Good Cellular Activity Research Articles

Enhanced surface antimicrobial, biocompatible and mechanical properties of 3D printed titanium alloys by electrophoretic deposition of chitosan/ZnO

In this study, to address the susceptibility of 3D-printed titanium implants to bacterial infection, we propose to form a chitosan/ZnO composite coating by electrophoretic deposition to enhance its antimicrobial, biocompatible, and mechanical properties. The surface morphology of the composite coating is relatively flat, showing good hydrophilicity and coating adhesion, and the corrosion current density is significantly lower than that of the untreated titanium alloy. According to the results of the study, the composite coatings containing more than 0.1g of ZnO (Z2, Z3, Z4 groups) showed excellent antibacterial effects against Staphylococcus aureus and Escherichia coli, with antibacterial rates of more than 95%, and the medium-concentration ZnO coatings (Z2 group) showed good cellular activity, with cell viability rates of more than 80%. In contrast, the high-concentration ZnO coatings (Z3, Z4 groups) showed a certain degree of cytotoxicity. The inherent film-forming property of the composite coating enabled the cells to adhere well to the coating surface. It was found through SBF body fluid immersion that Zn²⁺ can increase the rate of hydroxyapatite precipitation and enhance bioactivity. These results emphasize the importance of precise control of the ZnO content in the improved antimicrobial and biocompatible chitosan-ZnO composite coatings to ensure excellent antimicrobial properties and necessary biocompatibility.

Customized 3D‐printed heterogeneous porous titanium scaffolds for bone tissue engineering

AbstractBone defect is a common clinical disease. Due to the uncertainty of trauma or infection areas, customized size features are often required for bone substitutes. By inspiration of the natural bone structure, this study designs porous scaffolds with a biomimetic design perspective by using different inner and outer pore units. The outer pore units adopt body‐centered cubic (BCC) structure to simulate the weight‐bearing function of human cortical bone, while inner pore units using I‐Wrapped Package structure, a kind of three periods minimum surface, to obtain a good permeability and simulates the inner layer of cancellous bone. To further regulate the overall modulus of the scaffold within the range of natural bone modulus in the human body, the scaffold was designed to axial gradient structure. Compression experiments were conducted, and the results indicated that when the volume fraction linearly increased from 20% to 50%, the Young's modulus was close to the cortical bone modulus in the human body. In vitro cell experiments further proved that osteoblasts have good cellular activity and spreading morphology on the surface of this scaffold. The customized 3D‐printed heterogeneous porous titanium scaffold has great application potential in bone tissue engineering.

Development of a Biodegradable, Cytocompatible, Antibacterial, and Biofilm-Controlling Chitosan Sulfobetaine Derivative Film as a Biological Material

The purpose of this research was to develop a chitosan sulfobetaine (CS-SNCC) film via the solution-casting method as a biodegradable antibacterial material for biomedical applications. Chitosan and monochloro-triazine sulfobetaine were used as the raw materials for CS-SNCC preparation, and Fourier-transform infrared (FTIR), ultraviolet–visible (UV-Vis), energy-dispersive X-ray (EDX), and X-ray photoelectron spectroscopy (XPS) spectra were used to characterize and analyze the structure of the synthesized CS-SNCC. Furthermore, the swelling property, thermal stability, biodegradability, cytocompatibility, and antibacterial properties of the CS-SNCC film were comprehensively investigated and compared with those of the chitosan film. The results for the film’s enzymatic biodegradation behavior show that the CS-SNCC film undergoes a weight loss of 45.54% after 21 days of incubation. In addition, the CS-SNCC film effectively resists bacterial adhesion, prevents the formation of bacteria biofilms, and exhibits high antibacterial activity, with inactivation rates of 93.43% for Escherichia coli and 91.00% for Staphylococcus aureus. Moreover, the CS-SNCC film shows good cellular activity and cytocompatibility according to the cytotoxicity results. Therefore, the prepared biodegradable, cytocompatible, antibacterial, and biofilm-controlling CS-SNCC film has potential for biomedical applications.

Exploring the novel antioxidant peptides in low-salt dry-cured ham: Preparation, purification, identification and molecular docking

Dry-cured ham is important source of bioactive peptides. In this study, the antioxidant activities of peptides and components from low and fully salted dry-cured hams were compared by peptidomics. And novel antioxidant peptides were identified and characterized. The results showed that the peptides (<3 KDa) extracted from low-salt dry-cured ham had higher antioxidant activity. Therefore, the antioxidant peptides in low-salt dry-cured ham were further characterized and the mechanism of their antioxidant activity was investigated. From the five candidate peptides selected, we found DWPDARGIWHND (DD12) to be highly stable, non-sensitizing, and non-toxic with the highest free radical scavenging activity. Molecular docking predicted that DD12 interacted with Keap1 through hydrogen-bond formation and hydrophobic interactions, suggesting that DD12 had good cellular antioxidant activity. DD12 peptide can bind to DPPH• and ABTS•+, resulting in strong free radical scavenging activity. Our findings support the development and application of natural antioxidant peptides in dry-cured ham.

Bioactive compound composition and cellular antioxidant activity of fig (Ficus carica L.).

Fig (Ficus carica L.) fruit is consumed worldwide as a functional food. It contains phytochemicals that have been related to health benefits. However, the characteristic chemicals remain unclear. In this work, phytochemicals were prepared from figus by ultrasound-assisted extraction under optimized conditions. The chemical composition of fig fruit and leaves was characterized by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). One hundred and fifty-seven compounds were identified, including 58 flavonoids, 29 coumarins, 19 acids, 15 terpenoids, 11 alkaloids, and 25 other compounds. The mass spectrum (MS) fragmentation pathways of representative chemicals were elucidated. Flavonoid glycosides and prenylated flavonoids were mainly present in fig fruit, whereas coumarins were abundant in leaves. Both fig fruit and leaf extracts showed good cellular antioxidant activity. The full phytochemical profile of fig was revealed by UPLC-MS/MS. Prenylated flavonoids and prenylated coumarins were the characteristic phytochemicals. These data provided useful information for the extensive utilization of fig fruit in functional food. © 2023 Society of Chemical Industry.

Nondestructive separation/enrichment and rolling circle amplification-powered sensitive SERS enumeration of circulating tumor cells via aptamer recognition

Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.

Permethrin as a Potential Furin Inhibitor through a Novel Non-Competitive Allosteric Inhibition

Furin is a potential target protein associated with numerous diseases; especially closely related to tumors and multiple viral infections including SARS-CoV-2. Most of the existing efficient furin inhibitors adopt a substrate analogous structure, and other types of small molecule inhibitors need to be discovered urgently. In this study, a high-throughput screening combining virtual and physical screening of natural product libraries was performed, coupled with experimental validation and preliminary mechanistic assays at the molecular level, cellular level, and molecular simulation. A novel furin inhibitor, permethrin, which is a derivative from pyrethrin I generated by Pyrethrum cinerariifolium Trev. was identified, and this study confirmed that it binds to a novel allosteric pocket of furin through non-competitive inhibition. It exhibits a very favorable protease-selective inhibition and good cellular activity and specificity. In summary, permethrin shows a new parent nucleus with a new mode of inhibition. It could be used as a highly promising lead compound against furin for targeting related tumors and various resistant viral infections, including SARS-CoV-2.

Novel Antioxidant Peptides from Pearl Shell Meat Hydrolysate and Their Antioxidant Activity Mechanism

Free radicals are associated with aging and many diseases. Antioxidant peptides with good antioxidant activity and absorbability are one of the hotspots in antioxidant researches. In our study, pearl shell (Pinctada martensii) meat hydrolysate was purified, and after identification by proteomics, six novel antioxidant peptides SPSSS, SGTAV, TGVAS, GGSIT, NSVAA, and GGSLT were screened by bioinformatics analysis. The antioxidant peptides exhibited good cellular antioxidant activity (CAA) and the CAA of SGTAV (EC50: 0.009 mg/mL) and SPSSS (EC50: 0.027 mg/mL) were better than that of positive control GSH (EC50: 0.030 mg/mL). In the AAPH-induced oxidative damage models, the antioxidant peptides significantly increased the viability of HepG2 cells, and the cell viability of SGTAV, SPSSS, and NAVAA were significantly restored from 79.41% to 107.43% and from 101.09% and 100.09%, respectively. In terms of antioxidant mechanism by molecular docking, SGTAV, SPSSS, and NAVAA could tightly bind to free radicals (DPPH and ABTS), antioxidant enzymes (CAT and SOD), and antioxidant channel protein (Keap1), suggesting that the antioxidant peptides had multiple antioxidant activities and had structure-activity linkages. This study suggests that the antioxidant peptides above are expected to become new natural materials for functional food industries, which contribute to the high-value applications of pearl shell meat.

Screening of Rosemary Essential Oils with Different Phytochemicals for Antioxidant Capacity, Keratinocyte Cytotoxicity, and Anti-Proliferative Activity.

Nowadays, the demand for rosemary essential oils (REOs) in the cosmetic, food, and pharmaceutical industries is increasing, and the abundant germplasm resources of rosemary provide more possibilities for functional applications. The REOs from six cultivars were selected to evaluate and compare their bioactivities. REOs have good cellular antioxidant activity in scavenging reactive oxygen species, and the technology for order preference by similarity to an ideal solution (TOPSIS)-random forest multivariate model indicated that 'Dutch Mill' REO has the best antioxidant activity, which is closely related to its verbenone content. In addition, α-pinene-dominant REOs are more toxic to human keratinocytes, which is closely related to the content of α-pinene, as revealed by multivariate analyses. Moreover, anti-proliferative assays on six cancer cell lines showed that all REOs have a higher anti-proliferative ability against human pancreatic cancer cell line SW1990 and gastric epithelial cell line NCI-N87. Among them, 'Miss Jessopp's Upright' and 'Blue Lagoon' REOs exhibit more prominent anti-proliferative activity. Our study provides a reference value for exploring the application potential of different REOs by evaluating their differences in chemical composition and bioactivity.

Differences between water-soluble and water-insoluble melanin derived from Inonotus hispidus mushroom.

Melanin is a natural pigment with a high content and a complex polymer structure. In this study, both water-soluble and water-insoluble melanin were obtained from the fruiting bodies of the mushroom Inonotus hispidus, and scanning electron microscopy (SEM), ultraviolet (UV) absorption, Fourier transform infrared (FTIR) spectroscopy, elemental analysis, nuclear magnetic resonance (NMR) spectroscopy, pyrolysis gas chromatography mass spectroscopy (Py-GCMS), and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS/MS) were used to analyze the water-soluble and water-insoluble I. hispidus fungal melanin (IHFM). Differences in the chemical composition and structure of I. hispidus fungal melanin (IHFM) were found. Studies showed that both have a maximum absorbance at 226nm, and the maximum mass-to-charge ratios are [M+H] + m/z=272.2434 and 272.0308, respectively. Elemental analysis revealed that they both contain C, H, N, O, and do not contain sulfur, which is only found in eumelanin. The differences between water-soluble and water-insoluble IHFM may relate to the following factors: (i) the microscopic particles of water-soluble IHFM are relatively small, the arrangement is relatively neat, and they contain a large quantity of benzene, phenol, and indole compounds; and (ii) the microscopic particles of water-insoluble IHFM are larger and the arrangement is irregular. The types and quantities of benzene, phenol, and indole compounds are relatively small, rich in a variety of sugar monomers, and may be insoluble in water due to the presence of a greater number of aliphatic groups. Studies have found that water-soluble IHFM has good cellular antioxidant activity, can reduce oxidative damage by hydrogen peroxide (H2O2) on LO2 cells effectively and can significantly reduce the level of intracellular reactive oxygen species (ROS) to protect the liver. These results are beneficial in order to develop applications of melanin in the food industry and in other fields.

Surface coating prolongs the degradation and maintains the mechanical strength of surgical suture in vivo

Biodegradable and absorbable sutures have been widely used in surgical procedures. However, for the repair of ligament and tendon injures, the biodegradable suture cannot provide sufficient mechanical support to close the wound for a long period of time which is important to completely heal the tissue. Herein, we develop a simple method that makes a surface coating to prolong the degradation of the suture in vivo. Polylactic acid (PLLA) and Polycaprolactone (PCL) were successfully coated to a commercial degradable polydioxanone (PDO) suture in this study, which was confirmed by Fourier transform infrared spectra (FTIR). Scanning electron microscopy (SEM) was used to observe the smooth surface of the coated sutures. Moreover, live/dead assay of human fibroblasts after co-culturing with the modified/unmodified sutures showed fairly good cellular activity. In vivo study demonstrates the degradation properties of sutures were significantly changed after the surface coating. The raw suture exhibited the fastest degradation in 12 weeks, showing significantly decline in mechanical strength. Interestingly, the PCL-coated suture was able to maintain more than 20% of its original tensile strength after 12 weeks' implantation. In addition, in vivo results of PCL-coated sutures also showed less inflammatory cell infiltration and less surface inflammation. These findings indicate the one step suture-coating method could be feasibly for the development of clinical equipment.

Discovery of IDO1 inhibitors containing a decahydroquinoline, decahydro-1,6-naphthyridine, or octahydro-1H-pyrrolo[3,2-c]pyridine scaffold

A series of IDO1 inhibitors containing a decahydroquinoline, decahydro-1,6-naphthyridine, or octahydro-1H-pyrrolo[3,2-c]pyridine scaffold were identified with good cellular and human whole blood activity against IDO1. These inhibitors contain multiple chiral centers and all diastereomers were separated. The absolute stereochemistry of each isomers were not determined. Compounds 15 and 27 stood out as leads due to their good cellular as well as human whole blood IDO1 inhibition activity, low unbound clearance, and reasonable mean residence time in rat cassette PK studies.

3D Printing of Bone-Mimetic Scaffold Composed of Gelatin/β-Tri-Calcium Phosphate for Bone Tissue Engineering.

3D printed scaffolds composed of gelatin and β-tri-calcium phosphate (β-TCP) as a biomimetic bone material are fabricated, thereby providing an environment appropriate for bone regeneration. The Ca2+ in β-TCP and COO- in gelatin form a stable electrostatic interaction, and the composite scaffold shows suitable rheological properties for bioprinting. The gelatin/β-TCP scaffold is crosslinked with glutaraldehyde vapor and unreacted aldehyde groups which can cause toxicity to cells is removed by a glycine washing. The stable binding of the hydrogel is revealed as a result of FTIR and degradation rate. It is confirmed that the composite scaffold has compressive strength similar to that of cancellous bone and 60wt% β-TCP groups containing 40wt% gelatin have good cellular activity with preosteoblasts. Also, in the animal experiments, the gelatin/β-TCP scaffold confirms to induce bone formation without any inflammatory responses. This study suggests that these fabricated scaffolds can serve as a potential bone substitute for bone regeneration.

Discovery of CA-4948, an Orally Bioavailable IRAK4 Inhibitor for Treatment of Hematologic Malignancies.

Small molecule potent IRAK4 inhibitors from a novel bicyclic heterocycle class were designed and synthesized based on hits identified from Aurigene's compound library. The advanced lead compound, CA-4948, demonstrated good cellular activity in ABC DLBCL and AML cell lines. Inhibition of TLR signaling leading to decreased IL-6 levels was also observed in whole blood assays. CA-4948 demonstrated moderate to high selectivity in a panel of 329 kinases as well as exhibited desirable ADME and PK profiles including good oral bioavailability in mice, rat, and dog and showed >90% tumor growth inhibition in relevant tumor models with excellent correlation with in vivo PD modulation. CA-4948 was well tolerated in toxicity studies in both mouse and dog at efficacious exposure. The overall profile of CA-4948 prompted us to select it as a clinical candidate for evaluation in patients with relapsed or refractory hematologic malignancies including non-Hodgkin lymphoma and acute myeloid leukemia.

Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A.

Botulinum neurotoxins have remarkable persistence (∼weeks to months in cells), outlasting the small-molecule inhibitors designed to target them. To address this disconnect, inhibitors bearing two pharmacophores-a zinc binding group and a Cys-reactive warhead-were designed to leverage both affinity and reactivity. A series of first-generation bifunctional inhibitors was achieved through structure-based inhibitor design. Through X-ray crystallography, engagement of both the catalytic Zn2+ and Cys165 was confirmed. A second-generation series improved on affinity by incorporating known reversible inhibitor pharmacophores; the mechanism was confirmed by exhaustive dialysis, mass spectrometry, and in vitro evaluation against the C165S mutant. Finally, a third-generation inhibitor was shown to have good cellular activity and low toxicity. In addition to our findings, an alternative method of modeling time-dependent inhibition that simplifies assay setup and allows comparison of inhibition models is discussed.

Selective targeting of the αC and DFG-out pocket in p38 MAPK

The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.

Design, synthesis, and biological evaluation of tetrahydroquinolin derivatives as potent inhibitors of CBP bromodomain

CREB-binding protein (CBP) is a large multi-domain protein containing a HAT domain catalyzing transacetylation and a bromodomain responsible for acetylated lysine recognition. CBPs could act as transcription co-activators to regulate gene expression and have been shown to play a significant role in the development and progression of many cancers. Herein, through in silico screening two hit compounds with tetrahydroquinolin methyl carbamate scaffold were discovered, among which DC-CPin7 showed an in vitro inhibitory activity with the TR-FRET IC50 value of 2.5 ± 0.3 μM. We obtained a high-resolution co-crystal structure of the CBP bromodomain in complex with DC-CPin7 to guide following structure-based rational drug design, which yielded over ten DC-CPin7 derivatives with much higher potency, among which DC-CPin711 showed approximately 40-fold potency compared with hit compound DC-CPin7 with an in vitro TR-FRET IC50 value of 63.3 ± 4.0 nM. Notably, DC-CPin711 showed over 150-fold selectivity against BRD4 bromodomains. Moreover, DC-CPin711 showed micromolar level of anti-leukemia proliferation through G1 phase cell cycle arrest and cell apoptosis. In summary, through a combination of computational and crystal-based structure optimization, DC-CPin711 showed potent in vitro inhibitory activities to CBP bromodomain with a decent selectivity towards BRD4 bromodomains and good cellular activity to leukemia cells, which could further be applied to related biological and translational studies as well as serve as a lead compound for future development of potent and selective CBP bromodomain inhibitors.

Polymer Membranes Sonocoated and Electrosprayed with Nano-Hydroxyapatite for Periodontal Tissues Regeneration.

Diseases of periodontal tissues are a considerable clinical problem, connected with inflammatory processes and bone loss. The healing process often requires reconstruction of lost bone in the periodontal area. For that purpose, various membranes are used to prevent ingrowth of epithelium in the tissue defect and enhance bone regeneration. Currently-used membranes are mainly non-resorbable or are derived from animal tissues. Thus, there is an urgent need for non-animal-derived bioresorbable membranes with tuned resorption rates and porosity optimized for the circulation of body nutrients. We demonstrate membranes produced by the electrospinning of biodegradable polymers (PDLLA/PLGA) coated with nanohydroxyapatite (nHA). The nHA coating was made using two methods: sonocoating and electrospraying of nHA suspensions. In a simulated degradation study, for electrosprayed membranes, short-term calcium release was observed, followed by hydrolytic degradation. Sonocoating produced a well-adhering nHA layer with full coverage of the fibers. The layer slowed the polymer degradation and increased the membrane wettability. Due to gradual release of calcium ions the degradation-associated acidity of the polymer was neutralized. The sonocoated membranes exhibited good cellular metabolic activity responses against MG-63 and BJ cells. The collected results suggest their potential use in Guided Tissue Regeneration (GTR) and Guided Bone Regeneration (GBR) periodontal procedures.

Additively manufactured biodegradable porous zinc

Additively manufacturing (AM) opens up the possibility for biodegradable metals to possess uniquely combined characteristics that are desired for bone substitution, including bone-mimicking mechanical properties, topologically ordered porous structure, pore interconnectivity and biodegradability. Zinc is considered to be one of the promising biomaterials with respect to biodegradation rate and biocompatibility. However, no information regarding the biodegradability and biocompatibility of topologically ordered AM porous zinc is yet available. Here, we applied powder bed fusion to fabricate porous zinc with a topologically ordered diamond structure. An integrative study was conducted on the static and dynamic biodegradation behavior (in vitro, up to 4 weeks), evolution of mechanical properties with increasing immersion time, electrochemical performance, and biocompatibility of the AM porous zinc. The specimens lost 7.8% of their weight after 4 weeks of dynamic immersion in a revised simulated body fluid. The mechanisms of biodegradation were site-dependent and differed from the top of the specimens to the bottom. During the whole in vitro immersion time of 4 weeks, the elastic modulus values of the AM porous zinc (E = 700–1000 MPa) even increased and remained within the scope of those of cancellous bone. Indirect cytotoxicity revealed good cellular activity up to 72 h according to ISO 10,993–5 and -12. Live-dead staining confirmed good viability of MG-63 cells cultured on the surface of the AM porous zinc. These important findings could open up unprecedented opportunities for the development of multifunctional bone substituting materials that will enable reconstruction and regeneration of critical-size load-bearing bone defects. Statement of significanceNo information regarding the biodegradability and biocompatibility of topologically ordered AM porous zinc is available. We applied selective laser melting to fabricate topologically ordered porous zinc and conducted a comprehensive study on the biodegradation behavior, electrochemical performance, time-dependent mechanical properties, and biocompatibility of the scaffolds. The specimens lost 7.8% of their weight after4 weeks dynamic biodegradation while their mechanical properties surprisingly increased after 4 weeks. Indirect cytotoxicity revealed good cellular activity up to 72 h. Intimate contact between MG-63 cells and the scaffolds was also observed. These important findings could open up unprecedented opportunities for the development of multifunctional bone substituting materials that mimic bone properties and enable full regeneration of critical-size load-bearing bony defects.

1973P - Targeting ARG2 as a novel therapeutic approach for cancer

BackgroundImmunotherapies are considered the most promising therapeutic approach for cancer and the immunosuppressive activity of ARG1 has been recognized as an important mechanism of the tumor immune evasion. This prompted the development of arginase inhibitors, which in preclinical models, enhanced antitumor immunity as a monotherapy and in combination with other immune checkpoint inhibitors. On the other hand, ARG2, but not ARG1, is highly expressed in neoplastic cells in many tumors and its expression is correlated with malignant phenotype. Preclinical studies confirmed that ARG2 promotes the proliferation of cancer cells and the growth of tumor xenografts independently of its immunosuppressive activity. Generation of polyamines to facilitate the growth of hypoxic and nutrient-deprived tumors, as well as specific metabolic adaptations including increased reliance on protein catabolism are the major mechanisms underlying the tumorigenic activity of ARG2. Hence, the tumor cell intrinsic activity of ARG2 represents an attractive intracellular target for novel therapies with arginase inhibitors. MethodsThe compound activity was determined using human ARG1 and ARG2, and in CHO-K cells expressing ARG1 and ARG2. ARG1 and ARG2 expression in cancer cell lines and dissected tumors was assessed by WB and qPCR. CellTiter-Glo was used to assess the antiproliferative activity of the compound. In vivo antitumor activity was evaluated in murine CT26 (syngeneic) and human K562 (xenograft) subcutaneous mouse models. ResultsThe expression of the endogenous ARG2 was confirmed in multiple human cancer cell lines and xenografts. We developed a highly potent dual ARG1 and ARG2 inhibitor, OATD-02, with a good cellular activity. We demonstrated that OATD-02 inhibited proliferation of multiple human cancer cell lines expressing ARG2 and suppressed the growth of human xenografts. OATD-02 also strongly inhibited the growth of the syngeneic CT26 tumors. ConclusionsOATD-02, a potent ARG1 and ARG2 inhibitor, exerts its antitumor efficacy not only by the reactivation of the immune response but also by directly suppressing the ARG2-dependent proliferation of cancerous cells. Thus, OATD-02 is a very promising compound for the treatment of hypoxic tumors which are particularly resistant to therapies. Legal entity responsible for the studyOncoArendi Therapeutics SA. FundingNational Centre for Research and Development in Poland. DisclosureM.M. Grzybowski: Full / Part-time employment: OncoArendi Therapeutics SA. J. Pęczkowicz-Szyszka: Full / Part-time employment: OncoArendi Therapeutics SA. P. Wolska: Full / Part-time employment: OncoArendi Therapeutics SA. P.S. Stańczak: Full / Part-time employment: OncoArendi Therapeutics SA. M. Welzer: Full / Part-time employment: OncoArendi Therapeutics SA. E. Nikolaev: Full / Part-time employment: OncoArendi Therapeutics SA. A.M. Siwińska: Full / Part-time employment: OncoArendi Therapeutics SA. R. Błaszczyk: Full / Part-time employment: OncoArendi Therapeutics SA. B. Borek: Full / Part-time employment: OncoArendi Therapeutics SA. M. Dzięgielewski: Full / Part-time employment: OncoArendi Therapeutics SA. A. Gzik: Full / Part-time employment: OncoArendi Therapeutics SA. J. Nowicka: Full / Part-time employment: OncoArendi Therapeutics SA. J. Brzezińska: Full / Part-time employment: OncoArendi Therapeutics SA. K. Jędrzejczak: Full / Part-time employment: OncoArendi Therapeutics SA. J. Chrzanowski: Full / Part-time employment: OncoArendi Therapeutics SA. A. Gołębiowski: Leadership role, Full / Part-time employment: OncoArendi Therapeutics SA. J. Olczak: Leadership role, Full / Part-time employment: OncoArendi Therapeutics SA. P. Dobrzański: Leadership role, Full / Part-time employment: OncoArendi Therapeutics SA. All other authors have declared no conflicts of interest.