Degraders in epigenetic therapy: PROTACs and beyond.

Epigenetics refers to the reversible process through which changes in gene expression occur without changing the nucleotide sequence of DNA. The process is currently gaining prominence as a pivotal objective in the treatment of cancers and other ailments. Numerous drugs that target epigenetic mechanisms have obtained approval from the Food and Drug Administration (FDA) for the therapeutic intervention of diverse diseases; many have drawbacks, such as limited applicability, toxicity, and resistance. Since the discovery of the first proteolysis-targeting chimeras (PROTACs) in 2001, studies on targeted protein degradation (TPD)-encompassing PROTACs, molecular glue (MG), hydrophobic tagging (HyT), degradation TAG (dTAG), Trim-Away, a specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein eraser (SNIPER), antibody-PROTACs (Ab-PROTACs), and other lysosome-based strategies-have achieved remarkable progress. In this review, we comprehensively highlight the small-molecule degraders beyond PROTACs that could achieve the degradation of epigenetic proteins (including bromodomain-containing protein-related targets, histone acetylation/deacetylation-related targets, histone methylation/demethylation related targets, and other epigenetic targets) via proteasomal or lysosomal pathways. The present difficulties and forthcoming prospects in this domain are also deliberated upon, which may be valuable for medicinal chemists when developing more potent, selective, and drug-like epigenetic drugs for clinical applications.

Neddylation-dependent LSD1 destabilization inhibits the stemness and chemoresistance of gastric cancer.

Histone lysine-specific demethylase 1 (LSD1) expression has been evaluated in multiple tumors, including gastric cancer (GC). However, the mechanisms underlying LSD1 dysregulation in GC remain largely unclear. In this study, neural precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) was identified to be conjugated to LSD1 at K63 by ubiquitin-conjugating enzyme E2 M (UBE2M), and this neddylated LSD1 could promote LSD1 ubiquitination and degradation, leading to a decrease of GC cell stemness and chemoresistance. Herein, our findings revealed a novel mechanism of LSD1 neddylation and its contribution to decreasing GC cell stemness and chemoresistance. Taken together, our findings may whistle about the future application of neddylation inhibitors.

Exploring the synthetic approaches and clinical prowess of established macrocyclic pharmaceuticals.

Macrocyclic compounds, characterized by cyclic structures, often originate from either modified forms of unicyclic canonical molecules or natural products. Within the field of medicinal chemistry, there has been a growing fascination with drug-like macrocycles in recent years, primarily due to compelling evidence indicating that macrocyclization can significantly influence both the biological and physiochemical properties, as well as the selectivity, when compared to their acyclic counterparts. The approval of contemporary pharmaceutical agents like Lorlatinib underscore the notable clinical relevance of drug-like macrocycles. Nonetheless, the synthesis of these drug-like macrocycles poses substantial challenges, primarily stemming from the complexity of ring-closing reactions, which are inherently dependent on the size and geometry of the bridging linker, impacting overall yields. Nevertheless, macrocycles offer a promising avenue for expanding the synthetic toolkit in medicinal chemistry, enabling the creation of bioactive compounds. To shed light on the subject, we delve into the clinical prowess of established macrocyclic drugs, spanning various therapeutic areas, including oncology, and infectious diseases. Case studies of clinically approved macrocyclic agents illustrate their profound impact on patient care and disease management. As we embark on this journey through the world of macrocyclic pharmaceuticals, we aim to provide a comprehensive overview of their synthesis and clinical applications, shedding light on the pivotal role they play in modern medicine.

Unlocking the synthetic approaches and clinical application of approved small-molecule drugs for gastrointestinal cancer treatment: A comprehensive exploration.

Gastrointestinal (GI) cancers encompass a group of malignancies affecting the digestive system, including the stomach, esophagus, liver, colon, rectum and pancreas. These cancers represent a significant global health burden, necessitating effective treatment strategies. Small-molecule drugs have emerged as crucial therapeutic options in the fight against GI cancers due to their oral bioavailability, targeted mechanisms of action, and well-established safety profiles. The review then elucidates the clinical applications and synthetic methods of clinically approved small-molecule drugs for the treatment of GI cancer, shedding light on their mechanisms of action and their potential in mitigating GI cancer progression. The review also discusses future prospects and the evolving landscape of small-molecule drug development in GI oncology, highlighting the potential for personalized medicine. In summary, this review provides valuable insights into cutting-edge strategies for harnessing clinically approved small-molecule drugs to combat GI cancer effectively.

Synthetic approaches and application of clinically approved small-molecule drugs to treat hepatitis.

Hepatitis, a global public health concern, presents a significant burden on healthcare systems worldwide. Particularly, hepatitis B and C are viral infections that can lead to severe liver damage, cirrhosis, and even hepatocellular carcinoma (HCC). The urgency to combat these diseases has driven researchers to explore existing small-molecule drugs as potential therapeutics. This comprehensive review provides a systematic overview of synthetic routes to key antiviral agents used to manage hepatitis. Furthermore, it elucidates the mechanisms of action of these drugs, shedding light on their interference with viral replication and liver disease progression. The review also discusses the clinical applications of these drugs, including their use in combination therapies and various patient populations. By evaluating the synthetic pathways and clinical utility of these drugs, this review not only consolidates current knowledge but also highlights potential future directions for research and drug development in the fight against hepatitis, ultimately contributing to improved patient outcomes and reduced global disease burden.

Advances of clinically approved small-molecule drugs for the treatment of non-small cell lung cancer.

Lung cancer continues to pose a significant challenge as a prominent contributor to global cancer-related mortality. Despite the considerable strides made in therapeutic interventions within the past decade, a substantial population of patients diagnosed with non-small cell lung cancer (NSCLC) still face the grim reality of an incurable condition. In the realm of optimal management strategies for individuals afflicted with locally advanced, yet amenable to surgical resection, NSCLC, a therapeutic approach encompassing chemoradiation stands as a fundamental component. Significant strides have been made in the therapeutic landscape of NSCLC during the preceding two decades, facilitating an enhanced comprehension of the underlying disease biology, and mechanisms governing tumor progression, as well as advancements in early detection modalities and multimodal therapeutic interventions. Nevertheless, the overall rates of curative interventions and survival outcomes for NSCLC continue to exhibit a discouragingly low trajectory, particularly in the context of metastatic disease. Hence, the imperative for sustained research endeavors in the realm of novel pharmaceutical agents and combinatorial therapeutic approaches remains paramount, with the overarching objective of broadening the scope of clinical advantages conferred upon a wider demographic of patients, thereby fostering tangible improvements in outcomes pertaining to NSCLC. The primary objective of this review is to provide an all-encompassing examination encompassing the clinical application and synthetic routes of specific drugs, with the explicit aim of disseminating invaluable knowledge that can inform future research and development endeavors focused on NSCLC.

Synthesis and clinical application of small-molecule inhibitors of Janus kinase.

Janus kinase (JAK) plays a crucial role in intracellular signaling pathways, particularly in cytokine-mediated signal transduction, making them attractive therapeutic targets for a wide range of diseases, including autoimmune disorders, myeloproliferative neoplasms, and inflammatory conditions. The review provides a comprehensive overview of the development and therapeutic potential of small-molecule inhibitors targeting JAK family of proteins in various clinical trials. It also discusses the mechanisms of action, specificity, and selectivity of these inhibitors, shedding light on the challenges associated with achieving target selectivity while minimizing off-target effects. Moreover, the review offers insights into the clinical applications of JAK inhibitors, summarizing the ongoing clinical trials and the Food and Drug Administration (FDA)-approved JAK inhibitors currently available for various diseases. Overall, this review provides a thorough examination of the synthesis and clinical use of typical small-molecule JAK inhibitors in different clinical stages and offers a bright future for the development of novel small-molecule JAK inhibitors.

Synthetic approaches and application of clinically approved small-molecule Anti-HIV drugs: An update.

Application of chemotherapeutic agents to inhibit the HIV replication process has brought about a significant metamorphosis in the landscape of AIDS. Substantial declines in morbidity and mortality rates have been attained, accompanied by notable decreases in healthcare resource utilization. However, treatment modalities do not uniformly inhibit HIV replication in every patient, while the emergence of drug-resistant viral strains poses a substantial obstacle to subsequent therapeutic interventions. Furthermore, chronic administration of therapy may lead to the manifestation of toxicities. These challenges necessitate the exploration of novel pharmacological agents and innovative therapeutic approaches aimed at effectively managing the persistent viral replication characteristic of chronic infection. This review examines the role of clinically approved small-molecule drugs in the treatment of HIV/AIDS, which provides an in-depth analysis of the major classes of small-molecule drugs, including nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors, entry inhibitors, and pharmacokinetic enhancers. The review mainly discusses the application, synthetic routes, and mechanisms of action of small-molecule drugs employed in the treatment of HIV, as well as their use in combination with antiretroviral therapy, presenting viewpoints on forthcoming avenues in the development of novel anti-HIV drugs.

A microbiological system for screening the interference of XNA monomers with DNA and RNA metabolism.

We explored the toxicity and mutagenicity of a wide range of xenobiotic nucleoside triphosphates to an Escherichia coli strain equipped with a nucleoside triphosphate transporter. This bacterial test provides a tool to evaluate and guide the synthesis of nucleotides for applications such as the propagation of non-natural genetic information or the selection of potential drugs.

Design, synthesis, and anti-respiratory syncytial virus potential of novel 3-(1,2,3-triazol-1-yl)furoxazine-fused benzimidazole derivatives.

Respiratory syncytial virus (RSV) is a major cause of serious lower respiratory tract infections in infants, children, and older persons. Currently, the only approved anti-viral chemotherapeutic drug for RSV treatment is ribavirin aerosol; however, its significant toxicity has led to restricted clinical use. In a previous study, we developed various benzimidazole derivatives against RSV. In this study, we synthesised 3-azide substituted furoxazine-fused benzimidazole derivatives by sulfonylation and azide substitution of the 3-hydroxyl group of the furoxazine-fused benzimidazole derivatives. Subsequently, a series of 3-(1,2,3-triazol-1-yl)-substituted furoxazine-fused benzimidazole derivatives were synthesised using the classical click reaction. Biological evaluations of the target compounds indicated that compound 4a-2 had higher activity against RSV (EC50 = 12.17 µM) and lower cytotoxicity (CC50 = 390.64 µM). Compound 4a-2 exerted anti-viral effects against the RSV Long strain by inhibiting apoptosis and the elevation of reactive oxygen species (ROS) and inflammatory factors caused by viral infection in vitro. Additionally, the clinical symptoms of the virus-infected mice were markedly relieved, and the viral load in the lung tissues was dramatically decreased. The biosafety profile of compound 4a-2 was also favourable, showing no detectable adverse effects on any of the major organs in vivo. These findings underscore the potential of compound 4a-2 as a valuable therapeutic option for combating RSV infections while also laying the foundation for further research and development in the field.

Conformational Morphing by a DNA Analogue Featuring 7-Deazapurines and 5-Halogenpyrimidines and the Origins of Adenine-Tract Geometry.

Several efforts are currently directed at the creation and cellular implementation of alternative genetic systems composed of pairing components that are orthogonal to the natural dA/dT and dG/dC base pairs. In an alternative approach, Watson-Crick-type pairing is conserved, but one or all of the four letters of the A, C, G, and T alphabet are substituted by modified components. Thus, all four nucleobases were altered to create halogenated deazanucleic acid (DZA): dA was replaced by 7-deaza-2'-deoxyadenosine (dzA), dG by 7-deaza-2'-deoxyguanosine (dzG), dC by 5-fluoro-2'-deoxycytidine (FdC), and dT by 5-chloro-2'-deoxyuridine (CldU). This base-pairing system was previously shown to retain function in Escherichia coli. Here, we analyze the stability, hydration, structure, and dynamics of a DZA Dickerson-Drew Dodecamer (DDD) of sequence 5'-FdC-dzG-FdC-dzG-dzA-dzA-CldU-CldU-FdC-dzG-FdC-dzG-3'. Contrary to similar stabilities of DDD and DZA-DDD, osmotic stressing revealed a dramatic loss of hydration for the DZA-DDD relative to that for the DDD. The parent DDD 5'-d(CGCGAATTCGCG)-3' features an A-tract, a run of adenosines uninterrupted by a TpA step, and exhibits a hallmark narrow minor groove. Crystal structures─in the presence of RNase H─and MD simulations show increased conformational plasticity ("morphing") of DZA-DDD relative to that of the DDD. The narrow dzA-tract minor groove in one structure widens to resemble that in canonical B-DNA in a second structure. These changes reflect an indirect consequence of altered DZA major groove electrostatics (less negatively polarized compared to that in DNA) and hydration (reduced compared to that in DNA). Therefore, chemical modifications outside the minor groove that lead to collapse of major groove electrostatics and hydration can modulate A-tract geometry.

Synthesis of mizoribine prodrugs and their in vivo evaluation as immunosuppressive agents.

Mizoribine is a well-known immunosuppressive drug, based on a nucleoside scaffold, that targets inosine-monophosphate dehydrogenase (IMPDH). In an effort to increase its in vivo efficacy, three different types of prodrugs (a phosphoramidate prodrug, a lipophilic ester derivative and an amino acid conjugate) were prepared. Screening of these prodrugs in a rapid whole blood assay revealed that the two ester-based mizoribine prodrugs potently inhibited interleukin 2 secretion. Moreover, these prodrugs were able to prolong graft survival, when evaluated in a mouse model of cardiac allograft transplantation. Strikingly, a combination therapy of these mizoribine prodrugs with tacrolimus had a synergistic in vivo effect.

Recent advance of clinically approved small-molecule drugs for the treatment of myeloid leukemia.

Myeloid leukemia denotes a hematologic malignancy characterized by aberrant proliferation and impaired differentiation of blood progenitor cells within the bone marrow. Despite the availability of several treatment options, the clinical outlook for individuals afflicted with myeloid leukemia continues to be unfavorable, making it a challenging disease to manage. Over the past, substantial endeavors have been dedicated to the identification of novel targets and the advancement of enhanced therapeutic modalities to ameliorate the management of this disease, resulting in the discovery of many clinically approved small-molecule drugs for myeloid leukemia, including histone deacetylase inhibitors, hypomethylating agents, and tyrosine kinase inhibitors. This comprehensive review succinctly presents an up-to-date assessment of the application and synthetic routes of clinically sanctioned small-molecule drugs employed in the treatment of myeloid leukemia. Additionally, it provides a concise exploration of the pertinent challenges and prospects encompassing drug resistance and toxicity. Overall, this review effectively underscores the considerable promise exhibited by clinically endorsed small-molecule drugs in the therapeutic realm of myeloid leukemia, while concurrently shedding light on the prospective avenues that may shape the future landscape of drug development within this domain.

Synthesis and anticancer evaluation of acetylated-lysine conjugated gemcitabine prodrugs.

Gemcitabine is an antimetabolite drug approved for the treatment of various cancers. However, its use is limited due to several issues such as stability, toxicity and drug resistance. Herein, we present the design and synthesis of a series of gemcitabine prodrugs with modifications on the 4-N-amino group by employing an acetylated l- or d-lysine moiety masked by different substitutions. Prodrugs 1-3 and 6-8 showed up to 2.4 times greater anticancer activity than gemcitabine in A549 lung cells, while they exhibited potent activity against BxPC-3 pancreatic cells with IC50 values in the range of 7-40 nM. Moreover, prodrugs 2-3 and 7-8 were found to be less potent against CTSL low expression Caco-2 cells and at least 69-fold less toxic towards human normal HEK-293T cells compared to gemcitabine, leading to improved selectivity and safety profiles. Further stability studies showed that representative prodrugs 2 and 7 exhibited enhanced metabolic stability in human plasma, human liver microsomes and cytidine deaminase. Prodrug 1 can be cleaved by tumor cell-enriched CTSL to release parent drug gemcitabine. Overall, these results demonstrated that acetylated lysine conjugated gemcitabine prodrugs could serve as promising leads for further evaluation as new anticancer drugs.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Characterization of an HNA aptamer suggests a non-canonical G-quadruplex motif.

Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.

Synthesis and anti-SARS-CoV-2 evaluation of lipid prodrugs of ß-D-N4-hydroxycytidine (NHC) and a 3'-fluoro-substituted analogue of NHC.

ß-D-N4-hydroxycytidine (NHC, EIDD-1931) is a nucleoside analogue that exhibits broad spectrum antiviral activity against a variety of RNA viruses. Herein, we report the synthesis of a series of lipid prodrugs of NHC and a novel 3'-fluoro modified NHC analogue, and evaluation of their antiviral activity against five variants of SARS-CoV-2. All lipid prodrugs showed potent antiviral activity against the tested SARS-CoV-2 variants with EC50 values in the range of 0.31-3.51 µM, which were comparable to those of NHC or higher than those of remdesivir and molnupiravir. An increase in the cytostatic activity of the lipid prodrugs was found, but prodrug 2d proved equally selective as molnupinavir. The 3'-F analogue of NHC (6) only displayed minor antiviral activity against the SARS-CoV-2 Omicron variant (EC50 = 29.91 µM), while no activity was found for other variants at the highest concentration tested. The promising antiviral data of the lipid prodrugs of NHC suggest that they deserve further investigation as new anti-SARS-CoV-2 drugs.

Synthesis, antiviral activity, and computational study of ß-d-xylofuranosyl nucleoside phosphonates.

Molecular dynamics (MD) simulations provided insights into the favorable interactions between xylose nucleosides bearing a phosphonate moiety at their 3'-position and specific residues at the active site of the archetypal RNA-dependent RNA-polymerase (RdRp) of Enterovirus 71. Therefore, a series of xylosyl nucleoside phosphonates with adenine, uracil, cytosine, guanosine, and hypoxanthine as nucleobases were synthesized through multistep sequences starting from a single common precursor. Following antiviral activity evaluation, the adenine containing analogue was found to possess good antiviral activity against RNA viruses displaying an EC50 of 12 and 16 µM against measles virus (MeV) and enterovirus-68 (EV-68), respectively, whereas lacking cytotoxicity.

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization.

Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5-anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.

An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.

With the advent of CRISPR-Cas9 gene editing, we now have to hand a simple two-component system amendable to silencing and knock-in editing effectively any gene. Yet we must not forget that the implications of immunotoxicity along with the poor stability and specificity of canonical nucleic acids hold enormous challenges for in vivo applications, especially in gene therapy. Our study endorses the feasibility of the enzymatic approach to incorporate nucleobase modifications into the CRISPR-Cas9 system unveiling the tolerance of Cas9 to N1-methylpseudouridine (m1Ψ)- and emissive thienoguanosine (thG)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.