Human Ca2 Research Articles

Phosphorylation strongly affects the inhibition of human carbonic anhydrase I CO2 hydration activity

Human carbonic anhydrases (hCAs) have essential roles in respiration, acid-base balance, and fluid secretion, with implications in diseases such as glaucoma, epilepsy, obesity, and cancer. Of the fifteen known hCAs, human CA I (hCA I) is particularly abundant in erythrocytes, playing a critical role in CO2 transport. Despite extensive research on hCA I, the impact of post-translational modifications (PTMs), particularly phosphorylation, on its catalytic activity and inhibitor binding remains poorly understood. Although multiple phosphorylation sites have been identified in hCA I in vivo through high-throughput proteomics studies including at the highly conserved Ser51 residue, the functional consequences of these modifications are not well characterized. We investigated the effects of a phosphomimetic mutation at Ser51 on hCA I, examining its catalytic efficiency and susceptibility to inhibition by sulfonamides and anions. Using a recombinant expression system and a stopped-flow kinetic assay, we characterized the CO2 hydration activity and inhibition profiles of S51E hCA I compared to the wild type enzyme. Our results demonstrate that the S51E mutation increases the catalytic turnover rate (kcat) from 2.0 × 105 s−1 to 2.6 × 105 s−1 but significantly decreases substrate affinity, raising the Michaelis constant (KM) from 4.0 mM to 13.9 mM, reducing overall catalytic efficiency by over 50 %. Inhibition studies with a panel of 41 sulfonamides revealed that the S51E mutation dramatically alters inhibitor sensitivity, particularly for the most effective inhibitors. For example, 15 of the 16 most effective sulfonamide inhibitors for hCA I (with KIs <350 nM) were an average of over 35-fold less effective in inhibiting S51E hCA I than the wild type. The KI of the anticonvulsant zonisamide increased from 31 nM for the wild type hCA I to 4.0 μM. The inhibition profile with a panel of 37 small anions further indicated that the S51E mutant exhibited significantly reduced susceptibility to inhibition by 24 out of 37 tested anions, with some KI values increasing by up to 11,000-fold for inhibitors like hydrogen sulfide. This study underscores the significant impact that phosphorylation may have on hCA I function and inhibition. By characterizing the effects of phosphorylation on the CO2 hydration activity and inhibitor sensitivity of hCA I, these findings represent early steps in developing more selective proteoform-specific inhibitors, which could lead to more effective treatments for diseases involving carbonic anhydrases.

Abstract Mo059: Leucine-Rich Repeat-Containing Protein 10 Modulates Human Cardiac L-Type Ca 2+ Channels but not T-Type Ca 2+ Channels

Background: Leucine-rich repeat-containing protein 10 (LRRC10) is a novel regulator of the cardiac L-type Ca 2+ channel/current (LTCC/I Ca,L ). LRRC10 is crucial for cardiac regeneration in multiple species, and LRRC10 variants have been associated with dilated cardiomyopathy in humans. LRRC10 increases I Ca,L in HEK-293 cells expressing rabbit Ca V 1.2 subunit, and knockout of LRRC10 in zebrafish and mouse cardiomyocytes (CM) reduces I Ca,L . However, LRRC10-mediated regulation of human cardiac LTCC has not been demonstrated, and the mechanism by which LRRC10 modulates LTCC is not known. Moreover, the effect of LRRC10 on the T-type Ca 2+ channel/current (TTCC/I Ca,T ), which has been implicated in CM cell cycle regulation, has never been tested. Methods: LRRC10 knockout ( LRRC10 –/– ) human induced-pluripotent stem cells (hiPSC) were generated from wild-type (WT) DF19-9-11T hiPSCs using CRISPR-Cas9 techniques. LRRC10 –/– and WT hiPSCs were differentiated to CMs using the standard GiWi protocol. Whole-cell I Ca,L and I Ca,T were examined in LRRC10 –/– and WT hiPSC-CMs. Whole-cell I Ca,L was also assessed in HEK-293 cells expressing human cardiac LTCC subunits (Ca V 1.2, β 2 , and α 2 δ) with or without human LRRC10. To identify the LRRC10-interacting site of Ca V 1.2, GST-pulldown assays were performed using purified GST-fusion constructs of the intracellular domains of rabbit Ca V 1.2 and lysates from HEK-293 cells expressing human LRRC10. Ca V protein sequences were analyzed using the Clustal Omega program. Results: Genetic ablation of LRRC10 in hiPSC-CMs decreased peak I Ca,L (WT -29.4±2.2 pA/pF, LRRC10 –/– -15.0±1.8 pA/pF, p&lt;0.01) but did not significantly affect I Ca,T magnitude (WT -3.9±0.5 pA/pF, LRRC10 –/– -4.0±0.7 pA/pF, p&gt;0.05 [NS]). LRRC10 increased peak I Ca,L in HEK-293 cells expressing human LTCC (with LRRC10 -156.0±19.8 pA/pF, without LRRC10 -60.2±12.0 pA/pF, p&lt;0.05). LRRC10 was pulled down by the GST-Ca V 1.2-N-terminus but not by other GST-Ca V 1.2 constructs. Sequence analyses revealed an invariant exon 2-encoded segment of the N-terminus (NT) that was present in both human and rabbit LTCC Ca V 1.2 but not in TTCC Ca V 3.1 and Ca V 3.2. Conclusions: The Ca 2+ influx-potentiating effect of LRRC10 is conserved in human LTCC and is specific to regulate I Ca,L , not I Ca,T . The invariant Ca V 1.2 NT segment is a potential LRRC10-interacting site. The LRRC10 –/– hiPSC line is a promising tool for future investigations into the roles of LRRC10-LTCC interaction in human cardiac biology and disease.

맹자와 장자의 감정 이해

In today's increasingly uncertain society, Zhuangzi is often praised as a great individualist who worships absolute freedom. Some call him an optimistic nihilist. However, if you focus on the Zhuangzi's argument about emotions, you doubt that he is a person who has neglected the feelings of himself and others. Because of this, it is very important to properly grasp the understanding of Zhuangzi’s opiniom towards emotions. As far as emotion is concerned, Mengzi and Zhuangzi are in different positions. Mengzi positively and actively embraced emotions, and furthermore laid the foundations of his studies on emotions. He saw heaven as a nature within man, and that nature manifests itself with emotion. Because of this, he argued that humans should live according to their emotions, and that social problems should also be dealt with based on emotions. On the other hand, Zhuangzi had an indifferent attitude toward emotions. He also saw that human beings can't be without emotions. At the same time he felt that the act of suppressing emotions was not appropriate. However, he insisted that because emotion interferes with mental and physical tranquility, it must be let go constantly. Modern emotional psychology determines that Zhuangzi's attitude toward emotion is unhealthy. Even if emotion makes me tired, I must actively embrace it and examine its cause and meaning. Instead of letting go of my feelings, I have to move in the direction it dictates. Only then will I be able to lead an emotional life with abundant emotions and empathy for others.

Activation Effect of 2-β-hydroxy Manoyl Oxide Isolated from Sideritis perfoliata on Carbonic Anhydrase Isoenzymes I and II

Background: Sideritis species were used for the treatment of mental disorders such as Alzheimer’s and dementia traditionally in Turkey. Several in vivo studies report that the mid-polar extract of Sideritis species can develop the brain functions of mice. 2-β-hydroxy manoyl oxide, isolated from ethyl acetate extract of Sideritis perfoliata, was assayed in vitro and in silico on human erythrocytes CA I and CA II. The compound was found to be an activator on two isoenzymes. It has been reported that activators of carbonic anhydrases may be used as a novel approach to treating disorders such as Alzheimer’s and age-related diseases. This study aimed to investigate the activity effect of 2-β-hydroxy manoyl oxide in vitro and in silico on human erythrocytes CA I and CA II (hCA I and hCA II) and to elucidate its pharmacokinetic and physicochemical characteristics. Methods: The test compound was isolated from ethyl acetate extract of Sideritis perfoliata using chromatographic techniques and identified with spectroscopic evidence. Carbonic anhydrase activities were assayed using CO2 substrates. Docking studies were carried out with Molegro Virtual Docker. The compound underwent ADME-Tox prediction by using AdmetSAR and SwissADME software. Results: 2-β-hydroxy manoyl oxide was found to increase the hCA-l and hCAII activity with AC50 values 9 and 19 μM, respectively. These results were further confirmed in silico molecular modeling. It showed favorable pharmacokinetic and physicochemical characteristics as a new drug candidate. Conclusion: These findings demonstrated that 2-β-hydroxy manoyl oxide activated the hCA-l and hCA II. These results provide a novel and alternative activator for the carbonic anhydrase and confirm the traditional usage of the Sideritis perfoliata.

Compression of Color Image Using Butterworth Low Pass Filtering

The human eye can’t detect the high-frequency components of the image. Therefore, it is unnecessary to carry the high-frequency components as it consumes too much storage space as well as bandwidth. To overcome this problem, filtering of the image is performed which eliminates the high-frequency components from the image. In this article, we propose a novel method of color image filtering using Butterworth Low Pass Filtering (LPF). The selected color image is divided into three planes- Red, Green, and Blue. Butterworth Low Pass Filtering is applied to each plane separately and then they are joined together. The cut-off frequency is varied to improve the quality of the output image. Quality is checked by the PSNR method. The total operation is performed using MATLAB software and it provides a satisfactory result. This system is useful for all types of images. Keywords: Image, Fourier Transform, Low Pass Filtering, Cut-off frequency, PSNR

NPM1c Mutations Are Associated with Reduced Levels of Multiple Ribosome Biogenesis Factors Leading to Therapeutic Vulnerabilities

Objectives: Mutations in the NPM1 gene are found in more than 30% of acute myeloid leukaemia (AML) cases. The mutations disrupt a nucleolar localization signal (NoLS) and create a novel nuclear export signal (NES), leading to cytoplasmic displacement of the protein (NPM1c). NPM1cmutations prime haematopoietic progenitors to leukaemic transformation, but their precise molecular consequences remain elusive. Here, we first examine the effects of isolated NPM1c mutations on mouse haematopoietic cells and explore their impact on their transcriptome, including at single-cell resolution, and their proteome. Then, using several murine and human AML models and datasets we investigate the role of such NPM1c-associated changes in fully transformed AML cells and explore their potential as therapeutic vulnerabilities. Methods: We performed RNA-seq, CITE-seq and TMT global quantitative proteomics using lineage-depleted bone marrow progenitor cells from NPM1 flox-cA/+;Mx1-cre mice ( Vassiliou et al, Nat Genet, 2011). Proteomic changes were contrasted to those identified in a recently published dataset of 44 human AMLs, including 8 NPM1-mutant AMLs. Candidate vulnerabilities were identified using datasets from CRISPR-Cas9 drop-out screens and were validated in NPM1 cA/+-harbouring cell lines and primary mouse NPM1 cA/+;FLT3 ITD/+ AMLs. Results: We found a global increase in the protein abundance of several members of the KPN family in pre-leukaemic Npm1 cA/+ cells, an effect also observed in human NPM1c+ AMLs. Moreover, we observed a decrease in the relative abundance of several ribosome biogenesis-related proteins in pre-leukaemic Npm1 cA/+ cells and confirmed that this effect persists in human NPM1c+ AML patient samples. We go on to show that, when compared to NPM1 +/+ cells, pre-leukaemic NPM1 cA/+cells show high sensitivity to Actinomycin D and other RNA pol I inhibitors, but not to the translation inhibitor cycloheximide. Combination treatment with Actinomycin D and Venetoclax inhibited the growth and colony forming ability of both pre-leukaemic NPM1 cA/+cells and primary mouse Npm1 cA/+;Flt3 ITD/+AMLs. By contrasting our findings with data from CRISPR drop-out screens, including a genome-wide screen in primary mouse Npm1 cA/+;Flt3 ITD/+/Cas9AMLcells, we identified and validated ribosome biogenesis factors, such as Tsr3, whose knock-out selectively affected cell growth, survival and clonogenicity of NPM1 +/+AML cells, therefore representing novel potential therapeutic targets that could be exploited in the clinic. Conclusions: We show that NPM1c mutations are associated with reduced levels in several ribosome biogenesis factors resulting in therapeutic vulnerabilities of NPM1-mutant pre-leukaemic and leukaemic (AML) cells. The development of novel therapeutics to target these factors may help improve the survival of patients with NPM1-mutant AML. Figure Legend: (A) Volcano plot showing protein abundance in mouse NPM1 cA/+vs NPM1 +/+ pre-leukaemic haematopoetic progenitors. (B) Relative abundance of ribosome-related proteins in human NPM1 cA/+vs NPM1 +/+AML samples (dataset from Kramer et al., 2022).

Expanding and protecting human rights from space

The area of surveillance of human activities from above is fraught with concerns and controversy. Such surveillance is clearly being used in ways that invade human lives and often put those lives in danger. At the same time, there is an argument for more oversight, and the need to support more surveillance, in a controlled and monitored way, to expose and render access to sites of criminal activity, and particularly those of the most egregious crimes—slavery, genocide, environmental destruction, and the operation of ‘rebel’ military gangs or national militaries run by rogue states. If nature can be harried, harmed, and turned against human existence, it is also true that humans have now brought increasingly powerful diagnostic techniques to bear so that oceans, land, forests, fields, plains—all ecosystems—can be monitored for health as well as the scars of exploitation—including that of the human within the monitored ecosystem. The field of human rights now stretches far beyond humans to all life and nature—since useful and meaningful human rights can’t truly exist in a physical and natural world that is hostile to homo sapiens. Earth observation can be, and often is, a force for good.

Fatigue Strength of Rotary Bending Copper Materials

Non-ferrous metal is a type of metal that does not contain iron (Fe). Pure non-ferrous metals do not need alloying elements because of their chemical resistance and good electrical conductivity. The non-ferrous metal in question is copper. Almost every human need can’t be separated from the element of copper as a supporting material. In the industrial world, copper has an important role because many factory machinery construction utilizes this material. Copper is a material that conducts heat and electricity quite well and has smooth and soft properties. The durability of copper-based structures can be carried out by fatigue tests (fatigue). This study aims to analyze the fatigue strength of copper material with rotary bending type. The test was carried out on copper material without heat treatment. At each loading, the test was carried out five times on different test specimens (Sp1, Sp2, Sp3, Sp4, and Sp5) with variations in the load determined at 15 kg, 17 kg, and 19 kg. Preparation of test specimens using a lathe and photos of micro and macro structures using a microscope. While the fatigue test was carried out using a rotary bending type fatigue test machine. The results of microstructure observations for all loadings showed that the specimens had soft properties. Meanwhile, the observation of the macro structure shows that the specimen has brittle properties. The results of the fatigue test show that the flexural stresses for each load of 15 kg, 17, kg, and 19 kg are written as 33 kg/mm2, 37 kg/mm2, and 42 kg/mm2, respectively.

Isolation of CA 125 from Breast Tumor Tissue Using Gel Chromatography

Gel filtration chromatography technique was used (Sepharose CL - 68) of CA 125 from malignant breast tumor homogenate and the isolation of (CA 125 I 1251 - anti CA 125 antibody), complex was prepared from crude CA 125 , isolated CA 125 and standard human CA 125. The results revealed the presence of two forms of CA 125 (Agl &amp; AgII) and found to be 517 Kda and 38 Kda respectively; The isolation of CA 125 shov,:ed a purification fold of 17.5 Agl and 15 for AgII.

Forensic Analysis of Faces on Low-Quality Images using Detection and Recognition Methods

Facial recognition is an essential aspect of conducting criminal action investigations. Captured images from the camera or the recording video can reveal the perpetrator's identity if their faces are deliberately or accidentally captured. However, many of these digital imagery results display the results of image quality that is not good when seen by the human eye. Hence, the facial recognition process becomes more complex and takes longer. This research aims to analyze face recognition on a low-quality image with noise, blur and brightness problem to help digital forensic investigator do an investigation in recognizing faces that the human eye can’t do. The Viola-Jones algorithm method has several processes such as the Haar feature, integral image, adaboost, and cascade classifier for detecting a face in an image. Detected face will be passed to the next process for recognition call Fisher’s Linear Discriminant (FLD), Local Binary Pattern’s (LBP) and Principal Component analysis (PCA). The software's facial recognition feature shows one of the images in the database that the program suspects has the same face as the analyzed face image. In conclusion, from the analysis we determined that LBP approach is the best among the other recognition methods for blur and brightness problem, bet found PCA method is the best for recognize face in noise problem. The software's facial recognition feature shows one of the images in the database that the program suspects has the same face as the analyzed face image. The position of the face object in the image, whether or not there is an additional object that was not previously included in the image in the dataset, as well as the brightness level of an image and the color of the face's skin, all affect the accuracy rates.

A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens.

Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity.

Carbonic Anhydrase Inhibitors from Marine Natural Products.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes in organisms in all life kingdoms, being involved in pH regulation, metabolic processes and many other physiological and pathological conditions. CA inhibitors and activators thus possess applications as pharmacological agents in the management of a range of diseases. Marine natural products have allowed the identification of some highly interesting CA inhibitors, among which are sulfonamides, phenols, polyamines, coumarins and several other miscellaneous inhibitors, which are reviewed here. Psammaplin C and some bromophenols were the most investigated classes of such marine-based inhibitors and have been used as lead molecules for developing interesting types of potent and, in some cases, isoform-selective inhibitors, with applications as antitumor agents by inhibiting human CA XII and P-glycoprotein activities. Some phenols have shown interesting bacterial and fungal β-CA inhibitory effects. Marine natural products thus constitute a gold mine for identifying novel CA inhibitors, some of which may lead to the development of novel types of pharmacological agents.

May Sulfonamide Inhibitors of Carbonic Anhydrases from Mammaliicoccus sciuri Prevent Antimicrobial Resistance Due to Gene Transfer to Other Harmful Staphylococci?

Mammaliicoccus sciuri, previously known as Staphylococcus sciuri, is a Gram-positive bacterium involved in gene transfer phenomena that confer resistance to multiple antibiotics. These plasmid-encoded genes can be easily transferred to other pathogenic staphylococci. Because antibiotic resistance is rising, inhibiting M. sciuri proliferation may be a credible strategy for restricting antimicrobial resistance gene transfer to other pathogenic bacteria. Recently, it has been shown that blocking bacterial carbonic anhydrases (CAs, EC 4.2.1.1), metalloenzymes sustaining bacterial metabolic activities, can reduce pathogen survival and fitness. Here, the recombinant M. sciuri γ-CA (MscCAγ) has been cloned and purified, utilizing the DNA recombinant technology. Its kinetic properties for the CO2 hydration reaction, as well as the sulfonamide inhibition profile, were investigated and compared with those reported earlier for MscCAβ (previously described as SauBCA) and the two off-target human CA isoforms (hCA I and hCA II). The recombinant MscCAγ showed significant hydratase activity. Moreover, the MscCAγ sulfonamide inhibitory profile was different from that of MscCAβ, implying that a varied amino acid set typifies the catalytic pocket of the two enzymes. These differences provide additional evidence for the possibility of developing novel CA class-specific inhibitors.

Synthesis and anticancer activity of new benzensulfonamides incorporating s-triazines as cyclic linkers for inhibition of carbonic anhydrase IX

Limited presence of hCA IX in normal physiological tissues and their overexpression only in solid hypoxic tumors made this isoform excellent possible target for developing new anticancer agents. We reported designing and synthesis of two novel series of benzenesulfonamides derivatives as hCA IX inhibitors bearing rigid cyclic linkers (1,3,5-dihydrotriazine in series A and 1,3,5-triazine in series B) in replace of traditional linear linkers. Also, novel cyanoethenyl spacer was assembled next to the 1,3,5-triazine linker in series B. Target compounds of series (A) and (B) were screened against four hCA isoforms. Human CA IX efficiently inhibited in series (A) by compound 5a (KI = 134.8 nM). Meanwhile, in series (B) the most active inhibitor was 12i (KI = 38.8 nM). US-NCI protocol was followed to evaluate the anticancer activity of target compounds against panel of sixty cancer cell lines. Compound 12d, exposed the best activity towards breast cancer (MDA-MB-468) with GI% = 62%. The most active analogues, 12d and 12i were further screened for in vitro cytotoxic activity under hypoxic condition against breast cancer (MDA-MB-468) (IC50 = 3.99 ± 0.21 and 1.48 ± 0.08 µM, respectively) and leukemia (CCRF-CM) cell line (IC50 = 4.51 ± 0.24 and 9.83 ± 0.52 µM, respectively). In addition, 12d arrested breast cancer MDA-MB-468 cell cycle in G0-G1 and S phases and induced its apoptosis which indicated by increasing the level of cleaved caspases 3 and 9. Molecular docking was performed for selected analogues to understand their biological alterations. This study revealed that insertion of 1,3,5-triazines as cyclic linkers enhanced the significant anticancer and hCA IX inhibition activity of benzenesulfonamides.

Development of active laser reflector for alignment and monitoring of large-scale scientific devices

As large-scale scientific devices like particle accelerators and tokamaks become bigger and more complex, efficient measurements are needed for these devices. At present, when laser trackers are used to measure these devices, manually rotating reflectors is needed in many circumstances, but it's inefficient and it's inappropriate to be used in the areas where human can't approach. To solve the problem, we designed an active laser reflector, which can rotate to a target angle position controlled by a host computer software. We designed a fixture to embed and clamp a spherically mounted retro-reflector (SMR). And two custom-designed motors are used to drive the SMR to rotate horizontally and vertically respectively. The shaft of the motor has a flange to match another flange on the load, which can reduce the coaxiality error between them. The motor control system consists of two motor controllers and a host computer software, and they communicate through WIFI. We made a prototype of the active laser reflector based on the design scheme and measured the offset of the SMR center, which is around ±0.23 mm. The active laser reflector we designed can increase the measuring and monitoring efficiency for large-scale scientific devices.

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.

Novel Insights on Human Carbonic Anhydrase Inhibitors Based on Coumalic Acid: Design, Synthesis, Molecular Modeling Investigation, and Biological Studies.

Human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms IX and XII are overexpressed in solid hypoxic tumors, and they are considered as prognostic tools and therapeutic targets for cancer. Based on a molecular simplification of the well-known coumarin scaffold, we developed a new series of derivatives of the pyran-2-one core. The new compounds are endowed with potent and selective inhibitory activity against the tumor-related hCA isoforms IX and XII, in the low nanomolar range, whereas they are inactive against the two cytosolic off-targets hCA I and II. The compounds exhibiting the best hCA inhibition were further investigated against the breast adenocarcinoma cell line (MCF7) in hypoxic conditions, evaluating their ability to eventually synergize with doxorubicin. The compounds’ biocompatibility on healthy cells was also tested and confirmed on Human Gingival Fibroblasts (HGFs). Furthermore, the possible binding mode of all compounds to the active site of the tumor-associated human CA IX was investigated by computational techniques which predicted the binding conformations and the persistency of binding poses within the active site of the enzyme, furnishing relevant data for the design of tight binding inhibitors.

Design, synthesis, SAR, and biological evaluation of saccharin-based hybrids as carbonic anhydrase inhibitors.

Saccharin is a cyclic secondary sulfonamide, which is a selective inhibitor of the tumor-associated carbonic anhydrase (CA; EC 4.2.1.1) enzymes CA IX and CA XII compared to many primary sulfonamides. In this study, new saccharin-1,2,3-triazole and saccharin-1,2,4-oxadiazole hybrids were synthesized. All the newly synthesized molecules were screened for their CA-inhibitory activity against four important human CA (hCA) isoforms: hCA I, hCA II, hCA IX, and hCA XII. Compounds 8a and 8f emerged as potent hCA II inhibitors (Ki = 3 µM). Compounds 6d, 6e and 7a, 7b were highly selective against hCA IX (6d, 6e) and hCA II (7a, 7b), with moderate inhibitory activity. The activity of these compounds was further confirmed by performing in silico docking studies against hCA II and hCA IX.

SAR-based approach to explore in silico ferrocene analogues as the potential inhibitors of major viral proteins of SARS-CoV-2 virus and human Ca2+-channel blocker

Amid the pandemic COVID-19, there is a desperate and urgent need for a therapeutic solution for COVID-19. Our present studies have adapted the SAR-based approach to explore in silico several selected ferrocene-based complexes as the potential inhibitors of the major viral proteins (Spike, RdRp, M pro , N protein) of the SARS-CoV-2 virus. The SAR-based molecular docking studies have revealed that compound 1 is the strongest inhibitor of the major viral proteins with a binding energy of &gt;9.0 kcal/mol. Compound 1 is also able to inhibit the human Ca 2+ channel and thereby potentially able to prevent the strong inflammatory signalling cascades causing severe respiratory distress to the COVID-19 patients. Overall, our computational studies explored ferrocene-based compounds as the emerging multi-targeting therapeutic solution for COVID-19 by inhibiting viral replication as well as modulating the inflammatory signalling cascades.

Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus-mediated peripheral analgesia.

Ample data support a prominent role of peripheral T-type calcium channels 3.2 (Ca V 3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of Ca V 3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of Ca V 3.2 from the intrinsically disordered regions (IDRs) of Ca V 3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in Ca V 3.2 protein and identified several Ca V 3.2iPA candidates that significantly reduced Ca V 3.2 current in HEK293 cells stably expressing human wide-type Ca V 3.2. Two prototype Ca V 3.2iPAs (iPA1 and iPA2) derived from the IDRs of Ca V 3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by Ca V 3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated Ca V 3.2iPA expression suppressed neuronal excitability, suggesting that Ca V 3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral Ca V 3.2-targeting strategy for clinical treatment of pain.