Cytosolic Isoforms Research Articles

Benzimidazolium salts bearing ester group: Synthesis, characterization, crystal structure, molecular docking studies, and inhibitory properties against metabolic enzymes

In this work, the synthesis of several unsymmetrical benzimidazolium salts with an ester (2-ethoxy-2-oxoethyl) group on the nitrogen atom is described. The structures of all compounds were fully characterized by spectroscopic (1H, 13C NMR, FTIR) and analytical (elemental analysis) methods. However, single-crystal X-ray diffraction analysis elucidated the molecular and crystal structures of compounds 1a and 1c. Asymmetric units of both crystal structures contain two crystallographically independent molecules and two bromide anions. All compounds exhibited substantial inhibition against the cytosolic carbonic anhydrase isoforms (hCA I and hCA II) and acetylcholinesterase (AChE) with Ki values 51.00–45.18 nM, 46.94–305.65 nM, and 17.57–65.68 nM, respectively. Notably, compound 1d showed extreme inhibitory effect against hCA I with 51.00±5.31 nM (AZA: 275.44±13.32 nM), hCA II with 46.94±5.44 nM (AZA: 236.55±17.88 nM) and AChE with 17.57±3.14 nM (TAC: 80.44±6.88 nM). In silico approach showed that compound 1d could form stable complexes with target enzymes with a different binding affinity (BE:9.01 kcal/mol for AChE; -7.22 kcal/mol for hCA II and -6.51 kcal/mol for hCA I). The results supported the medical potential of benzimidazolium salts containing ester group. They significantly lighted our knowledge about the chemistry of side groups on phenyl ring especially in the para position as compared to ortho and meta positions.

Novel thiazolotriazole and triazolothiadiazine scaffolds as selective tumor associated carbonic anhydrase inhibitors endowed with cathepsin B inhibition.

The present research focused on the tail-approach synthesis of novel extended thiazolotriazoles (8a-8j) and triazolothiadiazines (11a-11j) including aminotriazole intermediate 10. After successful synthesis, all the compounds were evaluated for their inhibition potential against cytosolic isoforms of human carbonic anhydrase (hCA I, II), tumor-linked transmembrane isoforms (hCA IX, XII), and cathepsin B. As per the inhibition data, the newly synthesized compounds showed poor inhibition against hCA I. Many of the compounds showed effective inhibition toward hCA IX and/or XII in low nanomolar concentration. Despite the strong to moderate inhibition of hCA II by these compounds, more than half of them demonstrated better inhibition against hCA IX and/or XII, comparatively. Further, insights of CA inhibition data of these extended analogs and their comparison with earlier reported thiazolotriazole and triazolothiadiazine derivatives might help in the rational design of novel potent and selective hCA IX and XII inhibitors. The novel compounds were also found to possess anti-cathepsin B potential at a low concentration of 10-7 M. Broadly, compounds of series 11a-11j presented more effective inhibition against cathepsin B than their counterparts in series 8a-8j. Moreover, these in vitro results with respect to cathepsin B inhibition were also supported by the in silico insights obtained via molecular modeling studies.

Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024.

Suppression of Plastidial Glucan Phosphorylase (PHO1) Increases Drought Tolerance in Potato (Solanum tuberosum L.)

Glucan phosphorylase is present in plants in two isozymes, namely, a plastidial isoform (PHO1) and a cytosolic isoform (PHO2), and is involved in starch-related carbohydrate metabolism. The aim of this study was to determine whether mutations in the genes encoding glucan phosphorylase caused these plants to have increased resistance to short-term drought. One of the strategies plants use to defend themselves against drought stress is to change their starch content, which may be due to changes in glucan phosphorylase activity. In our greenhouse pot experiment, we used potato leaves from wild-type plants and transgenic mutant lines with reduced expression of genes encoding both PHO isozymes. The plants were exposed to drought or were grown under optimal conditions. A lack of water strongly affected the water saturation deficit (WSD) and leaf protein content. The activity of the plastidial glucan phosphorylase isoform (PHO1) in mutant plants increased under drought stress, in contrast to its activity in wild-type plants. After analyzing several physiological parameters, we found that suppressed expression of the gene encoding one of the subunits of plastidial glucan phosphorylase, PHO1a, resulted in increased tolerance to drought in potatoes.

Exploring Mutation-Driven Changes in the ATP-ADP Conformational Cycle of Human Hsp70 by All-Atom MD Adaptive Sampling.

Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.

The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Ritlecitinib, a Janus Kinase 3 and Tyrosine-Protein Kinase Family Inhibitor, in Humans.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (∼30%), with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione-related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (∼71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 (CYP) fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata in humans, as well as characterization of clearance pathways and pharmacokinetics of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters, such as clearance, volume of distribution, and bioavailability, allowing for a more comprehensive understanding of drug disposition.

Role of Mitochondrial and Cytosolic Folylpolyglutamate Synthetase in One-Carbon Metabolism and Antitumor Efficacy of Mitochondrial-Targeted Antifolates.

Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange among serine, glycine, and formate. In both the cytosol and mitochondria, folates exist as polyglutamates, with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT)2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-day]pyrimidine antifolate that targets SHMT2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens, and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ∼19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (∼30-fold) more than cFPGS (∼4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the nonclassical SHMT2/SHMT1 inhibitor SHIN1. These results suggest a critical role of mFPGS levels in determining antitumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. SIGNIFICANCE STATEMENT: AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT)2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347. Mitochondrial FPGS levels play important roles in determining the antitumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.

Protein Kinase A Regulates Platelet Phosphodiesterase 3A through an A-Kinase Anchoring Protein Dependent Manner.

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.

Synthesis and Carbonic Anhydrase I, II, IX, and XII Inhibition Studies with a Series of Cyclic Sulfonyl Guanidines.

A series of thirteen cyclic sulfonyl guanidines were prepared and evaluated against tumor-associated human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA IX and hCA XII, as well as against off-target cytosolic isoforms hCA I and hCA II. The compounds reported here were generally inactive against both off-target isoforms (KI>100 μM), while all of them moderately inhibited both target isoforms hCA IX and XII in the submicromolar to micromolar ranges in which KI values spanned from 0.57 to 8.4 μM against hCA IX and from 0.34 to 9.7 against hCA XII. Due to the notable selectivity of the title compounds toward isoforms hCA IX and XII, they can be considered as useful scaffolds for further chemical optimization to develop new highly selective antitumor agents.

Exploration of the binding determinants of protein phosphatase 5 (PP5) reveals a chaperone-independent activation mechanism

The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C-termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves auto-inhibition in PP5’s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5’s enzymatic activity. The enhanced affinity for PP5’s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry (paDSF) assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C-termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (ELP1/IKBKAP) as a putative partner and, indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5’s TPR domain in vitro. Consistent with this idea, mutation of ELP1’s terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5’s TPR domain and expand the scope of PP5’s functions to include chaperone-independent complexes.

Isocoumarins incorporating chalcone moieties act as isoform selective tumor-associated carbonic anhydrase inhibitors.

Aim: A series of isocoumarin-chalcone hybrids were prepared and assays for the inhibition of four isoforms of human carbonic anhydrase (hCA; EC 4.2.1.1), hCA I, II, IX and XII. Materials & methods: Isocoumarin-chalcone hybrids were synthesized by condensing acetyl-isocoumarin with aromatic aldehydes. They did not significantly inhibit off-target cytosolic isoforms hCA I and II (KI>100μM) but acted as low micromolar or submicromolar inhibitors for the tumor-associated isoforms hCA IX and XII. Results & conclusion: Our work provides insights into a new and scarcely investigated chemotype which provides interesting tumor-associated CA inhibitors, considering that some such derivatives like sulfonamide SLC-0111 are in advanced clinical trials for the management of metastatic advanced solid tumors.

In-vitro and in-silico investigations of SLC-0111 hydrazinyl analogs as human carbonic anhydrase I, II, IX, and XII inhibitors.

Two novel series of hydrazinyl-based benzenesulfonamides 9a-j and 10a-j were designed and synthesized using SLC-0111 as the lead molecule. The newly synthesized compounds were evaluated for their inhibitory activity against four different human carbonic anhydrase (hCA) isoforms I, II, IX, and XII. Both the series reported here were practically inactive against the off-target isozyme hCA I. Notably, derivative 10a exhibited superior potency (Ki of 10.2 nM) than acetazolamide (AAZ) against the cytosolic isoform hCA II. The hCA IX and XII isoforms implicated in tumor progression were effectively inhibited with Kis in the low nanomolar range of 20.5-176.6 nM and 6.0-127.5 nM, respectively. Compound 9g emerged as the most potent and selective hCA IX and XII inhibitor with Ki of 20.5 nM and SI of 200.1, and Ki of 6.0 nM and SI of 683.7, respectively, over hCA I. Furthermore, six compounds (9a, 9h, 10a, 10g, 10i, and 10j) exhibited significant inhibition toward hCA IX (Kis = 27.0, 41.1, 27.4, 25.9, 40.7, and 30.8 nM) relative to AAZ and SLC-0111 (Kis = 25.0 and 45.0 nM, respectively). These findings underscore the potential of these derivatives as potent and selective inhibitors of hCA IX and XII over the off-target hCA I and II.

Synthesis, carbonic anhydrase inhibition studies and modelling investigations of phthalimide–hydantoin hybrids

A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with K Is values in the submicromolar to micromolar ranges. The salts 3a and 3b, followed by derivative 5, displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico. These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.

Abstract 1814: Development and evaluation of Hsp90β-selective inhibitors to overcome the safety challenges associated with pan-Hsp90 inhibitors

Abstract The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. More than 20 small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms (Hsp90a, Hsp90β, Grp94 and TRAP-1). Most of these clinical trials have failed as pan-Inhibition of Hsp90 appears to be detrimental with toxicities reported alongside induction of the pro-survival heat shock response, which leads to additional dosing challenges/toxicities. The cytosolic Hsp90 isoforms Hsp90a and Hsp90β are the major contributors to cancer growth/survival. It has been established that inhibition of Hsp90a is associated with cardiac toxicity, retinal toxicity, and induction of the heat shock response. Therefore, the development of Hsp90β -selective inhibitors represents an alternative approach for the treatment of cancer with limited side effects. Towards this goal, Hsp90β-selective inhibitors that exhibit >150-fold selectivity over Hsp90a have been developed, which exhibit efficacy against select cancers while overcoming the safety challenges associated with previous Hsp90 inhibitors. The results from such studies will be presented. Citation Format: Sanket Mishra, Tyelor Reynolds, Brian Blagg. Development and evaluation of Hsp90β-selective inhibitors to overcome the safety challenges associated with pan-Hsp90 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1814.

Sulfonamide-incorporated bis(α-aminophosphonates) as promising carbonic anhydrase inhibitors: Design, synthesis, biological evaluation, and X-ray crystallographic studies.

A novel series of sulfonamide-incorporated bis(α-aminophosphonates) acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. The synthesized bivalent ligands were tested against five human (h) isoforms, hCA I, hCA II, hCA VII, hCA IX, and hCA XIII. Such derivatives showed high activity and selectivity against the cancer-related, membrane-bound isoform hCA IX, and among them, compound 5h, tetraisopropyl (1,3-phenylenebis{[(4-sulfamoylphenyl)amino]methylene})bis(phosphonate)showed a KI of 15.1 nM, being highly selective against this isoform over all other investigated ones (hCA I/IX = 42; hCA II/IX = 6, hCA VII/IX = 3, hCA XIII/IX = 5). Therefore, compound 5h could be a potential lead for the development of selective anticancer agents. The newly developed sulfonamides were also found effective inhibitors against the cytosolic hCA XIII isoform. Compound 5i displayed the best inhibition against this isoform with a KI of 17.2 nM, equal to that of the well-known inhibitor acetazolamide (AAZ), but significantly more selective over all other tested isoforms (hCA I/XIII = 239; hCA II/XIII = 23, hCA VII/XIII = 2, hCA IX/XIII = 3) compared to AAZ.

Multiple Roles of Glycerate Kinase-From Photorespiration to Gluconeogenesis, C4 Metabolism, and Plant Immunity.

Plant glycerate kinase (GK) was previously considered an exclusively chloroplastic enzyme of the glycolate pathway (photorespiration), and its sole predicted role was to return most of the glycolate-derived carbon (as glycerate) to the Calvin cycle. However, recent discovery of cytosolic GK revealed metabolic links for glycerate to other processes. Although GK was initially proposed as being solely regulated by substrate availability, subsequent discoveries of its redox regulation and the light involvement in the production of chloroplastic and cytosolic GK isoforms have indicated a more refined regulation of the pathways of glycerate conversion. Here, we re-evaluate the importance of GK and emphasize its multifaceted role in plants. Thus, GK can be a major player in several branches of primary metabolism, including the glycolate pathway, gluconeogenesis, glycolysis, and C4 metabolism. In addition, recently, the chloroplastic (but not cytosolic) GK isoform was implicated as part of a light-dependent plant immune response to pathogen attack. The origins of glycerate are also discussed here; it is produced in several cell compartments and undergoes huge fluctuations depending on light/dark conditions. The recent discovery of the vacuolar glycerate transporter adds yet another layer to our understanding of glycerate transport/metabolism and that of other two- and three-carbon metabolites.

The cytosolic isoform of triosephosphate isomerase, ZmTPI4, is required for kernel development and starch synthesis in maize (Zea mays L.)

Triosephosphate isomerase (TPI) is an enzyme that functions in plant energy production, accumulation, and conversion. To understand its function in maize, we characterized a maize TPI mutant, zmtpi4. In comparison to the wild type, zmtpi4 mutants showed altered ear development, reduced kernel weight and starch content, modified starch granule morphology, and altered amylose and amylopectin content. Protein, ATP, and pyruvate contents were reduced, indicating ZmTPI4 was involved in glycolysis. Although subcellular localization confirmed ZmTPI4 as a cytosolic rather than a plastid isoform of TPI, the zmtpi4 mutant showed reduced leaf size and chlorophyll content. Overexpression of ZmTPI4 in Arabidopsis led to enlarged leaves and increased seed weight, suggesting a positive regulatory role of ZmTPI4 in kernel weight and starch content. We conclude that ZmTPI4 functions in maize kernel development, starch synthesis, glycolysis, and photosynthesis.

A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

BackgroundHeat shock proteins (HSPs) are present throughout the brain. They function as molecular chaperones, meaning they help with the folding and unfolding of large protein complexes. These chaperones are vital in the development of neuropathological conditions such as Alzheimer’s disease and Lewy body disease, with HSP90, a specific subtype of HSP, playing a key role. Many studies have shown that drugs that inhibit HSP90 activity have beneficial effects in the neurodegenerative diseases. Therefore, HSP90 PET imaging ligand can be used effectively to study HSP90 in neurodegenerative diseases. Among four HSP90 isoforms, two cytosolic isoforms (HSP90α and HSP90β) thought to be involved in the structural homeostasis of the proteins related to the neurodegenerative diseases. Currently, no useful PET imaging ligands selectively targeting the two cytosolic isoforms of HSP90 have been available yet.ResultsIn this study, we developed a novel positron emission tomography (PET) imaging ligand, [11C]BIIB021, by 11C-radiolabeling (a positron emitter with a half-life of 20.4 min) 6-Chloro-9-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-9H-purin-2-amine (BIIB021), an inhibitor with a high affinity for and selectivity to HSP90α and HSP90β. [11C]BIIB021 was synthesized with a high yield, molar activity and radiochemical purity. [11C]BIIB021 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90β proteins. Radioactivity was well detected in the rat brain (SUV 1.4). It showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections. Radiometabolite was detected in the brain, however, total distribution volume was well quantified using dual-input graphical model. Inhibition of p-glycoprotein increased brain radioactivity concentrations. However, total distribution volume values with and without p-glycoprotein inhibition were nearly the same.ConclusionsWe have developed a new PET imaging agent, [11C]BIIB021, specifically targeting HSP90α/β. We have been successful in synthesizing [11C]BIIB021 and in vitro and in vivo imaging HSP90α/β. However, the quantification of HSP90α/β is complicated by the presence of radiometabolites in the brain and the potential to be a substrate for p-glycoprotein. Further efforts are needed to develop radioligand suitable for imaging of HSP90α/β.

Sulfonyl thiourea derivatives from 2-aminodiarylpyrimidines: Invitro and in silico evaluation as potential carbonic anhydrase I, II, IX, and XII inhibitors.

A series of synthesized sulfonyl thiourea derivatives (7a-o) of substituted 2-amino-4,6-diarylpyrimidines (4a-o) exhibited the remarkable inhibitory activity against some the human carbonic anhydrases (hCAs), including hCA I, II, IX, and XII isoforms. The inhibitory efficacy of synthesized sulfonyl thiourea derivatives were experimentally validated by invitro enzymatic assays. 7a (KI = 46.14 nM), 7j (KI = 48.92 nM), and 7m (KI = 62.59 nM) (for isoform hCA I); 7f (KI = 42.72 nM), 7i (KI = 40.98 nM), and 7j (KI = 33.40 nM) (for isoform hCA II); 7j (KI = 228.5 nM), 7m (KI = 195.4 nM), and 7n (KI = 210.1 nM) (for isoform hCA IX); 7l (KI = 116.9 nM), 7m (KI = 118.8 nM), and 7n (KI = 147.2 nM) (for isoform hCA XII) in comparison with KI values of 452.1, 327.3, 437.2, and 338.9 nM, respectively, of the standard drug AAZ. These compounds also had significantly more potent inhibitory action against cytosolic isoform hCA I and tumor-associated isoforms hCA IX and hCA XII. Furthermore, the potential inhibitory compounds were subjected to in silico screening for molecular docking and molecular dynamics simulations. The results of invitro and in silico studies revealed that compounds 7a, 7j, and 7m were the most promising derivatives in this series due to their significant effects on studied hCA I, II, IX, and XII isoforms, respectively. The results showed that the sulfonyl thiourea moiety was accommodated deeply in the active site and interacted with the zinc ion in the receptors.

Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and β) in sustaining malignant cells’ growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90β isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.