Activation and differentiation of cognate T cells by a dextran-based antigen-presenting system for cancer immunotherapy.

Immunotherapeutic modulation of antigen-specific T-cell responses instead of the whole repertoire helps avoid immune-related adverse events. We have developed an artificial antigen-presenting system (aAPS) where multiple copies of a multimeric peptide-MHC class I complex presenting a murine class I MHC restricted ovalbumin-derived peptide (signal 1), along with a costimulatory ligand (signal 2) are chemically conjugated to a dextran backbone. Cognate naive CD8+ T cells, when treated with this aAPS underwent significant expansion and showed an activated phenotype. Furthermore, elevated expression of effector cytokines led to the differentiation of these cells to cytotoxic T lymphocytes which resulted in target cell lysis, indicative of the functional efficacy of the aAPS. CD8+ T cells with decreased proliferative potential due to repeated antigenic stimulation could also be re-expanded by the developed aAPS. Thus, the developed aAPS warrants further engineering for future application as a rapidly customizable personalized immunotherapeutic agent, incorporating patient-specific MHC-restricted tumor antigens and different costimulatory signals to modulate both naive and antigen-experienced but exhausted tumor-specific T cells in cancer.

Angle distortion model for predicting enediyne activation towards Bergman cyclization: an alternate to the distance theory.

The kinetics of Bergman cyclization (BC) of enediynes into 1,4-benzene diradicals (also known as p-benzynes) have attracted interest ever since the discovery of natural enediynes which pointed out a surprising reactivity profile difference across enediynes with varying structural architectures. From the analysis of experimental kinetic data, several models were proposed to have a structure-kinetics correlation, out of which, the cd-distance model and the transition state model are the most accepted ones. Recently, Houk et al. introduced a distortion model to explain the regioselectivity of nucleophilic addition to unsymmetrical o-benzynes based on the geometry of the transition state. In the case of BC, since the reaction is endothermic, the transition state geometrically resembles the product structure which implies that in the reaction pathway, the sp-carbons of enediynes are transformed into the trigonal sp2 carbons of the benzenoid product. Thus, greater bending of the interior angles at the proximal alkyne carbons in the enediynes will lead to a lower activation barrier for the BC and hence faster cyclization. This hypothesis has been tested on a series of enediynes including natural product surrogates and the extent of deviation correlates well with the kinetic results. A cut-off value for the average internal proximal angles has been proposed to categorize enediynes as per their reactivity under ambient conditions. We believe that this distortion theory offers an alternative model in designing new unnatural enediynes with desired kinetic stabilities.

Targeting an evolutionarily conserved "E-L-L" motif in the spike protein to develop a small molecule fusion inhibitor against SARS-CoV-2.

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved E-L-L motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing the post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this E-L-L motif resulting in effective inhibition of SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy towards blocking S protein-mediated viral entry, mutations within the E-L-L motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential E-L-L motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

The C-terminal end of mycobacterial HadBC regulates AcpM interaction during the FAS-II pathway: a structural perspective.

Comprehending the molecular strategies employed by Mycobacterium tuberculosis (Mtb) in FAS-II regulation is of paramount significance for curbing tuberculosis progression. Mtb employs two sets of dehydratases, namely HadAB and HadBC (ß-hydroxyacyl acyl carrier protein dehydratase), for the regulation of the fatty acid synthase (FAS-II) pathway. We utilized a sequence similarity network to discern the basis for the presence of two copies of the dehydratase gene in Mtb. This analysis groups HadC and HadA in different clusters, which could be attributed to the variability in their physiological role with respect to the acyl chain uptake. Our study reveals structural details pertaining to the crystal structure of the last remaining enzyme of the FAS-II pathway. It also provides insights into the highly flexible hot-dog helix and substrate regulatory loop. Additionally, mutational studies assisted in establishing the role of the C-terminal end in HadC of HadBC in the regulation of acyl carrier protein from Mtb-mediated interactions. Complemented with surface plasmon resonance and molecular dynamics simulation studies, the present study provides the first evidence of the molecular mechanisms involved in the differential binding affinity of the acyl carrier protein from Mtb towards both mtbHadAB and mtbHadBC.

Fluorescently labelled thioacetazone for detecting the interaction with Mycobacterium dehydratases HadAB and HadBC.

Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes.

Structural and dynamic studies of the human RNA binding protein RBM3 reveals the molecular basis of its oligomerization and RNA recognition.

Human RNA-binding motif 3 protein (RBM3) is a cold-shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N-terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a ßαßßαß topology, which is found in many RNA-binding proteins. NMR-monitored titration experiments and molecular dynamic simulations show that the beta-sheet and two loops form the RNA-binding interface. Hydrogen bond, pi-pi and pi-cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross-linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold-shock protein. Temperature-dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature-dependent oligomerization.

Targeting an evolutionarily conserved "E-L-L" motif in spike protein to identify a small molecule fusion inhibitor against SARS-CoV-2.

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key toward sustenance. Here, we identify an evolutionarily conserved "Ex3Lx6L" ("E-L-L") motif present within the HR2 domain of all human and nonhuman coronavirus spike (S) proteins that play a crucial role in stabilizing its postfusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this "E-L-L" motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the "E-L-L" motif rendered the protein completely resistant to the drug, establishing its specificity toward this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective toward all major variants of concerns of SARS-CoV-2, including Beta, Kappa, Delta, and Omicron. Together, we show that this conserved essential "E-L-L" motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

Mycobacterial crypto-AcpM as a tool to investigate the consequence of drug binding on its key FAS II partner enzyme HadAB.

Background Mycobacterial FASII pathway is governed by the Protein-Protein Interaction mediated dynamics existent between Acyl Carrier Protein and its partner enzymes. The dehydratase HadAB, involved in the third step of FASII synthesis has remained a key target of drugs like Thiacetazone (TAC) and its consequence on AcpM binding is yet to be deciphered. Owing to the transient nature of these interactions, analysing their implications as a drug target has been exhausting. Methods In this context, we have developed an in vitro method to study the effect of thiocarbamide-containing compounds, TAC and SPA0355 (a thiourea analogue) against mycobacterial HadAB. Additionally, by utilizing crypto-ACP (NBD-tagged Acyl Carrier Protein) as a tool of our choice, we attempted at exploring the effect of TAC and SPA0355 on mycobacterial HadAB. Results SPA0355 behaves at par with TAC and undergoes activation in the presence of monooxygenase EthA thus, bringing about a covalent modification in HadA subunit of HadAB. The crypto-ACP method provides insights into the altered substrate housing capability in HadAB associated with the impediment of its AcpM mediated functionality; an outcome attributed to the repercussions associated with the binding of the aforementioned thiourea compounds. Conclusion This investigation has assisted in unveiling a two-step mechanism undertaken by AcpM for interacting with its corresponding partner protein during acyl chain transfer. General significance This study highlights the alterations brought about by drug binding in the interplay between ACP and HadAB. Additionally, this work for the first time establishes the role of SPA0355 as a promising drug candidate against dehydratase HadAB.

Sterically hindering the trajectory of nucleophilic attack towards p-benzynes by a properly oriented hydrogen atom: an approach to achieve regioselectivity.

Nucleophilic addition to p-benzynes derived via Bergman cyclization has become a topic of keen interest. Studying the regioselectivity in such addition can reveal important information regarding the parameters controlling such addition. Recently, high regioselectivity has been achieved in nucleophilic addition to a p-benzyne derived from an ortho substituted benzo fused cyclic azaenediyne. Rather than having a freely rotating substitution, a rigid hydrogen atom coming from a suitable naptho fused enediyne and residing in the plane of the p-benzyne ring can offer hindrance to the trajectory of the nucleophile. This can lead to regioselectivity provided the other side remains relatively free of such hindrance. Based on that approach, halide addition to p-benzynes derived from naphtho fused cyclic azaenediynes was studied and a high level of regioselectivity was observed. Steric hindrance to the trajectory of nucleophile by the bay hydrogen was found to be the main cause of such regioselectivity; however, differential electrostatic potential as well as distortions at reactive centres have a minor role in controlling the regioselectivity. The products of such high yielding addition are the halo naphtho tetrahydroisoquinolines.

Label-Free Method Development for Hydroxyproline PTM Mapping in Human Plasma Proteome.

Post-translational modifications (PTMs) impart structural heterogeneities that can alter plasma proteins' functions in various pathophysiological processes. However, the identification and mapping of PTMs in untargeted plasma proteomics is still a challenge due to the presence of diverse components in blood. Here, we report a label-free method for identifying and mapping hydroxylated proteins using tandem mass spectrometry (MS/MS) in the human plasma sample. Our untargeted proteomics approach led us to identify 676 de novo sequenced peptides in human plasma that correspond to 201 proteins, out of which 11 plasma proteins were found to be hydroxylated. Among these hydroxylated proteins, Immunoglobulin A1 (IgA1) heavy chain was found to be modified at residue 285 (Pro285 to Hyp285), which was further validated by MS/MS study. Molecular dynamics (MD) simulation analysis demonstrated that this proline hydroxylation in IgA1 caused both local and global structural changes. Overall, this study provides a comprehensive understanding of the protein profile containing Hyp PTMs in human plasma and shows the future perspective of identifying and discriminating Hyp PTM in the normal and the diseased proteomes.

Regioselective Synthesis of Benzo-Fused Tetrahydroisoquinoline-Based Biaryls through a Tandem One-Pot Halogenation of p-Benzynes from Enediynes and Suzuki-Miyaura Coupling.

A regioselective halogenation of p-benzyne derived from a nonaromatic enediyne core via Bergman cyclization and Suzuki-Miyaura coupling of the resulting haloarene in one-pot is disclosed. For the one-pot protocol to work, the reaction conditions were modified compared to an earlier reported procedure ( J. Org. Chem. 2019 , 84 , 2911 - 2921 ) by reducing the amount of lithium iodide and exclusion of pivalic acid. Under these modified conditions, the products, benzo-fused tetrahydroisoquinoline-based biaryl derivatives were obtained in overall high to excellent yields (71-90%).

Staphylococcal superantigen-like proteins interact with human MAP kinase signaling protein ERK2.

This study aimed to identify the intracellular binding partner of a unique class of staphylococcal secreted exotoxins called superantigen-like proteins (SSL) from human macrophage and keratinocyte cell lysates. Here, we report that SSL1 specifically binds to human extracellular signal-regulated kinase 2 (hERK2), an important stress-activated kinase in mitogen-activated protein kinase signaling pathways. Western blot and in vitro binding studies with recombinant hERK2 confirmed the binding interaction of SSL1, SSL7, and SSL10 with hERK2. Moreover, the SSLs-hERK2 interaction was validated biochemically by ELISA. Our finding shows that SSLs play a novel role by binding with host cell MAP kinase signaling pathway protein. Understanding the SSL-hERK2 interaction will also provide a basis for designing SSL-based peptide inhibitors of hERK2 in cancer therapy.

Identification of a novel humoral antifungal defense molecule in the hemolymph of tasar silkworm Antheraea mylitta.

Humoral defenses are the major components of insect innate immune system that include the production of several soluble effector molecules from fat body and hemocytes, and released in to the hemolymph upon microbial infection. Hemolymph was collected from the fungal immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, extracted with a mixture of solvent (methanol/glacial acetic acid/water) and fractionated through RP-HPLC. Several fractions were collected, lyophilized and their antifungal activity was tested against Candida albicans. Only the fraction showing strong antifungal activity was further purified via gel filtration chromatography and the purity of active compound was confirmed by thin layer chromatography which showed only single spot after staining with ninhydrin. The molecular mass of this purified compound was determined by high resolution mass spectrometry as 531 Da and analysis of 1H and 13C NMR spectral data along with mass fragmentation pattern indicated the probable structure of the isolated compound as symmetric bis-decanoate derivative. Scanning electron microscopic study revealed that the compound degraded fungal cell wall leading to its lysis and may be the major target for its antifungal activity. These results indicate that presence of this compound in the hemolymph of A. mylitta provides defense against fungal infection.

Rapid Fluorescent-Based Detection of New Delhi Metallo-ß-Lactamases by Photo-Cross-Linking Using Conjugates of Azidonaphthalimide and Zinc(II)-Chelating Motifs.

A method for rapid detection of metallo-ß-lactamases NDM-5 and NDM-7 using conjugates of azidonaphthalimide and Zn(II) chelating motifs (like sulfonamides, hydroxamate, and terpyridine) is described. Incubation and irradiation, followed by gel electrophoresis, clearly show the presence of NDMs. The o-sulfonamide-based probe has the highest efficiency of detection for both the NDMs. The proteins are detectable at nM concentrations, and the method is also selective, works both in vitro and in vivo, as revealed by cellular imaging and also with clinical isolates.

Naphthalimide-Based Template for Inhibitor Screening via Cross-Linking and In-Gel Fluorescence: A Case Study against HCA II.

We describe a rapid electrophoresis-based method for profiling of carbonic anhydrase inhibitors. In addition to the pharmacophore moiety intended for reversible interaction with a target enzyme, a fluorescent template with a built-in azide group for photoaffinity labeling is also included as a part of the inhibitor design. Following incubation and irradiation, gel electrophoresis with visualization under UV allows assessment of the efficiency of cross-linking. The relative efficiency of cross-linking of various probes can be regarded as a reflection of their inhibition potencies, an assumption supported by the trend in their IC50/K i values. The method has the advantage of being applicable to impure enzyme preparations and also can be used to screen several inhibitors including their promiscuity in parallel in a short time as has been currently demonstrated with HCA II.

Decrypting the oscillating nature of the 4'-phosphopantetheine arm in acyl carrier protein AcpM of Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, acyl carrier protein (AcpM)-mediated fatty acid synthase type II is integral for the synthesis of mycolic acids. AcpM, designated as an atypical ACP, comprises of a putative 33 amino acid long C-terminal extension which is distinctive in nature. Here, we aimed at devising an 'easy-to-go' method for the generation of crypto-AcpM loaded with a solvatochromic probe 7-Nitrobenz-2-oxa-1,3-diazol-4-yl, which is linked to the 4'-phosphopantetheine (Ppant) prosthetic group of AcpM. The crypto-AcpM, coupled with fluorescence spectroscopy and molecular dynamics simulation studies, was employed to explore the elusive dynamics of Ppant arm in AcpM. This investigation establishes the role of the flexible C-terminal extension of AcpM in regulating the prosthetic group sequestration ability by modulating the 'Asp-Ser-Leu' motif.

How To Achieve High Regioselectivity in Barrier-less Nucleophilic Addition to p-Benzynes Generated via Bergman Cyclization of Unsymmetrical Cyclic Azaenediyne?

Inducing high regioselectivity in nucleophilic addition to p-benzynes, first reported by Perrin and O'Connor et al. ( J. Am. Chem. Soc. 2007 , 129 , 4795 - 4799 ) has been a challenge as the reaction involves a very fast barrier-less addition of nucleophile. On the other hand, achieving a high degree of regioselectivity is important as that will make the reaction synthetically useful. Recently, a study has been reported from our group ( J. Org. Chem. 2018 , 83 , 7730 - 7740 ), whereby it was shown that nucleophilic addition to p-benzynes derived from unsymmetrical N-substituted cyclic enediynes proceeds with low extent of selectivity by incorporation of groups with divergent electronic characters. Herein, we report that excellent regioselectivity (>99%) can be achieved keeping an ortho alkoxy group in unsymmetrical 1,2-dialkynylbenzene in the form of a cyclic enediyne in quantitative yields. High regioselectivity (∼84%) is also shown by pyridine based enediynes where the pyridine nitrogen is in a 1,3-relationship with the impending radical center, expanding the synthetic scope of this nucleophilic addition. The regioselectivity can be explained in terms of computed electrostatic potentials which are substantially different around two radical centers arising due to the "ortho effect" (conformational alignment of lone pair of the ortho alkoxy oxygen or the nitrogen in pyridine systems).

Laser desorption ionization mass spectrometry: Recent progress in matrix-free and label-assisted techniques.

The MALDI-based mass spectrometry, over the last three decades, has become an important analytical tool. It is a gentle ionization technique, usually applicable to detect and characterize analytes with high molecular weights like proteins and other macromolecules. The earlier difficulty of detection of analytes with low molecular weights like small organic molecules and metal ion complexes with this technique arose due to the cluster of peaks in the low molecular weight region generated from the matrix. To detect such molecules and metal ion complexes, a four-prong strategy has been developed. These include use of alternate matrix materials, employment of new surface materials that require no matrix, use of metabolites that directly absorb the laser light, and the laser-absorbing label-assisted LDI-MS (popularly known as LALDI-MS). This review will highlight the developments with all these strategies with a special emphasis on LALDI-MS.

Experiment and Computational Study on the Regioselectivity of Nucleophilic Addition to Unsymmetrical p-Benzynes Derived from Bergman Cyclization of Enediynes.

The regioselectivity in addition of nucleophiles to the p-benzyne intermediates derived from unsymmetrical aza-substituted enediynes via Bergman cyclization was studied. Computational studies [using UB3LYP/6-31G(d,p) level of theory] suggest that the p-benzyne intermediate retains its similar electrophilic character at the two radical centers even under unsymmetrical electronic perturbation, thus supporting the predicted model of nucleophilic addition to p-benzyne proposed by Perrin and co-workers (Perrin et al. J. Am. Chem. Soc. 2007, 129, 4795-4799) and later by Alabugin and co-workers (Peterson et al. Eur. J. Org. Chem. 2013, 2013, 2505-2527). However, observed experimental results suggest that there was small but definite regioselectivity (∼5-25%), the extent varying with the electronic nature of the substituents. Differential solvated halide ion concentrations around the vicinity of two radical centers arising due to surrounding surface electrostatic potential (computationally calculated) may be one of the possible factors for such selectivity in some of the examined p-benzynes. However, other complicated dynamical issues like the trajectory of the attacking nucleophile to the radical center which can be influenced by electronic and/or steric perturbation of starting enediyne conformation cannot be ruled out. The overall yield of the anionic addition was in the range of 80-99%.