Specific Conformation Research Articles

Roles of the gate loop in β-arrestin-1 conformational dynamics and phosphorylated receptor interaction

Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using β-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of β-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state.

First-in-human autologous CAR-T for metastatic breast cancers to target growth factor receptor MUC1*.

TPS2683 Background: MUC1 has been one of the most important therapeutic targets for solid tumor cancers for the past 20+ years. However, no MUC1 targeted therapeutic has yet succeeded or has been granted FDA approval. Although MUC1 is overexpressed on cancer cells, it is also widely expressed on normal epithelial cells. We previously reported that a MUC1 transmembrane cleavage product, MUC1* (muk 1 star), is a Class I growth factor receptor activated by ligand-induced dimerization of its truncated extra cellular domain. We generated monoclonal antibodies that recognize the specific conformation that is created when MUC1 is cleaved by specific enzymes expressed in the tumor microenvironment. 93% of human breast cancer tissues are recognized by one of these antibodies, huMNC2, which importantly does not bind to normal MUC1. We have incorporated this antibody into two CARs, wherein one bears the “1XX” mutations in CD3z that greatly increase CAR-T cell persistence, inhibit exhaustion and enable the killing of low antigen expressing cancer cells. Methods: Phase I for huMNC2-CAR44, an autologous frozen product, opened January, 2020, but as a 1st-in-human trial was paused during COVID. All patients receive 300 mg/m2 cyclophosphamide and 30 mg/m2 fludarabine for 3 days prior to CAR-T treatment. Dose escalation from 3.3x105 – 1x107 CAR+ cells/kg follows a standard 3 + 3 design. In addition to standard organ function inclusion criteria, the trial was first open to breast cancer patients whose cancer had progressed after at least 2 or 3 prior therapies while metastatic, yet there was no limit on the number of prior therapies. A recent patient biopsy had to be at least 30% positive for MUC1* in a CLIA validated IHC assay. Importantly, there were no limits on the vein-to-vein time. Dose levels 1 and 2 for huMNC2-CAR44 were completed without DLTs. Phase I for huMNC2-CAR22, which bore the 1XX mutations in CD3z to increase persistence and increase the killing of low antigen expressing cells opened September, 2023. Based on responses of initial huMNC2-CAR44 patients, inclusion criteria were amended to an Enrichment Trial Design. The number of prior therapies was limited to less than or equal to 10. Anticipated survival at the time of product infusion should be at least 3 months. Vein-to-vein time, which historically had been 15-16 days at one site and as high as 83 days at another site, would be targeted to 16- 22 days. The tumors of eligible patients need to have a MUC1* membrane positive H-score equal to or greater than 120 out of a possible 300, defined as high MUC1* positivity. Based on IHC analysis of human breast cancer TMAs, the H-score restriction would include roughly 40% of breast cancers. The increased ability of huMNC2-CAR22 to kill low antigen expressing cancer cells may support future studies of patients expressing low levels of MUC1*. Clinical trial information: NCT04020575 .

Study on the molecular interactions between givosiran and theophylline by surface-enhanced Raman spectroscopy, biolayer interferometry, and visible/fluorescence spectroscopy

Givosiran is a small-interfering ribonucleic acid (siRNA) drug used to treat acute hepatic porphyria. As an exogenous double-stranded RNA, givosiran has a specific spatial conformation. When they coexist, givosiran and small-molecule drugs may interact through non-covalent bonds, which may affect their efficacies. This study aimed to evaluate the molecular interaction between givosiran and three structurally-related small-molecule drugs. We combined surface-enhanced Raman spectroscopy, biolayer interferometry, and visible/fluorescence spectroscopy with a methylene blue probe to study the molecular interaction between givosiran and theophylline, caffeine, and theobromine. Molecular interactions were observed only between theophylline and givosiran, with an affinity constant (KD) of 64.2 μM. The main binding forces were hydrogen bonds and van der Waals forces, and the equilibrium binding constant (KA) was 1.855 × 104 L·mol−1. This study focused on the 5 W (Whether, Where, Why, hoW about the tendency, hoW strong) issues of the molecular interaction between givosiran and small-molecule chemical drugs. Our findings contradict the previous perception that the probability of drug–drug interactions (DDI) between nucleic acid and small-molecular drugs is low. Our study illustrates that a combination of direct and indirect analytical methods will pave a new way for future siRNA-related unknown interactions studies.

Targeted synthesis of a 3D covalent organic framework with fjh topology exhibiting high photoluminescence: A robust platform for sensitive detection of furantoin

The inappropriate use of antibiotics poses significant risks to both the environment and human health, necessitating the development of accurate and sensitive detection methods. Fluorescence detection, particularly utilizing three-dimensional (3D) covalent organic frameworks (COFs), stands out as a promising avenue. Unlike two-dimensional COFs, 3D COFs can mitigate fluorescence quenching resulting from non-radiative energy loss, primarily attributed to π-π interlayer stacking. In this study, by carefully selecting and designing the building blocks, we synthesized a 3D COF named PyTTA-TTFB-COF with fjh topology and remarkable photoluminescence. This was achieved through the combination of a 4-connected square chromophore, 1,3,6,8-Tetrakis(4-aminophenyl) pyrene, with a 3-connected triangle, 1,3,5-trimethyl-2,4,6-tris(4-formylphenyl) benzene, possessing a specific stereo conformation. Significantly, PyTTA-TTFB-COF exhibits a high photoluminescence quantum yield, notably reaching 60.1% in acetonitrile. Moreover, it displays substantial fluorescence quenching when exposed to furantoin, surpassing the performance observed with other antibiotics. Competitive experiments have confirmed that PyTTA-TTFB-COF can serve as a specific fluorescent probe for the interference-free detection of furantoin.

Expanding chalcogen bonds in thiophenes to interactions with halogens.

Compounds containing the thiophene moiety find several applications in physics and chemistry, such as electrical conduction, which depends on specific conformations to properly exhibiting the desired properties. In turn, chalcogen bonding has found to modulate the conformation of some N-thiophen-2-ylfomamides. Since halogens participate in a kin interaction (halogen bonding) and are abundant in agrochemicals, pharmaceuticals, and materials, we have quantum-chemically explored the interaction between organic halogen and thiophene as a conformational modulator in some model compounds. Although such interaction indeed appears, as demonstrated by atoms in molecules and natural bond orbital analysis, it is inefficient to control the conformational equilibrium. An energy decomposition analysis scheme demonstrated that halomethane and thiophene tend to move away from one another due to a core component (Pauli repulsion and exchange), which is mainly due to a deformation term. Therefore, chalcogen bonds with halogens appear weaker than with other chalcogens.

Design and synthesis of peptides as stabilizers of histone deacetylase 4.

Histone deacetylase 4 (HDAC4) contributes to gene repression by complex formation with HDAC3 and the corepressor silencing mediator for retinoid or thyroid hormone receptors (SMRT). We hypothesized that peptides derived from the class IIa specific binding site of SMRT would stabilize a specific conformation of its target protein and modulate its activity. Based on the SMRT-motif 1 (SM1) involved in the interaction of SMRT with HDAC4, we systematically developed cyclic peptides that exhibit Ki values that are 9 to 56 times lower than that of the linear SMRT peptide. The peptide macrocycles stabilize the wildtype of the catalytic domain of HDAC4 (cHDAC4) considerably better than its thermally more stable 'gain-of-function' (GOF) variant, cHDAC4-H976Y. Molecular docking and mutagenesis studies indicated that the cyclic peptides bind in a similar but not identical manner as the linear SMRT peptide to a discontinuous binding site. Ion mobility mass spectrometry showed no major changes in the protein fold upon peptide binding. Consistent with these results, preliminary hydrogen-deuterium exchange mass spectrometry measurements indicated only minor conformational changes. Taken together, the cyclic SMRT peptides most likely stabilize the apo form of cHDAC4.

Origin of Enantioselectivity in Engineered Cytochrome c-Catalyzed Carbon-Radical FePP Hydrolysis Revealed Using QM/MM (ABEEM Polarizable Force Field) and MD Simulations.

The origin of highly efficient asymmetric aminohydroxylation of styrene catalyzed by engineered cytochrome c is investigated by the developed Atom-Bond Electronegativity Equalization Method polarizable force field (ABEEM PFF), which is a combined outcome of electronic and steric effects. Model molecules were used to establish the charge parameters of the ABEEM PFF, for which the bond-stretching and angle-bending parameters were obtained by using a combination of modified Seminario and scan methods. The interactions between carbon-radical Fe-porphyrin (FePP) and waters are simulated by molecular dynamics, which shows a clear preference for the pre-R over the pre-S. This preference is attributed to the hydrogen-bond between the mutated 100S and 101P residues as well as van der Waals interactions, enforcing a specific conformation of the carbon-radical FePP complex within the binding pocket. Meanwhile, the hydrogen-bond between water and the nitrogen atom in the active intermediate dictates the stereochemical outcome. Quantum mechanics/molecular mechanics (QM/MM (ABEEM PFF)) and free-energy perturbation calculations elucidate that the 3RTS is characterized by sandwich-like structure among adjacent amino acid residues, which exhibits greater stability than crowed arrangement in 3STS and enables the R enantiomer to form more favorably. Thus, this study provides mechanistic insight into the catalytic reaction of hemoproteins.

Four Parallel Pathways in T4 Ligase-Catalyzed Repair of Nicked DNA with Diverse Bending Angles.

The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.

Deciphering the conformational preference and ionization dynamics of tetrahydrofuran: Insights from advanced spectroscopic techniques.

Tetrahydrofuran (THF) has garnered significant attention due to its pivotal role in biological and chemical processes. The diverse array of conformations exhibited by THF profoundly impacts its reactivity and interactions with other molecules. Understanding these conformational preferences is crucial for comprehending its molecular behavior. In this study, we utilize infrared (IR) resonant vacuum ultraviolet photoionization/mass-analyzed threshold ionization (VUV-PI/MATI) mass spectroscopies to capture distinctive vibrational spectra of individual conformers, namely, "twisted" and "bent," within THF. Our conformer-specific vibrational spectra provide valuable insights into the relative populations of these two conformers. The analysis reveals that the twisted (C2) conformer is more stable than the bent (CS) conformer by 17 ± 15cm-1. By precisely tuning the VUV photon energy to coincide with vibrational excitation via IR absorption, we selectively ionize specific conformers, yielding two-photon IR + VUV-PI/MATI spectra corresponding to the twisted and bent conformers. This investigation conclusively affirms that both the twisted and bent conformers coexist in the neutral state, while only the twisted conformer exists in the cationic state. These findings not only bridge gaps in existing knowledge but also provide profound insights into the behavior of this pivotal molecule in the realms of biology and medicine.

Ultrasensitive Solvatochirochromism of Single Benzene Chromophores.

Solvents influence the structure, aggregation and folding behaviors of solvatochromic compounds. Ultrasensitive solvent mediated chiroptical response is conducive to the fabrication of molecular platform for sensing and recognition, which however, remains great challenges in conceptual or applicable design. Here we report a cysteine-based single benzene chromophore system that shows ultrasensitivity to solvents. Compared to the ratiometrically responsive systems, the chiroptical activities could be triggered or inverted depending on the substituents of chiral entities with an ultralow solvent volume fraction (<1 vol %). One drop of dipolar solvents shall significantly induce the emergence or inversion of chiroptical signals in bulky phases. Based on the experimental and computational studies, the ultrasensitivity is contributed to the intimate interplay between solvents and chiral compounds that anchors the specific chiral conformation. It illustrates that structurally simple organic compounds without aggregation or folding behaviors possess pronounced solvatochiroptical properties, which sheds light on the next-generation of chiroptical sensors and switches.

Three-Dimensional Homochiral Covalent Organic Frameworks with Intrinsic Chiral qzd Topology.

Although a variety of chiral porous framework materials have been reported, there are few examples known to combine molecular chirality, helicity, and three-dimensional (3D) intrinsically chiral topology in one structure, which is beneficial for chirality transfer and amplification. Here, we report the synthesis of the first two 3D covalent organic frameworks (COFs) with an intrinsic chiral qzd topology, which exhibit unusual integration of various homochiral and homohelical features. By imine condensation of 4-connected porphyrin tetraamines and 2-connected enantiopure diene dialdehyde, we prepared two isostructural COFs with a noninterpenetrated qzd topology. The specific geometry and conformation flexibility of the V-shaped diene linker control the alignment of square-planar porphyrin units with rotational linkages and facilitate the creation of homochiral extended porous structures that feature a helical arrangement of porphyrins. Post-synthetic metalation of CCOF 23 with Rh(I) affords a heterogeneous catalyst for the asymmetric Michael addition reaction of aryl boronic acids to 2-cyclohexenone, which shows higher enantioselectivities compared to their homogeneous counterparts, presumably due to the confined effect of helical channels. This finding will provide an impetus to explore multichirality materials, offering new insights into the generation and control of helicity, homochirality, and enantioselectivity in the solid state.

Exploring the Specific Role of Iron Center in the Catalytic Activity of Human Serum Transferrin: CTAB-Induced Conformational Changes and Sequestration by Mixed Micelles.

Conformational changes play a seminal role in modulating the activity of proteins. This concept becomes all the more relevant in the context of metalloproteins, owing to the formation of specific conformation(s) induced by internal perturbations (like a change in pH, ligand binding, or receptor binding), which may carry out the binding and release of the metal ion/ions from the metal binding center of the protein. Herein, we investigated the conformational changes of an iron-binding protein, monoferric human serum transferrin (Fe-hTF), using several spectroscopic approaches. We could reversibly tune the cetyltrimethylammonium bromide (CTAB)-induced conformation of the protein, exploiting the concept of mixed micelles formed by three sequestrating agents: (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) hydrate (CHAPS) and two bile salts, namely, sodium cholate (NaC) and sodium deoxycholate (NaDC). The formation of mixed micelles between CTAB and these reagents (CHAPS/NaC/NaDC) results in the sequestration of CTAB molecules from the protein environment and aids the protein in reattaining its native-like structure. However, the guanidinium hydrochloride-induced denatured Fe-hTF did not acquire its native-like structure using these sequestrating agents, which substantiates the exclusive role of mixed micelles in the present study. Apart from this, we found that the conformation of transferrin (adopted in the presence of CTAB) displays pronounced esterase-like activity toward the para-nitrophenyl acetate (PNPA) substrate as compared to native transferrin. We also outlined the impact of the iron center and amino acids surrounding the iron center on the effective catalytic activity in the CTAB medium. We estimated ∼3 times higher specific catalytic efficiency for the iron-depleted Apo-hTF compared to the fully iron-saturated Fe2-hTF in the presence of CTAB.

Filament formation drives catalysis by glutaminase enzymes important in cancer progression

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the ‘glutamine addiction’ of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an ‘activation loop’ to assume a specific conformation that works together with a ‘lid’ to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets.

Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression. We use recently established rapid automated flow peptide synthesis combined with solid-phase late-stage modifications to rapidly generate a set of FK506-binding protein 51 constructs bearing defined intramolecular lactam bridges. This trapped an otherwise rarely populated transient pocket-as confirmed by crystal structures-which led to an up to 39-fold improved binding affinity for conformation-selective ligands and represents a unique system for the development of ligands for this rare conformation. Overall, our results show how rapid automated flow peptide synthesis can be applied to precision protein engineering.

Gluing GAP to RAS Mutants: A New Approach to an Old Problem in Cancer Drug Development.

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

Engineering Tunable Ratiometric Dual Emission in Single Emitter-based Amorphous Systems.

Molecular emitters with multi-emissive properties are in high demand in numerous fields, while these properties basically depend on specific molecular conformation and packing. For amorphous systems, special molecular arrangement is unnecessary, but it remains challenging to achieve such luminescent behaviors. Herein, we present a general strategy that takes advantage of molecular rigidity and S1 -T1 energy gap balance for emitter design, which enables fluorescence-phosphorescence dual-emission properties in various solid forms, whether crystalline or amorphous. Subsequently, the amorphism of the emitters based polymethyl methacrylate films endowed an in situ regulation of the dual-emissive characteristics. With the ratiometric regulation of phosphorescence by external stimuli and stable fluorescence as internal reference, highly controllable luminescent color tuning (yellow to blue including white emission) was achieved. There properties together with a persistent luminous behavior is of benefit for an irreplaceable set of optical information combination, featuring an ultrahigh-security anti-counterfeiting ability. Our research introduces a concept of eliminating the crystal-form and molecular-conformational dependence of complex luminescent properties through emitter molecular design. This has profound implications for the development of functional materials.

Engineering Tunable Ratiometric Dual Emission in Single Emitter‐based Amorphous Systems

AbstractMolecular emitters with multi‐emissive properties are in high demand in numerous fields, while these properties basically depend on specific molecular conformation and packing. For amorphous systems, special molecular arrangement is unnecessary, but it remains challenging to achieve such luminescent behaviors. Herein, we present a general strategy that takes advantage of molecular rigidity and S1‐T1 energy gap balance for emitter design, which enables fluorescence–phosphorescence dual‐emission properties in various solid forms, whether crystalline or amorphous. Subsequently, the amorphism of the emitters based polymethyl methacrylate films endowed an in situ regulation of the dual‐emissive characteristics. With the ratiometric regulation of phosphorescence by external stimuli and stable fluorescence as internal reference, highly controllable luminescent color tuning (yellow to blue including white emission) was achieved. There properties together with a persistent luminous behavior is of benefit for an irreplaceable set of optical information combination, featuring an ultrahigh‐security anti‐counterfeiting ability. Our research introduces a concept of eliminating the crystal‐form and molecular‐conformational dependence of complex luminescent properties through emitter molecular design. This has profound implications for the development of functional materials.

An optimized purification protocol for enzymatically synthesized S-adenosyl-L-methionine (SAM) for applications in solution state infrared spectroscopic studies

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM’s infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM’s structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm−1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

Expression of the O-Linked N-Acetylglucosamine-containing Epitope H (O-GlcNAcH) in Human Uterine Cervical Mucosa.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress. In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps. In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic 'reserve' cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic 'reserve' and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH. O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between 'reserve' (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.

Pregnenolone derivatives for the treatment of Alzheimer's disease: synthesis, and in vitro inhibition of amyloid β1-42 peptide aggregation, acetylcholinesterase and carbonic anhydrase-II.

The amyloid state, which is a specific conformation of proteins, offers valuable information about both functional protein structures and the pathological assemblies associated with various diseases. One of the major hallmarks of Alzheimer's disease includes primarily the extracellular build-up of a peptide known as amyloid-β, which has a sequence consisting of 39 to 42 amino acid residues, and the formation of intracellular neurofibrillary tangles mostly consisting of hyperphosphorylated tau protein. Drugs that are expected to reduce Aβ production, prevent Aβ aggregation, and promote Aβ clearance are promising approaches for treating AD. Current work is focused on identifying the compounds that have balanced even mild biological activities against multiple targets instead of finding one-target compound with high potency. We synthesized pregnenolone derivatives and evaluated their potential against inhibition of eeAChE/eqBChE, hCA-II and self-mediated Aβ1-42 peptide aggregation. Our synthesized derivatives 23, and 25-27 exhibited concomitant inhibition of all the tested macromolecular targets. All the active compounds were found to be BBB penetrants in the PAMPA assay. Furthermore, these selected compounds were found to be non-neurotoxic in the MTT assay on neuroblastoma SH-SY5Y cells. Docking studies support dual binding site (PAS and CAS) inhibition of AChE which showed Aβ1-42 aggregation and AChE inhibition. Moreover, docking studies carried out on the 3D crystallographic structure of Aβ1-42 peptide (PDB ID = 1IYT) showed significant interactions with amino acid residues Asp 23 and Lys 28, and hydrophobic interactions with the Phe19, Phe20, and Ala 30 effectively impeding the formation of β-sheet structures.