Structure-activity Relationship Studies Research Articles

Kinact/KI Value Determination for Penicillin-Binding Proteins in Live Cells.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, kinact/KI, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, kinact/KI values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell kinact/KI assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro kinact/KI data and show a comparable relationship to previously established IC50 values. These results support the validity of our in vivo kinact/KI method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

Novel Meta-Diamide Derivatives Bearing Triazole: Design, Synthesis and Bioactivity.

In searching for novel insecticide lead,20new meta-diamidecompounds containingtriazole were designed and synthesized regardingCyproflanilide aslead compound.All the compounds were characterized by 1HNMR,13CNMR and HRMS.In preliminary bioassay, we found that one of the compounds: N-(cyclopropylmethyl)-N-(5-((2,6-dibromo-4-(1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl)phenyl)carbamoyl)-2-(1H-1,2,4-triazol-1-yl)phenyl)-6-(trifluoromethyl) nicotinamide(16a) had high activity against the target organism Plutella xylostellaat 1 mg/L and against the target organism Mythimna separataat 2 mg/L. It is obviously superior to theknowncompoundsⅠaand Ⅰb, and itisequivalent to cyproflanilide, the LC50values of the compound against P. xylostella and M. separata were 0.90 mg/L and 0.64 mg/L respectively. It is providing important clues for subsequent structure-activity relationship studies. From this, we explored the structure-activity relationship of these compounds. Through comprehensive analysis of their structural characteristics and insecticidal activity data, we found that different halogen substituents on benzene ring and different aromatic substituents on amino group in the compound had significant effects on insecticidal activity. This has certain guiding significance for further structural optimization and activity enhancement.

4-Substituted-2-Thiazole Amides as Viral Replication Inhibitors of Alphaviruses.

2-(Methylthio)-N-(4-(naphthalen-2-yl)thiazol-2-yl)nicotinamide 1 was identified as an inhibitor against Chikungunya virus (CHIKV) with good antiviral activity [EC50 = 0.6 μM; EC90 = 0.93 μM and viral titer reduction (VTR) of 6.9 logs at 10 μM concentration] with no observed cytotoxicity (CC50 = 132 μM) in normal human dermal fibroblast (NHDF) cells. Structure-activity relationship (SAR) studies to further improve the potency, efficacy, and drug-like properties of 1 led to the discovery of a new potent inhibitor N-(4-(3-((4-cyanophenyl)amino)phenyl)thiazol-2-yl)-2-(methylthio)nicotinamide 26, which showed a VTR of 8.7 logs at 10 μM against CHIKV and an EC90 of 0.45 μM with considerably improved MLM stability (t1/2 = 74 min) as compared to 1. Mechanism of action studies show that 26 inhibits alphavirus replication by blocking subgenomic viral RNA translation and structural protein synthesis. The in vivo efficacy studies of compound 26 on CHIKV infection in mice are reported.

Design, Synthesis, Computational Studies and Evaluation of Novel 2, 4, 5-Trisubstituted Pyrimidine Derivatives for Anticancer Activity against MCF-7 and A549 Cell Lines

Synthesized novel trisubstituted pyrimidine derivatives (U 01-15) were tested for anti-cancer efficacy against two cancer cell line MCF-7 and A549. Several spectroscopic methods assisted to characterized and confirmed all synthesized compounds. Some of the synthesized pyrimidine derivatives showed strong effectiveness, with IC50 values comparable to the standard drug doxorubicin and gefitinib. Compounds U-02, U-04, U-07, U-10 and U-11 showed good anti-cancer activity for both cell lines having IC50 values in range from 12.06 µM to 22.45 µM, while compounds U-06 and U-03 showed moderate potency against both the cell lines, respectively. Using PyRx software (based on Auto Dock 4 and a genetic algorithm), all the synthesized compounds were put through a molecular docking with receptor tyrosine-protein kinase erbB-4 and docking score was compared to approved drugs, gefitinib and lapatinib. Interestingly almost all derivatives showed good docking score as compared to reference drugs. Molecular dynamics simulation of the U-11 protein-ligand complex over 100 ns revealed key insights into stability and conformational changes. RMSD analysis indicated stability between 10-20 ns with fluctuations up to 5.71 Å, while RMSF showed an average fluctuation of 1.74 Å, particularly in the loop regions. Molecular interactions, including hydrogen bonds, were observed with key residues such as ASP836, MET774, and LEU769.Future studies could focus on optimizing the most promising pyrimidine derivatives, such as U-02, U-04, U-07, U-10, and U-11, for enhanced potency and selectivity through advanced biological testing and structure-activity relationship studies may provide deeper insights into their therapeutic potential against cancer.

Imidazo[1,2-A]Pyridine: Potent Biological Activity, SAR and Docking Investigations (2017-2022).

Regarding scientific research, Imidazo[1,2-a] pyridine derivatives are constantly being developed due to the scaffold's intriguing chemical structure and varied biological activity. They are distinctive organic nitrogen-bridged heterocyclic compounds that have several uses in medicines, organometallics and natural products. It has become a vital tool for medicinal chemists. In order to gather scientific information on Imidazo[1,2-a] pyridines derivative, Google, PubMed, Scopus, Google Scholar, and other databases were searched. In the current study, the medicinal value and therapeutic effect of Imidazo[1,2-a] pyridines were investigated using above mentioned databases. The current study analyzed the detailed pharmacological activities of Imidazo[1,2-a] pyridine analogs through literature from diverse scientific research works. Due to its wide range of biological activities, including antiulcer, anticonvulsant, antiprotozoal, anthelmintic, antiepileptic, antifungal, antibacterial, analgesic, antiviral, anticancer, anti-inflammatory, antituberculosis, and antitumor properties, imidazopyridine is one of the most significant structural skeletons in the field of natural and pharmaceutical products. An imidazopyridine scaffold serves as the basis for a number of therapeutically utilized medications, including zolpidem, alpidem, olprinone, zolimidine, and necopidem. This comprehensive study covers the period of the last five years, and it sheds light on the developments and emerging pharmacological actions of Imidazo[1,2-a] pyridines. Additionally, the structure-activity relationship and molecular docking studies are carefully documented throughout the paper, providing medicinal chemists with a clear picture for developing new drugs.

2-Aminobenzothiazole based adjuvant of polymyxin E against Gram-negative bacteria

Antibiotic resistance has emerged as a pressing global health issue. Polymyxin E, commonly regarded as a clinically important antibiotic, faces limitations due to its dose-dependent toxicity. A crucial strategy to combat this is the development of an antibiotic adjuvant to enhance efficacy while reducing dosage. Screening from our extensive in-house compound library, KJ1071 emerged as a promising adjuvant candidate for polymyxin E. Subsequent structure–activity relationship studies led to the discovery of compound A35, which demonstrated superior synergistic activity. However, its limited aqueous solubility posed challenges for biological experiments. To address this, we carried out a structural derivatization aimed at enhancing its solubility. The amino acid derivative A59 notably improved aqueous solubility to 3.237 mg/mL, a substantial 3237-fold increase compared to compound A35. Moreover, A59 amplified the efficacy of polymyxin E in eradicating a variety of Gram-negative bacteria, including certain clinical strains that are resistant to polymyxin E. The mechanism studies indicated that A59 might target the bacterial membrane. These findings suggest that the discovery of this innovative scaffold could provide critical insights for new adjuvant therapies.

Chemically Synthesized 1,2,3,4,6-Pentakis-O-Galloyl-β-D-Glucopyranoside Blocks SARS-CoV-2 Spike Interaction with Host ACE-2 Receptor.

In the search for anti-COVID-19 therapy, 1,2,3,4,6-pentakis-O-galloyl-βD-glucopyranoside, a natural polyphenolic compound isolated from many traditional medicinal herbs, has been reported as an RBD-ACE2 binding inhibitor and as a broad-spectrum anticoronaviral inhibitor targeting the main protease and RNA-dependent RNA polymerase of SARSCoV-2. To facilitate the structure-activity relationship studies of 1,2,3,4,6-pentakis-O-galloyl-β-Dglucopyranoside, we describe its chemical synthesis and characterization, as well as its activity towards the SARS-CoV-2 spike interaction with host ACE2 receptor. 1,2,3,4,6-Pentakis-O-galloyl-β-D-glucopyranoside was synthesized in two quantitative steps from 3,4,5-tribenzyloxybenzoic acid and β-D-glucopyranoside: DCC-mediated esterification and palladium-catalyzed per-debenzylation. The synthesized molecule was evaluated using a SARS-CoV-2 spike trimer (S1 + S2) ACE2 inhibitor screening colorimetric assay kit, SARS-CoV2 spike S1 RBD ACE2 inhibitor screening colorimetric assay kit, and a cellular neutralization assay using the Spike (SARS-CoV-2) Pseudotyped Lentivirus, ACE2-HEK293 recombinant cell line. The chemically synthesized product blocked the binding of the spike trimer of SARSCoV-2 to the human ACE2 receptor with IC50=22±2 µM. It also blocked ACE2:spike RBD binding with IC50=27±3 µM. Importantly, it inhibited the infectivity of SARS2-CoV2-Spike pseudotyped lentivirus on the ACE2 HEK293 cell line with IC50=20±2 µM. Overall, the chemically synthesized 1,2,3,4,6-pentakis-O-galloyl-β-D-glucopyranoside represents a lead molecule to develop anti-SARS-CoV-2 therapies that block the initial stage of the viral infection by blocking the virus entry to the host cell.

Synthesis of Some Novel 4-bromobenzoic Acid Clubbed Hydrazone Schiff Base Derivatives as Potent α-amylase Inhibitors: In vitro and In silico Studies

Aims: Synthesis of novel 4-bromobenzoic acid-based hydrazone-Schiff base derivatives and to screen them for their α-amylase inhibitory activity. Objective: The biological activities of hydrazone-Schiff base compounds encouraged us to evaluate the synthesized derivatives (4-32) for in-vitro inhibition activity against the α-amylase enzyme. Methods: In current research work twenty-nine Schiff base derivatives (4-32) of 4-bromobenzoic acid were synthesized in worthy yields by treating various replaced aldehydes with 4- bromobenzohydrazide using methanol solvent in catalytic quantity of acetic acid. The products were structurally described through the support of several spectroscopic methods (EI-MS and 1HNMR) and finally evaluated against α-amylase enzyme. Results: All the made derivatives exhibited worthy inhibition potential from IC50 = 0.21 ± 0.01 to 5.50 ± 0.01 μM when equated to the usual acarbose drug having IC50 = 1.34 ± 0.01 μM. Compound 21 (IC50 = 0.21 ± 0.01 μM) was established as the most active inhibitor among the series better than standard. The structure-activity relationship study showed that the alteration in the activity of the produced products might be due to the attached position and nature of the substituents. Furthermore, in-silico study supported the effects of groups attached on the binding interaction with α-amylase enzyme. Conclusion: A series of substituted hydrazone Schiff bases based on 4-bromobenzoic acid were produced, confirmed the structures by EI-MS and 1H-NMR spectroscopic methods and lastly tested for their in-vitro α-amylase inhibitory potential. Among the series, twenty-four products indicated brilliant inhibition potential having IC50 values from 0.21 ± 0.01 to 1.30 ± 0.01 μM. The structure-activity relationship study showed that the alteration in the activity of the synthesized products might be due to the attached position and nature of the substituents. On the other hand, in silico studies advocated that the synthesized Schiff base derivatives have prevalent interactions of binding within the active site of the α-amylase enzyme, and because of their various attached substituent, their conformation is altered in the active site of the enzyme. The current study recognized a number of lead candidates derived from 4-bromobenzoic acid. Additional investigation of the synthesized derivatives for coming research to get novel α-amylase inhibitors.

Structure-activity relationship study of novel evodiamine amino acid conjugates with potent anti-colorectal cancer efficacy

Evodiamine has been a promising lead structure with broad-spectrum antitumor activity. Druggability optimization is the most challenging part of evodiamine-based lead-to-candidate campaign. Amino acids as building blocks for conjugates are widely incorporated into approved drug and drug candidates, demonstrating highly attractive druggability. Herein, a series of evodiamine amino acid conjugates were designed and synthesized based on the evodiamine lead compound (±)-8b discovered in our previous work. The structure−activity relationship (SAR) studies culminated in the identification of a promising conjugate (−)-15h featuring a N-Boc-L-glutamine group and a chiral carbon atom (sinister), which exhibited nanomolar antiproliferative activity against LoVo and RKO colorectal cancer cells. Moreover, (−)-15h could inhibit topoisomerases I, arrest the cell cycle in the G2/M phase, and induce apoptosis. Importantly, (−)-15h (tumor growth inhibition rate was 82.53% in 40 mpk) showed better efficacy and tolerability to that of parent compound (−)-8b (tumor growth inhibition rate was 51.22% in 40 mpk) in LoVo xenograft model. Further, (−)-15h (tumor growth inhibition rate was 70.09% in 40 mpk) showed comparable efficacy and better tolerability to that of topotecan (tumor growth inhibition rate was 70.67% in 0.5 mpk) in HT-29 xenograft model. Collectively, this study further provided a strong scientific basis for amino acid-based structural modifications and a drug lead for anti-colorectal cancer applications.

Synthesis, quantum chemical insights, vibrational spectral elucidation, insecticide likeness and docking analysis of the insecticide active novel molecule 2-(4-nitrophenyl)-(3,5-difluoro-pyridine-6-yl)-1,3,4-oxadiazole

Oxadiazole derivatives containing pyridine moiety were biologically active molecules that have been widely applied in recent years in research on pesticides, particularly insecticides. In this work, a newly synthesized 2-(4-nitrophenyl)-(3,5-difluoro-pyridine-6-yl)-1,3,4-oxadiazole compound was characterized using spectroscopic techniques. The quantum chemical computations based on density functional theory were employed to investigate the molecular configuration of title compound. The modes of vibrations of the compound were identified through potential energy distribution, and the results were consistent with the experimental infrared and Raman data. Natural bond orbital assessment evaluated the significant donor-acceptor interactions within the molecule. The frontier molecular orbital analysis was performed to obtain global reactivity parameters. The molecular electrostatic potential was used to visualize the chemical reactivity and charge distribution of the molecule. From the nuclear magnetic resonance spectra, the carbon atoms bonded with nitrogen and oxygen atoms show the up-shielded peaks. The chemical implication of molecule was explained using electron localization function and localized orbital locator analysis. Moreover, the molecular docking studies performed for the newly synthesized compound revealed an efficient interaction with the acetylcholine receptor and exhibited better binding affinity than commercialized insecticides. Whilst, the physicochemical properties were assessed using the insectiPAD webtool to comprehend the insecticidal properties of title compound. Finally, the quantitative structure activity relationship study of 2-(4-nitrophenyl)-(3,5-difluoro-pyridine-6-yl)-1,3,4-oxadiazole showed higher activity (pLC50 = −1.43) against Mythimna separata, as compared to commercial insecticide avermectin (pLC50 = −1.47). These results provided that title compound could be used as a template for future insecticide investigations.

Structure-Activity Relationship Study of Majusculamide D: Overcoming Metabolic Instability and Severe Toxicity with a Fluoro Analogue

Majusculamide D, isolated from the marine cyanobacterium Moorea producens, is an anticancer lipopentapeptide consisting of fatty acid, tripeptide, and pyrrolyl proline moieties. In this work, by utilizing a convergent synthetic approach, late-stage modification, and bioisostere strategy, 26 majusculamide D analogues were synthesized, and two (1i and 1j) demonstrated IC50 values < 1 nM against PANC-1 cancer cells. The results summarized a preliminary structure-activity relationship mainly at the C23, C4, C34, and C10 sites. A series of in vitro assays, including wound healing, transwell, clone formation, EdU, and western blot, confirmed that majusculamide D inhibited the migration, invasion, and proliferation of pancreatic cancer cells. The optimized fluorinated analogue 1n demonstrated a notable enhancement in stability during the mouse plasma assay (>50% left after 24 h), exhibited tumor-suppressive effects (51.5% at a dosage of 5 mg/kg), and successfully mitigated the severe toxicity (no mouse dead) observed in the group treated with majusculamide D (3 mice dead) in a xenografted mouse model.

Concise Affinity-Based Purification of Ligated mRNA for Structure-Activity Relationship Studies of Nucleosugar Modification Patterns.

Position-specific nucleoside sugar modifications have been shown to improve the translational activity and stability of chemically synthesized mRNA. For pharmaceutical applications of chemically modified mRNAs, a rapid purification methodology is imperative to identify the optimal modification pattern. However, while the chemical synthesis of mRNAs can be accomplished by splint ligation of oligonucleotide fragments, the current purification method for ligated mRNAs based on denaturing polyacrylamide gel electrophoresis tends to be time consuming. In this study, we developed a two-step affinity purification method for rapid sample preparation. In this method, ligated mRNA is captured by oligo dT magnetic beads and streptavidin magnetic beads with 3'-biotinylated oligo DNA, which are complementary to the 3'-poly(A) and 5' terminal sequences of the target mRNA, respectively. Therefore, the target mRNA can be isolated from a complex mixture of splint ligations. Using this method, six sugar-modified mRNAs were simultaneously purified, and the translational activities of these mRNAs were evaluated immediately after purification. The results demonstrate that this methodology is suitable for the rapid preparation of various chemically synthesized mRNAs to identify their optimal modification patterns.

Identification of tanshinone I as a natural Cu(II) ionophore

The development of Cu(II) ionophores for targeted disruption of aberrant redox homeostasis in cancer cells has been considered an appealing strategy in the field of anticancer research. This study presents the first identification of tanshinone I (Ts1), a natural o-quinone, as a Cu(II) ionophore. Structure-activity relationship studies on tanshinones and mechanistic investigations reveal that the presence of Cu(II) effectively promotes the tautomerization of Ts1 from its diketo to keto-enol forms, thereby facilitating its sequential proton-loss Cu(II) chelation, and enabling it to function as a Cu(II) ionophore due to its structural features including the presence of an o-quinone moiety, a benzyl hydrogen, and a large conjugated system. The unique property allows Ts1 to preferentially induce copper accumulation in human hepatoma HepG2 cells over human umbilical vein endothelial cells, by releasing copper driven by reduced glutathione (GSH). This copper accumulation leads to a reduction in the GSH-to-oxidized glutathione ratio and the generation of reactive oxygen species, ultimately triggering apoptosis of HepG2 cells. The findings not only provide support for o-quinones as innovative types of anticancer Cu(II) ionophores, but also shed light on the previously unrecognized role of Ts1 as a potent Cu(II) ionophore for eradicating cancer cells by selectively disrupting their redox regulation programs, resembling a "Trojan horse".

Discovery and optimization of tetrahydroacridine derivatives as a novel class of antibiotics against multidrug-resistant Gram-positive pathogens by targeting type I signal peptidase and disrupting bacterial membrane

Increasing antimicrobial resistance underscores the urgent need for new antibiotics with unique mechanisms. Type I signal peptidase (SPase I) is crucial for bacterial survival and a promising target for antibiotics. Herein we designed and synthesized innovative tetrahydroacridine-9-carboxylic acid derivatives by optimizing the initial hit compound SP11 based on virtual screening. Structure-activity relationship (SAR) studies and bioactivity assessments identified compound C09 as a standout, showing excellent in vitro antimicrobial activity against MRSA and other multidrug-resistant Gram-positive pathogens. C09 targets SPase I with a favorable affinity, disrupts bacterial cell membranes, and eradicates biofilms, reducing resistance risk. In vivo tests in a murine MRSA skin infection model demonstrated significant efficacy. Additionally, C09 has good liver microsome metabolic stability, safe hemolytic activity and mammalian cytotoxicity, as well as a good in vivo safety profile. Overall, our findings highlight the potential of tetrahydroacridine-9-carboxylic acid derivatives as a novel class of antibiotics against multidrug-resistant Gram-positive bacteria.

Design and synthesis of camphor-thiazole derivatives as potent antifungal agents: structure-activity relationship and preliminary mechanistic study.

Plant pathogenic fungi pose a severe threat to crop yield and food security. This study aims to investigate the potential antifungal activity and mechanism of action of camphor-thiazole derivatives against six plant pathogenic fungi. A novel series of camphor-thiazole derivatives were designed, synthesized and evaluated for their antifungal effects against Rhizoctonia solani, Fusarium graminearum, Valsa mali, Alternaria solani, Colletotrichum orbiculare and Botryitis cinerea. Most of the synthesized camphor-thiazole derivatives exhibited notable antifungal activity. Amongst them, compounds C5, C10 and C17 showed significant activity against R. solani with median effective concentrations (EC50) values in the range 3-4 μg mL-1, demonstrating superior antifungal efficacy to the control drug boscalid (EC50 = 1.23 μg mL-1). Structure-activity relationship and density functional theory analysis emphasized the critical role of substituent selection in optimizing the biological activity of these compounds. Moreover, preliminary mechanistic studies revealed that compound C5 induced abnormal mycelial and cellular morphology in R. solani as observed using scanning and transmission electron microscopy, and triggered the production and accumulation of reactive oxygen species. Additionally, the increased concentration of C5 resulted in enhanced cell membrane permeability. In this study, the designed and optimized compound C5 emerged as a promising candidate for potent antifungal agents. The results demonstrate that synthesized camphor-thiazole derivatives possess potent antifungal activity and can serve as lead compounds for further optimization in antifungal agent development. © 2024 Society of Chemical Industry.

SAR Study on Thiolato-Bridged Triosmium Carbonyl Clusters: Higher Reactivity Does Not Equal Higher Antiproliferative Activity.

A structure-activity relationship study on triosmium clusters of the general formula Os3(μ-H)(μ-SC6H4X)(CO)10 (2-X, where X = o-, m- or p-NH2; o-, m- or p- OH; p-H, -Br, -NO2, -COOH or -CH2COOH) show that their antiproliferative activity is through the cluster core, and the nature and position of the phenyl ring substituent X shows a significant impact on the activity. Clusters with an electron-withdrawing group are more reactive but are thus quickly consumed through reaction with serum, while those with an electron-donating group persists sufficiently to enter the cells and result in higher antiproliferative activity. Interestingly, m-substituted clusters and those with lipophilicity >6.0 also exhibit higher antiproliferative activity. In contrast, o-substituted clusters capable of intramolecular hydrogen bonding have lower cytotoxicity. The cluster 2-m-OH, with higher antiproliferative activity and lower reactivity with serum, is a potential lead compound for further mode of action studies.

Unraveling GPCRs Allosteric Modulation. Cannabinoid 1 Receptor as a Case Study.

G-protein-coupled receptors (GPCRs) constitute one of the most prominent families of integral membrane receptor proteins that mediate most transmembrane signaling processes. Malfunction of these signal transduction processes is one of the underlying causes of many human pathologies (Parkinson's, Huntington's, heart diseases, etc), provoking that GPCRs are the largest family of druggable proteins. However, these receptors have been targeted traditionally by orthosteric ligands, which usually causes side effects due to the simultaneous targeting of homologous receptor subtypes. Allosteric modulation offers a promising alternative approach to circumvent this problematic and, thus, comprehending its details is a most important task. Here we use the Cannabinoid type-1 receptor (CB1R) in trying to shed light on this issue, focusing on positive allosteric modulation. This is done by using the protein-dipole Langevin-dipole (PDLD) within the linear response approximation (LRA) framework (PDLD/S-2000) along with our coarse-grained (CG) model of membrane proteins to evaluate the dissociation constants (KBs) and cooperativity factors (αs) for a diverse series of CB1R positive allosteric modulators belonging to the 2-phenylindole structural class, considering CP55940 as an agonist. The agreement with the experimental data evinces that significantly populated allosteric modulator:CB1R and allosteric modulator:CP55940:CB1R complexes have been identified and characterized successfully. Analyzing them, it has been determined that CB1R positive allosteric modulation lies in an outwards displacement of transmembrane α helix (TM) 4 extracellular end and in the regulation of the range of motion of a compound TM7 movement for binary and ternary complexes, respectively. In this respect, we achieved a better comprehension of the molecular architecture of CB1R positive allosteric site, identifying Lys1923.28 and Gly1943.30 as key residues regarding electrostatic interactions inside this cavity, and to rationalize (at both structural and molecular level) the exhibited stereoselectivity in relation to positive allosteric modulation activity by considered CB1R allosteric modulators. Additionally, putative/postulated allosteric binding sites have been screened successfully, identifying the real CB1R positive allosteric site, and most structure-activity relationship (SAR) studies of CB1R 2-phenylindole allosteric modulators have been rationalized. All these findings point out towards the predictive value of the methodology used in the current work, which can be applied to other biophysical systems of interest. The results presented in this study contribute significantly to understand GPCRs allosteric modulation and, hopefully, will encourage a more thorough exploration of the topic.

Structure–Activity Relationship Study of Splicing Modulators on Hsh155/SF3B1 through Chemical Synthesis and Yeast Genetics

Structure–Activity Relationship Study of Splicing Modulators on Hsh155/SF3B1 through Chemical Synthesis and Yeast Genetics

Optimized Production of Concanamycins Using a Rational Metabolic Engineering Strategy

Plecomacrolides, such as concanamycin and bafilomycin, are potent and specific inhibitors of vacuolar-type ATPase. Concanamycins are 18-membered macrolides with promising therapeutic potential against multiple diseases, including viral infection, osteoporosis, and cancer. Due to the complexity of their total synthesis, the production of concanamycins is only achieved through microbial fermentation. However, the low titers of concanamycin A and its analogs in the native producing strains are a significant bottleneck for scale-up, robust structure-activity relationship studies, and drug development. To address this challenge, we designed a library of engineered Streptomyces strains for the overproduction of concanamycins by combining the overexpression of target regulatory genes with the optimization of fermentation media. Integration of two endogenous regulators from the concanamycin biosynthetic gene cluster (cms) and one heterologous regulatory gene from the bafilomycin biosynthetic gene cluster into the attB site significantly increased production of concanamycin A and its low abundant analog concanamycin B in Streptomyces eitanensis. The highest titers reported to date were observed in the engineered S. eitanensis DHS10676, which produced over 900 mg/L of concanamycin A and 300 mg/L of concanamycin B. Heterologous overexpression of the identified target regulatory genes across a panel of Streptomyces spp., harboring a putative concanamycin biosynthetic gene cluster confirmed its identity, and significantly improved concanamycin A production in all tested strains. Strain engineering, optimization of fermentation, and extraction purification protocols enabled swift access to these structurally complex plecomacrolides for semi-synthetic medicinal chemistry-based approaches. Together, this work established a platform for robust overproduction of concanamycin analogs across species.

Accelerating Antimalarial Drug Discovery with a New High-Throughput Screen for Fast-Killing Compounds.

The urgent need for rapidly acting compounds in the development of antimalarial drugs underscores the significance of such compounds in overcoming resistance issues and improving patient adherence to antimalarial treatments. The present study introduces a high-throughput screening (HTS) approach using 1536-well plates, employing Plasmodium falciparum lactate dehydrogenase (PfLDH) combined with nitroreductase (NTR) and fluorescent probes to evaluate inhibition of the growth of the asexual blood stage of malaria parasites. This method was adapted to efficiently assess the speed of action profiling (SAP) in a 384-well plate format, streamlining the traditionally time-consuming screening process. By successfully screening numerous compounds, this approach identified fast-killing hits early in the screening process, addressing challenges associated with artemisinin-based combination therapies. The high-throughput SAP method is expected to be of value in continuously monitoring fast-killing properties during structure-activity relationship studies, expediting the identification and development of novel, rapidly acting antimalarial drugs within phenotypic drug discovery campaigns.