Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

High-grade B-cell lymphoma (HGBCL), the subtype of non-Hodgkin lymphoma, to be relapsed or refractory in patients after initial therapy or salvage chemotherapy. Dual dysregulation of MYC and BCL2 is one of the important pathogenic mechanisms. Thus, combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine. It has been shown to be a potential therapeutic target for multiple diseases. In this study, the DHODH inhibitor brequinar exhibited growth inhibition, cell cycle blockade, and apoptosis promotion in HGBCL cell lines with MYC and BCL2 rearrangements. The combination of brequinar and BCL2 inhibitors venetoclax had a synergistic inhibitory effect on the survival of DHL cells through different pathways. Venetoclax could upregulate MCL-1 and MYC expression, which has been reported as a resistance mechanism of BCL2 inhibitors. Brequinar downregulated MCL-1 and MYC, which could potentially overcome drug resistance to venetoclax in HGBCL cells. Furthermore, brequinar could downregulate a broad range of genes, including ribosome biosynthesis genes, which might contribute to its anti-tumor effects. In vivo studies demonstrated synergetic tumor growth inhibition in xenograft models with brequinar and venetoclax combination treatment. These results provide preliminary evidence for the rational combination of DHODH and BCL2 blockade in HGBCL with abnormal MYC and BCL2.

Mitochondrial therapy is a promising new strategy that offers the potential to achieve precise disease diagnosis or maximum therapeutic response. However, versatile mitochondrial theranostic platforms that integrate biomarker detection and therapy have rarely been exploited. Here, we report a charge-reversal nanomedicine activated by an acidic microenvironment for mitochondrial microRNA (mitomiR) detection and ion-interference therapy. The transporter liposome (DD-DC) was constructed from a pH-responsive polymer and a positively charged phospholipid, encapsulating NaCl nanoparticles with coloading of the aggregation-induced emission (AIE) fluorogens AIEgen-DNA/G-quadruplexes precursor and brequinar (NAB@DD-DC). The negatively charged nanomedicine ensured good blood stability and high tumor accumulation, while the charge-reversal to positive in response to the acidic pH in the tumor microenvironment (TME) and lysosomes enhanced the uptake by tumor cells and lysosome escape, achieving accumulation in mitochondria. The subsequently released Na+ in mitochondria not only contributed to the formation of mitomiR-494 induced G-quadruplexes for AIE imaging diagnosis but also led to an osmolarity surge that was enhanced by brequinar to achieve effective ion-interference therapy.

Proteins play an important role in biological systems and several proteins are used in diagnosis, therapy, food industry etc. Thus, knowledge about the physical properties of the proteins is of utmost importance, which will aid in understanding their function and subsequent applications. The melting temperature (Tm) of a protein is one of the essential parameters which gives information about the stability of a protein under different conditions. In the present study, we have demonstrated a method for determining the Tm of proteins using the supramolecular interaction between Quinaldine Red (QR) and proteins. Using this method, we have determined the Tm of 5 proteins and compared our results with established protocols. Our results showed good agreement with the other methods and published values. The method developed in this study is inexpensive, quick, and devoid of complex instruments and pre/post-treatment of the samples. In addition, this method can be adopted for high throughput in multi-plate mode. Thus, this study projects a new methodology for Tm determination of various proteins with user friendly operation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and the associated global pandemic resulting in >400 million infections worldwide and several million deaths. The continued evolution of SARS-CoV-2 to potentially evade vaccines and monoclonal antibody (mAb)-based therapies and the limited number of authorized small-molecule antivirals necessitates the need for development of new drug treatments. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

African swine fever virus (ASFV) is a double-stranded DNA virus that causes an acute and hemorrhagic disease in domestic swine, resulting in significant economic losses to the global porcine industry. The lack of vaccines and antiviral drugs highlights the urgent need for antiviral studies against ASFV. Here, we report that brequinar (BQR), which is a specific inhibitor of dihydroorotate dehydrogenase, robustly inhibits ASFV replication in Vero cells, as well as in porcine macrophages. We demonstrate that BQR exerts its antiviral activity in a dose-dependent manner through the depletion of pyrimidine pool. Although BQR does not affect the synthesis of an early viral protein, pI215L, the synthesis of late viral proteins, p17 and p72, is suppressed in the presence of BQR. We also show that BQR is able to induce cellular antiviral response in ASFV-infected macrophages by enhancing the expression of interferon-stimulated genes. Taken together, our study reveals that targeting nucleotide biosynthesis represents a promising strategy for developing antiviral agents against ASFV.

Tuberculosis (TB) has been described as a global health crisis since the second half of the 1990s. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB in humans, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2019, it was estimated that around 10 million individuals were contaminated by this bacillus and about 1.2 million succumbed to the disease. In recent years, our research group has reported the design and synthesis of quinoline derivatives as drug candidates for the treatment of TB. These compounds have demonstrated potent and selective growth inhibition of drug-susceptible and drug-resistant Mtb strains. Herein, a new synthetic approach was established providing efficient and rapid access (15 min) to a series of 4-alkoxy-6-methoxy-2-methylquinolines using ultrasound energy. The new synthetic protocol provides a simple procedure utilizing an open vessel system that affords the target products at satisfactory yields (45-84%) and elevated purities (≥95%). The methodology allows the evaluation of a larger number of molecules in assays against the bacillus, facilitating the determination of the structure-activity relationship with a reduced environmental cost.

In this report, we describe a near-infrared fluorescent probe called quinaldine red (QR) which lights up the β-sheet structure of amyloid fibrils. The photochemical and biophysical properties of QR along with other canonical amyloid probes in the presence of protein fibrils were investigated by using fluorescence spectroscopy, confocal fluorescent microscopy and isothermal titration calorimetry. Moreover, the binding sites and interaction mode between QR and insulin fibrils were calculated based on molecule docking. Among these amyloid probes, QR showed several advantages including strong supramolecular force, near-infrared emission, high sensitivity and resistance to bleaching. A linear response of the fluorescence intensity of QR towards fibril samples in the presence of sera was visualized in the range of 1-30 μM, with the limit of detection (LOD) of 2.31 μM. The recovery and relative standard deviation (RSD) of the proposed method for the determination of protein fibrils was 90.4%-99.2% and 3.05%-3.47%, respectively. Finally, QR can be fluorescently lighted up when meeting the aberrant protein aggregates of pathogenic mice. We recommend QR as a novel and excellent alternative tool for monitoring conformational transition of amyloid proteins.

The chiral structure of antibiotic vancomycin (Van) was exploited as an innovative coordination sphere for the preparation of an IrCp* based hybrid catalysts. We found that Van is able to coordinate iridium (Ir(III)) and the complexation was demonstrated by several analytical techniques such as MALDI-TOF, UV, Circular dichroism (CD), Raman IR, and NMR. The hybrid system so obtained was employed in the Asymmetric Transfer Hydrogenation (ATH) of cyclic imines allowing to obtain a valuable 61% e.e. (R) in the asymmetric reduction of quinaldine 2. The catalytic system exhibited a saturation kinetics with a calculated efficiency of Kcat/KM = 0.688 h-1mM-1.

We report the synthesis and metabolic and biological evaluation of a series of 17 novel heterocyclic derivatives of isocombretastatin-A4 (iso-CA-4) and their structure-activity relationships. Among these derivatives, the most active compound, 4f, inhibited the growth of a panel of seven cancer cell lines with an IC50 in the low nanomolar range. In addition, 4f showed interesting activity against CA-4-resistant colon-carcinoma cells and multidrug-resistant leukemia cells. It also induced G2/M cell-cycle arrest. Structural data indicated binding of 4f to the colchicine site of tubulin, likely preventing the curved-to-straight tubulin structural changes that occur during microtubule assembly. Also, 4f disrupted the blood-vessel-like assembly formed by human umbilical-vein endothelial cells in vitro, suggesting its function as a vascular-disrupting agent. An in vitro metabolism study of 4f showed its high human-microsomal stability in comparison with that of iso-CA-4. The physicochemical properties of 4f may be conducive to CNS permeability, suggesting that this compound may be a possible candidate for the treatment of glioblastoma.