features and cell line features. XDDC improved the interpretability of drug combination features and outperformed other machine learning methods. It achieved an area under the curve (AUC) of 0.966 ± 0.002 and an AUPR of 0.957 ± 0.002 when cross-validated on NCI-ALMANAC data. Different types of omics data were evaluated and compared in the model. Literature and experimental verification confirmed some of our predictions. XDDC could help medical professionals to rapidly screen synergistic drug combinations against specific cancer cell lines.

It has been well accumulated that G-quadruplex (G4-DNA) has great anticancer relevance, and various heterocyclic moieties have been synthesized and examined as potent G4-DNA binders with promising anticancer activity. Here, we have synthesized a series of naphthalimide-triazole-coumarin conjugates by substituting various amines and further examine their anticancer activity against 60 human cancer cell lines at 10 μM. One and five dose concentration results reveal low values of MG-MID GI50 for compounds including 8a (3.18 μM), 8b (13.11 μM), 8e (7.68 μM) and 8f (1.75 μM). Further cell apoptosis manifests that all compounds can induce cell apoptosis in cancer cells by stabilizing the c-MYC promoter G-quadruplex. Therefore, the G-quadruplex-mediated pathway may be responsible for the apoptosis that these naphthalimide-coumarin compounds caused in cancer cells. Therefore, multispectroscopic techniques are employed to evaluate the binding of molecules with c-MYC G4-DNA where all four molecules readily bind to G4-DNA and stabilize it with a high binding constant, leading to inhibition of cancer cells and apoptosis of cancer cells. Binding studies toward ct-DNA disclose that these compounds do not interact with ds-DNA and thus selectively target G4-DNA to exert their anticancer activity. All the active compounds have greater affinity toward Human Serum Albumin (HSA) and can readily bind with HSA with a binding constant of 12 × 104 M-1 (8a), 13.0 × 104 M-1 (8b), 14.2 × 104 M-1 (8e), 16.3 × 104 M-1 (8f). Thus, the results disclose that inhibition and killing of cancer cells by these derivatives feasibly occur due to their ability to interact with c-MYC G-quadruplex forming promoters and their high affinity toward HSA unfold potent anticancer agents and can be taken further for clinical trials.

Poly(amino acids), polypeptides, and their derivatives have demonstrated significant potential as biodegradable biomaterials in the field of drug delivery. As degradable drug carriers, they can effectively load or conjugate drug molecules including small molecule drugs, nucleic acids, peptides, and protein-based drugs, enhancing the stability and targeting of the drugs in vivo. This strategy ultimately facilitates precise drug delivery and controlled release, thereby improving therapeutic efficacy and reducing side effects within the body. This review systematically describes the structural characteristics and preparation methods of poly(amino acids) and polypeptides, summarizes the advantages of poly(amino acids), polypeptides, and their derivatives in drug delivery, and detailedly introduces the latest advancements in this area. The review also discusses current challenges and opportunities associated with poly(amino acids), peptides, and their derivatives, and offers insights into the future directions for these biodegradable materials. This review aims to provide valuable references for scientific research and clinical translation of biodegradable biomaterials based on poly(amino acids) and peptides.

Podophyllotoxin is a natural compound belonging to the lignan family and is well-known for its great antitumor activity. However, it shows several limitations, such as severe side effects and some pharmacokinetics problems, including low water solubility, which hinders its application as an anticancer agent. Over the past few years, antitumor research has been focused on developing nanotechnology-based medicines or nanomedicines which allow researchers to improve the pharmacokinetic properties of anticancer compounds. Following this trend, podophyllotoxin nanoconjugates have been obtained to overcome its biopharmaceutical drawbacks and to enhance its antitumor properties. The objective of this review is to highlight the advances made over the past few years (2017-2023) regarding the inclusion of podophyllotoxin in different nanosystems. Among the huge variety of nanoconjugates of diverse nature, drug delivery systems bearing podophyllotoxin as cytotoxic payload are organic nanoparticles mainly based on polymer carriers, micelles, and liposomes. Along with the description of their pharmacological properties as antitumorals and the advantages compared to the free drug in terms of biocompatibility, solubility, and selectivity, we also provide insight into the synthetic procedures developed to obtain those podophyllotoxin-nanocarriers. Typical procedures in this regard are self-assembly techniques, nanoprecipitations, or ionic gelation methods among others. This comprehensive perspective aims to enlighten the medicinal chemistry community about the tendencies followed in the design of new podophyllotoxin-based drug delivery systems, their features and applications.

The phospatidylinositol-3 kinase (PI3K) pathway is a critical intracellular signalling mechanism that is changed or amplified in a variety of cancers, including breast, gastric, ovarian, colorectal, prostate, glioma, and endometrial. PI3K signalling is important for cancer cell survival, angiogenesis, and metastasis, making it a promising therapeutic target. The PI3K kinases in their different isoforms, namely α, β, δ, and γ, encode PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes. Specific gene mutation or overexpression of the protein is responsible for the therapeutic failure of current therapeutics. There are several current and completed clinical trials using PI3K inhibitors (pan, isoform-specific, and dual PI3K/mTOR) to develop effective PI3K inhibitors capable of overcoming resistance to existing drugs. However, the bulk of these inhibitors have had their indications revoked or voluntarily withdrawn due to concerns about their harmful consequences. Several inhibitors containing medicinally privileged scaffolds like thiazole, triazine, benzimidazole, podophyllotoxin, pyridine, quinazoline, thieno-triazole, pyrimidine, triazole, benzofuran, imidazo-pyridazine, oxazole, coumarin, and azepine derivatives have been explored to target the PI3K pathway and/or a specific isoform in the current overview. This article reviews the structure, biological activities, and clinical status of PI3K inhibitors. It focuses on the development techniques, docking insight, and structure-activity connections of PI3K-based inhibitors. The findings provide useful insights and future approaches for the development of promising PI3K-based inhibitors.

Because of the well-established link between angiogenesis and tumor development, the use of antiangiogenic therapeutics, such as those targeting VEGFR-2, presents a promising approach to cancer treatment. In the current study, a set of five hydrazine-1- carbothioamide (compounds 3a-e) and three hydrazine-1-carboxamide derivatives (compounds 4a-c) were successfully synthesized from 3-phenoxybenzoic acid. These compounds were specially created as antiproliferative agents with the goal of targeting cancer cells by inhibiting VEGFR- 2 tyrosine kinase.

The new derivatives were synthesized by conventional organic methods, and their structure was versified by IR, 1HNMR, 13CNMR, and mass spectroscopy. In silico investigation was carried out to identify the compounds' target, molecular similarity, ADMET, and toxicity profile. The cytotoxic activity of the prepared compounds was evaluated in vitro against three human cancer cell lines (DLD1 colorectal adenocarcinoma, HeLa cervical cancer, and HepG2 hepatocellular carcinoma). The effects of the leading compound on cell cycle progression and apoptosis induction were investigated by flow cytometry, and the specific apoptotic pathway triggered by the treatment was evaluated by RT-PCR and immunoblotting. Finally, the inhibitory activities of the new compounds against VEGFR-2 was measured.

The designed derivatives exhibited comparable binding positions and interactions to the VEGFR-2 binding site to that of sorafenib (a standard VEGFR-2 tyrosine kinase inhibitor), as determined by molecular docking analysis. Compound 4b was the most cytotoxic compound, achieving the lowest IC50 against HeLa cells. Compound 4b, a strong representative of the synthesized series, induced cell cycle arrest at the G2/M phase, increased the proportion of necrotic and apoptotic HeLa cells, and activated caspase 3. The EC50 value of compound 4b against VEGFR-2 kinase activity was comparable to sorafenib's.

Overall, the findings suggest that compound 4b has a promising future as a starting point for the development of new anticancer drugs.

Epigenetics mechanisms play a significant role in human diseases by altering DNA methylation status, chromatin structure, and/or modifying histone proteins. By modulating the epigenetic status, the expression of genes can be regulated without any change in the DNA sequence itself. Epigenetic drugs exhibit promising therapeutic efficacy against several epigenetically originated diseases including several cancers, neurodegenerative diseases, metabolic disorders, cardiovascular disorders, and so forth. Currently, a considerable amount of research is focused on discovering new drug molecules to combat the existing research gap in epigenetic drug therapy. A novel and efficient delivery system can be established as a promising approach to overcome the drawbacks associated with the current epigenetic modulators. Therefore, formulating the existing epigenetic drugs with distinct encapsulation strategies in nanocarriers, including solid lipid nanoparticles, nanogels, bio-engineered nanocarriers, liposomes, surface modified nanoparticles, and polymer-drug conjugates have been examined for therapeutic efficacy. Nonetheless, several epigenetic modulators are untouched for their therapeutic potential through different delivery strategies. This review provides a comprehensive up to date discussion on the research findings of various epigenetics mechanism, epigenetic modulators, and delivery strategies utilized to improve their therapeutic outcome. Furthermore, this review also highlights the recently emerged CRISPR tool for epigenome editing.

Cyclophilin A (CypA), a member of the immunophilin family, stands out as the most prevalent among the cyclophilins found in humans. Beyond serving as the intracellular receptor for the immunosuppressive drug cyclosporine A (CsA), CypA exerts critical functions within the cell via its peptidyl-prolyl cis-trans isomerase (PPIase) activity, which is crucial for processes, such as protein folding, trafficking, assembly, modulation of immune responses, and cell signaling. Increasing evidence indicates that CypA is up-regulated in a variety of human cancers and it may be a novel potential therapeutic target for cancer treatment. Therefore, gaining a thorough understanding of CypA's contribution to cancer could yield fresh perspectives and inform the development of innovative therapeutic approaches. This review delves into the multifaceted roles of CypA in cancer biology and explores the therapeutic potential of targeting CypA.

Hydrophobic chemotherapy drugs face significant challenges in cancer treatment, including low bioavailability, unavoidable toxic side effects, and the development of drug resistance. To address these issues, a multifunctional nanoplatform was developed for cancer therapy, aimed at achieving effective drug delivery and enhancing antitumor efficacy. Poria cocos polysaccharide (PCP), a natural polymer known for its immunomodulatory properties, was utilized as an immunoreactive vector for drug delivery after being cross-linked with 1,4-phenylenebisboronic acid (BDBA). Subsequently, a small-molecule chemotherapy drug, esculetin (EL), was confirmed through density functional theory (DFT) simulations to be encapsulated within the PCP-BDBA nanoparticles via weak interactions. The results demonstrated that the synthesized nanoparticles were spherical, with an average particle size of 162.0 nm. In addition to exhibiting excellent stability, the nanoparticles also displayed pH-responsive drug release properties. In vivo experiments indicated that EL@PCP-BDBA NPs exhibited antitumor effects. Furthermore, EL@PCP-BDBA NPs showed superior in vitro antitumor activity compared to EL at the cellular level. Additionally, EL@PCP-BDBA NPs were found to increase intracellular reactive oxygen species (ROS) levels, induce cell apoptosis, and suppress cell migration to combat cancer. Meanwhile, EL@PCP-BDBA NPs enhanced immune function in vivo. In summary, this study developed a nano-pharmaceutical that combined chemotherapy and immunotherapy functions, which was considered a promising tool for cancer therapy.

This study focuses on the development of a novel liposome-based nanodelivery system designed to encapsulate polyamine-1, a compound with potential anti-tumor properties. The main objective of this work was to enhance the therapeutic and imaging potential of polyamine-1 by incorporating it into liposome-based nanoparticles, which were functionalized with a gadolinium complex for imaging purposes and a fluorescent phospholipid for tracking applications. These nanoparticles were characterized by measuring their size, shape, polydispersity index, zeta potential and encapsulation efficiency. In vitro experiments were conducted to evaluate the antitumor activity, specifically determining the cytotoxicity of both free and encapsulated polyamine-1 in cancerous and non-cancerous cell lines. Additionally, the study shows the enhanced signal intensity of gadolinium-loaded liposomes by T1-weighted MRI, highlighting their imaging potential. The experimental results suggest that this liposome-based nanodelivery system not only has therapeutic potential in targeted cancer therapy but also could be advantageous for diagnostic imaging, particularly in MRI applications.

Liver cancer is one of the most prevalent malignant tumors worldwide. According to the Barcelona Clinic Liver Cancer staging criteria, clinical guidelines provide tutorials to clinical management of liver cancer at their individual stages. However, most patients diagnosed with liver cancer are at advanced stage; therefore, many researchers conduct investigations on targeted therapy, aiming to improve the overall survival of these patients. To date, small-molecule-based targeted therapies are highly recommended (first line: sorafenib and lenvatinib; second line: regorafenib and cabozantinib) by current the clinical guidelines of the American Society of Clinical Oncology, European Society for Medical Oncology, and National Comprehensive Cancer Network. Herein, we summarize the small-molecule-based targeted therapies in liver cancer, including the approved and preclinical therapies as well as the therapies under clinical trials, and introduce their history of discovery, clinical trials, indications, and molecular mechanisms. For drug resistance, the revealed mechanisms of action and the combination therapies are also discussed. In fact, the known small-molecule-based therapies still have limited clinical benefits to liver cancer patients. Therefore, we analyze the current status and give our ideas for the urgent issues and future directions in this field, suggesting clues for novel techniques in liver cancer treatment.

In the search for new anticancer agents, we synthesized a new series of thiazole derivatives carried on thiadiazole-oxadiazole hybrid. Final compounds (5a-5i) were analyzed via 1H NMR, 13C NMR, and HRMS. The pharmacokinetic profile of the targeted compounds was predicted via in silico calculations. Their anticancer properties were determined using MTT method against MCF7 and A549 cell lines. Compounds 5a, 5b and 5c were found more active against MCF7 cells than A549 cells while they were not cytotoxic on L929 healthy cells. Generally, it can be summarized that acetamide moiety has a pivotal role in anticancer activity. For further studies, their aromatase inhibitory activity was evaluated. After determination all these features, the binding modes of the active compounds and the stability and relation of the ligand-enzyme complex were investigated using molecular docking and molecular dynamics simulation studies, respectively. In vitro and in silico studies suggest two important structure-activity relationship (SAR) points that at least one azole ring is essential, and if there is approximately 8.0 ± 0.5 Å distance between the H-bond rich zone of ligand and the heteroaryl ring system of ligand has a major impact on aromatase inhibitory activity. Compounds with small group substitution on thiazole are found potentially may be used for the treatment of anti-breast cancer orally.Communicated by Ramaswamy H. Sarma.

Aim: Angiogenesis is the hallmark of cancer progression driven by VEGF/VEGFR-2 signalling pathway, inhibition of which could be a solution to tackle the progression of tumour cells and thus arresting their growth.Materials & methods: A novel class of pyrazoles was synthesized using arginine and dibromo ketones. Antiangiogenic activity was performed by in vivo yolk sac method. Antioxidant activity was evaluated by hydroxyl and superoxide radical scavenging assays. Docking studies were performed to determine the pyrazoles' binding potential with VEGFR-2 receptor and VEGF tyrosine kinase. ADMET properties were calculated using SwissADME and admetSAR for drug-likeness.Results: Compounds 5a-e showed significant antiangiogenic effects. Compound 5f exhibited effective hydroxyl and superoxide radical scavenging activities. Docking results confirmed the potential binding efficiency with VEGFR-2 receptor over VEGF tyrosine kinase, thus, functioning as competitive-inhibitors. ADMET studies revealed that the compounds possess favourable drug-like qualities.Conclusion: This study presents a novel class of pyrazoles as promising antioxidant and antiangiogenic agents with favourable drug-likeness properties.

Drug resistance in cancer treatment presents a significant challenge, necessitating innovative approaches to improve therapeutic efficacy. Integrating machine learning (ML) in cancer research is promising as ML algorithms outrival in analysing complex datasets, identifying patterns, and predicting treatment outcomes. Leveraging diverse data sources such as genomic profiles, clinical records, and drug response assays, ML uncovers molecular mechanisms of drug resistance, enabling personalised treatment, maximising efficacy and minimising adverse effects. Various ML algorithms contribute to the drug discovery process - Random Forest and Decision Trees predict drug-target interactions and aid in virtual screening, and SVM classify leads on bioactivity data. Neural Networks model QSAR to optimise lead compounds and K-means clustering group compounds with similar chemical properties aiding compound selection. Gaussian Processes predict drug responses, Bayesian Networks infer causal relationships, Autoencoders generate novel compounds, and Genetic Algorithms optimise molecular structures. These algorithms collectively enhance efficiency and success rates in drug design endeavours, from lead identification to optimisation and are cost-effective, empowering clinicians with real-time treatment monitoring and improving patient outcomes. This review highlights the immense potential of ML in revolutionising cancer care through effective drug design to reduce drug resistance, and we have also discussed various limitations and research gaps to understand better.

A series of tetrahydrobenzo[b]thiophene derivatives was designed and synthesized as dual topoisomerase (Topo) I/II inhibitors implicating potential DNA intercalation. Ethyl-2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylate (1) was prepared by modification of the Gewald reaction procedure using a Fe2O3 nanocatalyst and then it was used as a building block for the synthesis of tetrahydrobenzo[b]thiophene candidates (2-14). Interestingly, compound 14 showed the best cytotoxic potential against hepatocellular, colorectal, and breast cancer cell lines (IC50 = 7.79, 8.10, and 3.53 µM), respectively, surpassing doxorubicin at breast cancer (IC50 = 4.17 µM). Meanwhile, the Topo I and II inhibition assay displayed that compound 3 could exhibit the best inhibitory potential among the investigated candidates (IC50 = 25.26 and 10.01 nM), respectively, in comparison to camptothecin (IC50 = 28.34 nM) and doxorubicin (IC50 = 11.01 nM), as reference standards. In addition, the DNA intercalation assay showed that compound 14 could display the best binding affinity with an IC50 value of 77.82 µM in comparison to doxorubicin (IC50 = 58.03 µM). Furthermore, cell cycle and apoptosis analyses described that compound 3 prompts the G1 phase arrest in michigan cancer foundation-7 cancer cells and increases the apoptosis ratio by 29.31% with respect to untreated cells (2.25%). Additionally, the conducted molecular docking assured the promising binding of the investigated members toward Topo I and II with potential DNA intercalation. Accordingly, the synthesized compounds could be treated as promising anticancer candidates for future optimization.

The transition from conventional cytotoxic chemotherapy to targeted cancer therapy with small-molecule anticancer drugs has enhanced treatment outcomes. This approach, which now dominates cancer treatment, has its advantages. Despite the regulatory approval of several targeted molecules for clinical use, challenges such as low response rates and drug resistance still persist. Conventional drug discovery methods are costly and time-consuming, necessitating more efficient approaches. The rise of artificial intelligence (AI) and access to large-scale datasets have revolutionized the field of small-molecule cancer drug discovery. Machine learning (ML), particularly deep learning (DL) techniques, enables the rapid identification and development of novel anticancer agents by analyzing vast amounts of genomic, proteomic, and imaging data to uncover hidden patterns and relationships.

In this review, the authors explore the important landmarks in the history of AI-driven drug discovery. They also highlight various applications in small-molecule cancer drug discovery, outline the challenges faced, and provide insights for future research.

The advent of big data has allowed AI to penetrate and enable innovations in almost every stage of medicine discovery, transforming the landscape of oncology research through the development of state-of-the-art algorithms and models. Despite challenges in data quality, model interpretability, and technical limitations, advancements promise breakthroughs in personalized and precision oncology, revolutionizing future cancer management.

Precision medicine has revolutionized modern cancer therapeutic management by targeting specific molecular aberrations responsible for the onset and progression of tumorigenesis. ROS proto-oncogene 1 (ROS1) is a receptor tyrosine kinase (RTK) that can induce tumorigenesis through various signaling pathways, such as cell proliferation, survival, migration, and metastasis. It has emerged as a promising therapeutic target in various cancer types. However, there is very limited availability of specific ROS1 inhibitors for therapeutic purposes. Exploring repurposed drugs for rapid and effective treatment is a useful approach. In this study, we utilized an integrated approach of virtual screening and molecular dynamics (MD) simulations of repurposing existing drugs for ROS1 kinase inhibition. Using a curated library of 3648 FDA-approved drugs, virtual screening identified drugs capable of binding to ROS1 kinase domain. The results unveil two hits, Midostaurin and Alectinib with favorable binding profiles and stable interactions with the active site residues of ROS1. These hits were subjected to stability assessment through all-atom MD simulations for 200 ns. MD results showed that Midostaurin and Alectinib were stable with ROS1. Taken together, the study showed a rational framework for the selection of repurposed Midostaurin and Alectinib with ROS1 inhibitory potential for therapeutic management after further validation.

A nucleus-targeted nanocomposite was prepared by clickable amino acid-tuned one-step co-assembly of proteins and chemotherapeutics. The nanocomposite with favorable pharmacokinetic behavior can effectively accumulate in the nucleus, thereby significantly enhancing the anticancer therapeutic effect both in vitro and in vivo.

Immune checkpoint inhibitors (ICIs) are used to relieve and refuel anti-tumor immunity by blocking the interaction, transcription, and translation of co-inhibitory immune checkpoints or degrading co-inhibitory immune checkpoints. Thousands of small molecule drugs or biological materials, especially antibody-based ICIs, are actively being studied and antibodies are currently widely used. Limitations, such as anti-tumor efficacy, poor membrane permeability, and unneglected tolerance issues of antibody-based ICIs, remain evident but are thought to be overcome by small molecule drugs. Recent structural studies have broadened the scope of candidate immune checkpoint molecules, as well as innovative chemical inhibitors. By way of comparison, small molecule drug-based ICIs represent superior oral bioavailability and favorable pharmacokinetic features. Several ongoing clinical trials are exploring the synergetic effect of ICIs and other therapeutic strategies based on multiple ICI functions, including immune regulation, anti-angiogenesis, and cell cycle regulation. In this review we summarized the current progression of small molecule ICIs and the mechanism underlying immune checkpoint proteins, which will lay the foundation for further exploration.

Synthetic biology aims to engineer transgene switches for precise therapeutic protein control in cell-based gene therapies. However, off-the-shelf trigger-inducible gene circuits are usually switched on by single or structurally similar molecules. This study presents a mammalian gene-switch platform that controls therapeutic gene expression by a wide range of molecules generating low, non-toxic levels of reactive oxygen species (ROS). In this system, KEAP1 (Kelch-like ECH-associated protein 1) serves as ROS sensor, regulating the translocation of NRF2 (nuclear factor erythroid 2-related factor 2) to the nucleus, where NRF2 binds to  antioxidant response elements (ARE) to activate the expression of a gene of interest. It is found that a promoter containing eight-tandem ARE repeats is highly sensitive to the low ROS levels generated by the soluble and volatile molecules, which include food preservatives, food additives, pharmaceuticals, and signal transduction inducers. In a proof-of-concept study, it is shown that many of these compounds can independently trigger microencapsulated engineered cells to produce sufficient insulin to restore normoglycemia in experimental type-1 diabetic mice. It is believed that this system greatly extends the variety of small-molecule inducers available to drive therapeutic gene switches.

Sensitive detection of small molecules with biological and environmental interests is important for many applications, such as food safety, disease diagnosis, and environmental monitoring. Herein, we propose a highly selective antibody-bridged DNAzyme walker to sensitively detect small molecules. The antibody-bridged DNAzyme walker consists of a track, small-molecule-labeled DNAzyme walking strand, and antibody against small molecules. The track is built by co-modifying fluorophore-labeled substrates and small-molecule-labeled DNA linkers onto a gold nanoparticle (AuNP). In the absence of the target molecule, the antibody binds small molecule labels at the DNAzyme walking strand and the DNA linker, driving the DNAzyme walking strand on the surface of the AuNP. The attached DNAzyme walking strand moves along the track and cleaves substrates to generate high fluorescence signals to achieve signal amplification. As target molecules exist, they competitively bind with antibody to displace the small-molecule-labeled linker and DNAzyme walking strand, rendering the DNAzyme walker inactive in substrate cleavage and causing weak fluorescence. By using this antibody-bridged DNAzyme walker, we achieved sensitive detection of two biologically important small molecules, digoxin and folic acid. This work provides a new paradigm by combining the signal amplification strategy of a DNA walker and immunorecognition for sensitive and selective detection of small molecules.

Despite the worldwide prevalence and huge burden of pain, pain is an undertreated phenomenon. Currently used analgesics have several limitations regarding their efficacy and safety. The discovery of analgesics possessing a novel mechanism of action has faced multiple challenges, including a limited understanding of biological processes underpinning pain and analgesia and poor animal-to-human translation. Computational pharmacology is currently employed to face these challenges. In this review, we discuss the theory, methods, and applications of computational pharmacology in pain research. Computational pharmacology encompasses a wide variety of theoretical concepts and practical methodological approaches, with the overall aim of gaining biological insight through data acquisition and analysis. Data are acquired from patients or animal models with pain or analgesic treatment, at different levels of biological organization (molecular, cellular, physiological, and behavioral). Distinct methodological algorithms can then be used to analyze and integrate data. This helps to facilitate the identification of biological molecules and processes associated with pain phenotype, build quantitative models of pain signaling, and extract translatable features between humans and animals. However, computational pharmacology has several limitations, and its predictions can provide false positive and negative findings. Therefore, computational predictions are required to be validated experimentally before drawing solid conclusions. In this review, we discuss several case study examples of combining and integrating computational tools with experimental pain research tools to meet drug discovery challenges.

Breast cancer invasive 2.3 million women worldly and second prominent factor of cancer-related mortality. Finding a new site-specific and safe small molecule is a current need in this field. With the aid of deep learning Algorithms, we analyzed the published big database from cancer CBioportal to find the best target protein. Further, Multi-omics analysis such as enrichment analysis, scores of molecular, RNA biological function at a cellular level, and protein domain were obtained and matched to find the better hit molecules. The gene analysis output shows nucleolar protein 6 plays a significant responsibility in breast carcinoma and 354 natural and synthetic lead molecules are docked inside the active site. Docking result gave the output hit molecule falavan-3-ols with a binding score of -5.325 (Kcal/mol) and interaction analysis illustrates, 13 active amino acids favoring the binding interaction with functional groups of the hit molecule compared to the standard molecule Abemacilib (-2.857 (Kcal/mol)). Best docked complex of flavan-3-ols and NOL6 protein subjected to dynamic simulation 100 ns to study the stability. The results proved that π-π stacked, carbon‑hydrogen and electrostatic interactions are stable throughout the 100 ns simulation. The overall results conclude the hit molecule flavan-3-ol will be a safe and potent lead molecule to generate and treat breast carcinoma patients.

A new class of benzimidazole-based derivatives (4a-j, 5, and 6) with potential dual inhibition of EGFR and BRAFV600E has been developed. The newly synthesized compounds were submitted for testing for antiproliferative activity against the NCI-60 cell line. All newly synthesized compounds 4a-j, 5, and 6 were selected for testing against a panel of sixty cancer cell lines at a single concentration of 10 µM. Some compounds tested demonstrated remarkable antiproliferative activity against the cell lines tested. Compounds 4c, 4e, and 4g were chosen for five-dose testing against 60 human tumor cell lines. Compound 4c demonstrated strong selectivity against the leukemia subpanel, with a selectivity ratio of 5.96 at the GI50 level. The most effective in vitro anti-cancer assay derivatives (4c, 4d, 4e, 4g, and 4h) were tested for EGFR and BRAFV600E inhibition as potential targets for antiproliferative action. The results revealed that compounds 4c and 4e have significant antiproliferative activity as dual EGFR/BRAFV600E inhibitors. Compounds 4c and 4e induced apoptosis by increasing caspase-3, caspase-8, and Bax levels while decreasing the anti-apoptotic Bcl2 protein. Moreover, molecular docking studies confirmed the potential of compounds 4c and 4e to act as dual EGFR/BRAFV600E inhibitors.

Intracellular protein-protein interactions provide a major therapeutic target for the development of peptide-based anticancer therapeutic agents. MDM2 is the 491-residue protein encoded by the MDM2 oncogene. Being a ubiquitin-protein ligase, MDM2 represses the transcription ability of the tumor suppressor p53 by proteasome-mediated degradation. Under typical cellular circumstances, a sustained p53 expression level is maintained by negative regulation of MDM2, whereas under stress conditions, this is alleviated to increase the p53 level. Modulation of MDM2-p53 interaction via fabrication of an MDM2-interacting peptide could be a useful strategy to inhibit subsequent proteasomal degradation of p53 and initiation of p53 signaling leading to the initiation of p53-mediated apoptosis of tumor cells. Here, in this research work, a novel anticancer peptide mPNC-NLS targeting the nucleus and the MDM2 protein (p53 negative regulator) was designed to promote the p53 protein activity for the prevention of cancer. It induces effective apoptosis in both A549 and U87 cells and remains non-cytotoxic to normal lung fibroblast cells (WI38). Further, immunocytochemistry and Western blot results confirm that the designed mPNC-NLS peptide induces the apoptotic death of lung cancer cells via activation of p53 and p21 proteins and remarkably stifled the in vitro growth of 3D multicellular spheroids composed of A549 cells.

Cancer is one of the most common reasons for mortality in the world. A continuous effort to develop effective anti-cancer drugs with minimum side effects has become necessary. The use of small-molecule drugs has revolutionized cancer research by inhibiting cancer cell survival and proliferation. Quinazolines are a class of bioactive heterocyclic compounds with active pharmacophores in several anti-cancer drugs. Such small molecule inhibitors obstruct the significant signals responsible for cancer cell development, thus blocking these cell signals to prevent cancer development and spread.

In the current study, novel quinazoline derivatives structurally similar to erlotinib were synthesized and explored as novel anti-cancer agents.

All the synthesized molecules were confirmed by spectroscopic techniques like 1H NMR, 13C NMR, and ESI-MS. Various techniques were applied to study the protein-drug interaction, DFT analysis, Hirshfeld surface, and target prediction. The molecules were screened in vitro for their anti-cancer properties against 60 human tumor cell lines. The growth inhibitory properties of a few compounds were studied against the MCF7 breast cancer cell line.

The activity of compounds 9f, 9o, and 9s were found to be active. However, compound 9f is more active when compared with other compounds.

Some synthesized compounds were active against different cancer cell lines. The in-vitro study results were found to be in agreement with the predictions from in-silico data. The selected molecules were further subjected to get the possible mechanism of action against different cancer cells.

Chemotherapy is an effortless and frequently used approach in cancer therapy. However, in most cases, it can only prolong life expectancy and does not guarantee a complete cure. Furthermore, chemotherapy is associated with severe adverse effects, one of the major complications of effective cancer therapy. In addition, newly published research outputs show that cancer stem cells are involved in cancer disease progression, drug resistance, metastasis, and recurrence and that they are functional in the trans-differentiation capacity of cancer stem cells to cancer cells in response to treatments. Novel strategies are therefore required for better management of cancer therapy. The prime approach would be to synthesize and develop novel drugs that need extensive resources, time, and endurance to be brought into therapeutic use. The subsequent approach would be to screen the anti-cancer activity of available non-cancerous drugs. This concept of repurposing non-cancer drugs as an alternative to current cancer therapy has become popular in recent years because using existing anticancer drugs has several adverse effects. Micronutrients have also been investigated for cancer therapy due to their significant anti-cancer effects with negligible or no side effects and availability in food sources. In this paper, we discuss an ideal hypothesis for screening available non-cancerous drugs with anticancer activity, with a focus on cancer stem cells and their clinical application for cancer treatment. Further, drug repurposing and the combination of micronutrients that can target both cancers and cancer stem cells may result in a better therapeutic approach leading to maximum tumor growth control.

The porous coordination cage PCC-1 represents a new platform potentially useful for the cellular delivery of drugs with poor cell permeability and solubility. PCC-1 is a metal-organic polyhedron constructed from zinc metal ions and organic ligands through coordination bonds. PCC-1 possesses an internal cavity that is suitable for drug encapsulation. To better understand the biocompatibility of PCC-1 with human cells, the cell entry mechanism, disassembly, and toxicity of the nanocage were investigated. PCC-1 localizes in the nuclei and cytoplasm within minutes upon incubation with cells, independent of endocytosis and cargo, suggesting direct plasma membrane translocation of the nanocage carrying its guest in its internal cavity. Furthermore, the rates of cell entry correlate to extracellular concentrations, indicating that PCC-1 is likely diffusing passively through the membrane despite its relatively large size. Once inside cells, PCC-1 disintegrates into zinc metal ions and ligands over a period of several hours, each component being cleared from cells within 1 day. PCC-1 is relatively safe for cells at low micromolar concentrations but becomes inhibitory to cell proliferation and toxic above a concentration or incubation time threshold. However, cells surviving these conditions can return to homeostasis 3-5 days after exposure. Overall, these findings demonstrate that PCC-1 enters live cells by crossing biological membranes spontaneously. This should prove useful to deliver drugs that lack this capacity on their own, provided that the dosage and exposure time are controlled to avoid toxicity.

Thiazolopyridines are a highly relevant class of small molecules, which have previously shown a wide range of biological activities. Besides their anti-tubercular, anti-microbial and anti-viral activities, they also show anti-cancerogenic properties, and play a role as inhibitors of cancer-related proteins. Herein, the biological effects of the thiazolopyridine AV25R, a novel small molecule with unknown biological effects, were characterized. Screening of a set of lymphoma (SUP-T1, SU-DHL-4) and B- acute leukemia cell lines (RS4;11, SEM) revealed highly selective effects of AV25R. The selective anti-proliferative and metabolism-modulating effects were observed in vitro for the B-ALL cell line RS4;11. Further, we were able to detect severe morphological changes and the induction of apoptosis. Gene expression analysis identified a large number of differentially expressed genes after AV25R exposure and significant differentially regulated cancer-related signaling pathways, such as VEGFA-VEGFR2 signaling and the EGF/EGFR pathway. Structure-based pharmacophore screening approaches using in silico modeling identified potential biological AV25R targets. Our results indicate that AV25R binds with several proteins known to regulate cell proliferation and tumor progression, such as FECH, MAP11, EGFR, TGFBR1 and MDM2. The molecular docking analyses indicates that AV25R has a higher binding affinity compared to many of the experimentally validated small molecule inhibitors of these targets. Thus, here we present in vitro and in silico analyses which characterize, for the first time, the molecular acting mechanism of AV25R, including cellular and molecular biologic effects. Additionally, this predicted the target binding of the molecule, revealing a high affinity to cancer-related proteins and, thus, classified AVR25 for targeted intervention approaches.

Gene expression at the transcriptional level is altered by epigenetic modifications such as DNA methylation, histone methylation, and acetylation, which can upregulate, downregulate, or entirely silence genes. Pathological dysregulation of epigenetic processes can result in the development of cancer, neurological problems, metabolic disorders, and cardiovascular diseases. It is of promising therapeutic interest to find medications that target these epigenetic alterations. Despite the enormous amount of work that has been done in this area, very few molecules have been approved for clinical purposes. This article provides a comprehensive review of recent advances in epigenetic therapeutics for cancer, with a specific focus on emerging delivery and development strategies. Various delivery systems, including pro-drugs, conjugated molecules, nanoparticles (NPs), and liposomes, as well as remedial strategies such as combination therapies, and epigenetic editing, are being investigated to improve the efficacy and specificity of epigenetic drugs (epi-drugs). Furthermore, the challenges associated with available epi-drugs and the limitations of their translation into clinics have been discussed. Target selection, isoform selectivity, physiochemical properties of synthesized molecules, drug screening, and scalability of epi-drugs from preclinical to clinical fields are the major shortcomings that are addressed. This Review discusses novel strategies for the identification of new biomarkers, exploration of the medicinal chemistry of epigenetic modifiers, optimization of the dosage regimen, and design of proper clinical trials that will lead to better utilization of epigenetic modifiers over conventional therapies. The integration of these approaches holds great potential for improving the efficacy and precision of epigenetic treatments, ultimately benefiting cancer patients.

In the design of delivery strategies for anticancer therapeutics, the controlled release of intact cargo at the destined tumor and metastasis locations is of particular importance. To this end, stimuli-responsive chemical linkers have been extensively investigated owing to their ability to respond to tumor-specific physiological stimuli, such as lowered pH, altered redox conditions, increased radical oxygen species and pathological enzymatic activities. To prevent premature action and off-target effects, anticancer therapeutics are chemically modified to be transiently inactivated, a strategy known as prodrug development. Prodrugs are reactivated upon stimuli-dependent release at the sites of interest. As most drugs and therapeutic proteins have the optimal activity when released from carriers in their native and original forms, traceless release mechanisms are increasingly investigated. In this review, we summarize the chemical toolkit for developing innovative traceless prodrug strategies for stimuli-responsive drug delivery and discuss the applications of these chemical modifications in anticancer treatment including cancer immunotherapy.

Harnessing surface engineering strategies to functionalize nucleic acid-lipid nanoparticles (LNPs) for improved performance has been a hot research topic since the approval of the first siRNA drug, patisiran, and two mRNA-based COVID-19 vaccines, BNT162b2 and mRNA-1273. Currently, efforts have been mainly made to construct targeted LNPs for organ- or cell-type-specific delivery of nucleic acid drugs by conjugation with various types of ligands. In this review, we describe the surface engineering strategies for nucleic acid-LNPs, considering ligand types, conjugation chemistries, and incorporation methods. We then outline the general purification and characterization techniques that are frequently used following the engineering step and emphasize the specific techniques for certain types of ligands. Next, we comprehensively summarize the currently accessible organs and cell types, as well as the other applications of the engineered LNPs. Finally, we provide considerations for formulating targeted LNPs and discuss the challenges of successfully translating the "proof of concept" from the laboratory into the clinic. We believe that addressing these challenges could accelerate the development of surface-engineered LNPs for targeted nucleic acid delivery and beyond.

The tumor microenvironment (TME) is well-defined target for understanding tumor progression and various cell types. Major elements of the tumor microenvironment are the followings: endothelial cells, fibroblasts, signaling molecules, extracellular matrix, and infiltrating immune cells. MicroRNAs (miRNAs) are a group of small noncoding RNAs with major functions in the gene expression regulation at post-transcriptional level that have also appeared to exerts key functions in the cancer initiation/progression in diverse biological processes and the tumor microenvironment. This study summarized various roles of miRNAs in the complex interactions between the tumor and normal cells in their microenvironment.

With the objective to identify novel anticancer leads, herein ruthenium-catalyzed electrochemical homo- and heterocoupling reactions of terminal alkynes have been developed for the synthesis of the desired products. Among the synthesized 1,3-diynes, some of them were rigorously examined for possible in vitro anticancer activity against HeLa (human cervical cancer) and L6 normal (rat skeletal muscle) cell lines. Additionally, the docking study was also performed toward 16 ovarian cancer targets with binding affinity calculations with respect to the standard. To the best of our knowledge, this is the first scientific report on the ruthenium-catalyzed electrochemical homocoupling reaction between terminal alkynes with its in vitro anticancer and in silico docking studies.

Sine oculis homeobox 4 (SIX4), a critical transcription factor modulating organ development, potentially participates in tumorigenesis through numerous pathways. Here, we investigated siRNA-mediated knockdown effects of SIX4 on pancreatic cancer cells and underlying molecular mechanisms. The expression of SIX4 in pancreatic cancer and adjacent tissues were investigated in clinical tissue samples and bioinformatically approved by gene expression omnibus (GEO) database. Appropriate siRNA transfected into PANC1 pancreatic cancer cells in order to SIX4 knockdown. The survival, migration, invasion, colony formation, mitochondrial membrane potential, apoptosis, autophagy, and cell cycle in the cancer cells were investigated after knockdown of SIX4. In addition, expression of genes involved in apoptosis and metastasis were assessed in the transfected cancer cells in mRNA and protein levels. High-throughput analysis using GEO database confirmed the overexpression of SIX4 in pancreatic cancer tissues by six independent pancreatic cancer microarrays. Knockdown of SIX4 by specific siRNA significantly decreased survival, colony formation, and mitochondrial membrane potential of the cancer cells. Further assessments demonstrated that knockdown of SIX4 increases the apoptosis and autophagy rates in the cancer cells through modifying the expression of related genes. Moreover, a significant decrease in migration and invasion rates were observed in SIX4 suppressed group. Furthermore, frequency of the cells transfected with SIX4 siRNA increased slightly in G1 and Sub-G1 phases of cell cycle. Our study suggested that siRNA-mediated knockdown of SIX4 increases the pancreatic cancer cells death and reduces the invasion and migration of the cancer cells through different molecular pathways.

Breast cancer is the most common malignancy in women worldwide and despite significant advancements in detection, treatment, and management of cancer, it is still the leading cause of malignancy related deaths in women. Understanding the fundamental biology of breast cancer and creating fresh diagnostic and therapeutic strategies have gained renewed focus in recent studies. In the onset and spread of breast cancer, a group of enzymes known as kinases are extremely important. Small-molecule kinase inhibitors have become a promising class of medications for the treatment of breast cancer owing to their capacity to specifically target kinases involved in the growth and progression of cancer. The creation of targeted treatments that block these kinases and the signalling pathways that they activate has completely changed how breast cancer is treated. Many of these targeted treatments have been approved for the treatment of breast cancer as clinical trials have demonstrated their great efficacy. CDK4/6 inhibitors, like palbociclib, abemaciclib, and ribociclib, EGFR inhibitors such as gefitinib and erlotinib and HER2-targeting small-molecule kinases like neratinib and tucatinib are some examples that have shown potential in treating breast cancer. Yet, there are still difficulties in the development of targeted medicines for breast cancer, such as figuring out which patient subgroups may benefit from these therapies and dealing with drug resistance problems. Notwithstanding these difficulties, kinase-targeted treatments for breast cancer still have a lot of potential. The development of tailored medicines will continue to be fuelled by the identification of novel targets and biomarkers for breast cancer as a result of advancements in genomic and proteomic technology.

Cancer treatment has improved over the past decades, but a major challenge lies in drug formulation, specifically for oral administration. Most anticancer drugs have poor water solubility which can affect their bioavailability. This causes suboptimal pharmacokinetic performance, resulting in limited efficacy and safety when administered orally. As a result, it is essential to develop a strategy to modify the solubility of anticancer drugs in oral formulations to improve their efficacy and safety. A promising approach that can be implemented is amorphous solid dispersion (ASD) which can enhance the aqueous solubility and bioavailability of poorly water-soluble drugs. The addition of a polymer can cause stability in the formulations and maintain a high supersaturation in bulk medium. Therefore, this study aimed to summarize and elucidate the mechanisms and impact of an amorphous solid dispersion system on cancer therapy. To gather relevant information, a comprehensive search was conducted using keywords such as "anticancer drug" and "amorphous solid dispersion" in the PubMed, Scopus, and Google Scholar databases. The review provides an overview and discussion of the issues related to the ASD system used to improve the bioavailability of anticancer drugs based on molecular pharmaceutics. A thorough understanding of anticancer drugs in this system at a molecular level is imperative for the rational design of the products.

A range of natural products have been extensively studied for their chemopreventive potential for cancer, including those that inhibit growth and induce apoptosis. Sidr honey derived from the Ziziphus or Lote tree (Ziziphus spina-christi, Ziziphus lotus, or Ziziphus jujuba) is used in a wide range of traditional medicine practices. In the current study, the Saudi Sidr honey was analyzed by means of a GC-MS chromatogram and investigated for its antiproliferative effects on colorectal cancer cells (HCT-116), breast cancer cells (MCF-7), and lung cancer cells (A-549), as well as its apoptosis induction and cell cycle arrest potentials against human colorectal cancer cells (HCT-116). The effects of Saudi Sidr honey on cells were determined using the MTT assay and the clonogenic assay. The induction of apoptosis was studied using Annexin V-FITC flow cytometry analysis. The propidium iodide staining method was used to detect cell cycle arrest via flow cytometry. By means of performing GS-MS and HR-LCMS analysis, 23 different chemical components were identified from Saudi Sidr honey. A dose-response analysis showed that Saudi Sidr honey was more effective against HCT-116 (IC50 = 61.89 ± 1.89 µg/mL) than against MCF-7 (IC50 = 78.79 ± 1.37 µg/mL) and A-549 (IC50 = 94.99 ± 1.44 µg/mL). The antiproliferation activity of Saudi Sidr honey has been found to be linked to the aggregation of cells during the G1 phase, an increase in early and late apoptosis, and necrotic cell death in HCT-116 cells. Considering these promising findings that highlight the potential use of Saudi Sidr honey as an antitumor agent, further research should be carried out with the aim of isolating, characterizing, and evaluating the bioactive compounds involved in Sidr honey's antiproliferative activity to better understand the mechanism of their action.

Multifunctional nanocarriers enhance the treatment efficacy for modern therapeutics and have gained increasing importance in biomedical research. Codelivery of multiple bioactive molecules enables synergistic therapies. Coencapsulation of cargo molecules into one nanocarrier system is challenging due to different physicochemical properties of the cargo molecules. Additionally, coencapsulation of multiple molecules simultaneously shall proceed with high control and efficiency. Orthogonal approaches for the preparation of nanocarriers are essential to encapsulate sensitive bioactive molecules while preserving their bioactivity. Preparation of nanocarriers by physical processes (i.e., self-assembly or coacervation) and chemical reactions (i.e., click reactions, polymerizations, etc.) are considered as orthogonal methods to most cargo molecules. This review shall act as a guideline to allow the reader to select a suitable preparation protocol for a desired nanocarrier system. This article helps to select for combinations of cargo molecules (hydrophilic-hydrophobic, small-macro, organic-inorganic) with nanocarrier material and synthesis protocols. The focus of this article lies on the coencapsulation of multiple cargo molecules into biocompatible and biodegradable nanocarriers prepared by orthogonal strategies. With this toolbox, the selection of a preparation method for a known set of cargo molecules to prepare the desired biodegradable and loaded nanocarrier shall be provided.