Novel hybrid compounds of sclareol and doxorubicin as potential anticancer nanotherapy for glioblastoma.

Two novel hybrid compounds, CON1 and CON2, have been developed by combining sclareol (SC) and doxorubicin (DOX) into a single molecular entity. These hybrid compounds have a 1:1 molar ratio of covalently linked SC and DOX. They have demonstrated promising anticancer properties, especially in glioblastoma cells, and have also shown potential in treating multidrug-resistant (MDR) cancer cells that express the P-glycoprotein (P-gp) membrane transporter. CON1 and CON2 form nanoparticles, as confirmed by Zetasizer, transmission electron microscopy (TEM), and chemical modeling. TEM also showed that CON1 and CON2 can be found in glioblastoma cells, specifically in the cytoplasm, different organelles, nucleus, and nucleolus. To examine the anticancer properties, the U87 glioblastoma cell line, and its corresponding multidrug-resistant U87-TxR cell line, as well as patient-derived astrocytoma grade 3 cells (ASC), were used, while normal human lung fibroblasts were used to determine the selectivity. CON1 and CON2 exhibited better resistance and selectivity profiles than DOX, showing less cytotoxicity, as evidenced by real-time cell analysis, DNA damage determination, cell death induction, mitochondrial respiration, and mitochondrial membrane depolarization studies. Cell cycle analysis and the ß-galactosidase activity assay suggested that glioblastoma cells die by senescence following CON1 treatment. Overall, CON1 and CON2 showed great potential as they have better anticancer features than DOX. They are promising candidates for additional preclinical and clinical studies on glioblastoma.

Development of iminosugar-based glycosidase inhibitors as drug candidates for SARS-CoV-2 virus via molecular modelling and in vitro studies.

We developed new iminosugar-based glycosidase inhibitors against SARS-CoV-2. Known drugs (miglustat, migalastat, miglitol, and swainsonine) were chosen as lead compounds to develop three classes of glycosidase inhibitors (α-glucosidase, α-galactosidase, and mannosidase). Molecular modelling of the lead compounds, synthesis of the compounds with the highest docking scores, enzyme inhibition tests, and in vitro antiviral assays afforded rationally designed inhibitors. Two highly active α-glucosidase inhibitors were discovered, where one of them is the most potent iminosugar-based anti-SARS-CoV-2 agent to date (EC90 = 1.94 µM in A549-ACE2 cells against Omicron BA.1 strain). However, galactosidase inhibitors did not exhibit antiviral activity, whereas mannosidase inhibitors were both active and cytotoxic. As our iminosugar-based drug candidates act by a host-directed mechanism, they should be more resilient to drug resistance. Moreover, this strategy could be extended to identify potential drug candidates for other viral infections.

Influence of cation-π interactions to the structural stability of phycocyanin proteins: A computational study.

The influences of cation-π interactions in phycocyanin proteins and their environmental preferences were analyzed. The number of interactions formed by arginine showed to be higher than those formed by the lysine in the cationic group, while histidine is comparatively higher than phenylalanine and N-terminal residue in the π group. Arg-Tyr and Arg-Phe interacting pairs are predominant among the various pairs analyzed. Cation-π interactions are distance-dependent and can be realized above a wider area above the π ring. We analyzed the energy contribution resulting from cation-π interactions using ab initio calculations. The energy contribution resulting from the most frequent cation-π interactions was in the lower range of strong hydrogen bonds. The results showed that, while most of their interaction energies lay ranged from - 2 to - 8 kcal/mol, those energies could be up to -12- 12 kcal/mol. Stabilization centers for these proteins showed that all residues found in cation-π interactions are important in locating one or more of such centers. In the cation-π interacting residues, 54% of the amino acid residues involved in these interactions might be conserved in phycocyanins. From this study, we infer that cation-π forming residues play an important role in the stability of the multiply commercially used phycocyanin proteins and could help structural biologists and medicinal chemists to design better and safer drugs.

4-Aminoquinoline-Based Adamantanes as Potential Anticholinesterase Agents in Symptomatic Treatment of Alzheimer's Disease.

Considering that acetylcholinesterase (AChE) inhibition is the most important mode of action expected of a potential drug used for the treatment of symptoms of Alzheimer's disease (AD), our previous pilot study of 4-aminoquinolines as potential human cholinesterase inhibitors was extended to twenty-two new structurally distinct 4-aminoquinolines bearing an adamantane moiety. Inhibition studies revealed that all of the compounds were very potent inhibitors of AChE and butyrylcholinesterase (BChE), with inhibition constants (Ki) ranging between 0.075 and 25 µM. The tested compounds exhibited a modest selectivity between the two cholinesterases; the most selective for BChE was compound 14, which displayed a 10 times higher preference, while compound 19 was a 5.8 times more potent inhibitor of AChE. Most of the compounds were estimated to be able to cross the blood-brain barrier (BBB) by passive transport. Evaluation of druglikeness singled out fourteen compounds with possible oral route of administration. The tested compounds displayed modest but generally higher antioxidant activity than the structurally similar AD drug tacrine. Compound 19 showed the highest reducing power, comparable to those of standard antioxidants. Considering their simple structure, high inhibition of AChE and BChE, and ability to cross the BBB, 4-aminoquinoline-based adamantanes show promise as structural scaffolds for further design of novel central nervous system drugs. Among them, two compounds stand out: compound 5 as the most potent inhibitor of both cholinesterases with a Ki constant in low nano molar range and the potential to cross the BBB, and compound 8, which met all our requirements, including high cholinesterase inhibition, good oral bioavailability, and antioxidative effect. The QSAR model revealed that AChE and BChE inhibition was mainly influenced by the ring and topological descriptors MCD, Nnum, RP, and RSIpw3, which defined the shape, conformational flexibility, and surface properties of the molecules.

Novel TEAD1 gene variant in a Serbian family with Sveinsson's chorioretinal atrophy.

Sveinsson's chorioretinal atrophy (SCRA) or helicoidal peripapillary chorioretinal degeneration (HPCD) as previously referred, is a rare ocular disease with autosomal dominant pattern of inheritance. The vast majority of reported cases were of Icelandic origin but the characteristic clinical picture of SCRA was also described in patients of non-Icelandic descent. Here, we report a novel disease-causing variant c.1261T>A, p.Tyr421Asn in TEAD1, detected in a Serbian family from Bosnia diagnosed with SCRA. The newly discovered change occurred at the same position as the "Icelandic mutation" (c.1261T>C, p.Tyr421His). According to our findings, this position in the exon 13 of the TEAD1 gene, at base pair 94, should be considered as a mutation hotspot and a starting point for future genetic analyses of patients with SCRA diagnosis.

Cross-Reactive Effects of Vaccines: Heterologous Immunity between Tetanus and Chlamydia.

Vaccines can have heterologous effects on the immune system, i.e., effects other than triggering an immune response against the disease targeted by the vaccine. We investigated whether monoclonal antibodies (mAbs) specific for tetanus could cross-react with Chlamydia and confer heterologous protection against chlamydial infection. The capability of two tetanus-specific mAbs, namely mAb26 and mAb51, to prevent chlamydial infection has been assessed: (i) in vitro, by performing a neutralization assay using human conjunctival epithelial (HCjE) cells infected with Chlamydia trachomatis serovar B, and (ii) in vivo, by using a guinea pig model of Chlamydiacaviae-induced inclusion conjunctivitis. The mAb26 has been superior in comparison with mAb51 in the prevention of chlamydial infection in HCjE cells. The mAb26 has conferred ≈40% inhibition of the infection, compared to less than 5% inhibition in the presence of the mAb51. In vivo, mAb26 significantly diminished ocular pathology intensity in guinea pigs infected with C. caviae compared to either the mAb51-treated or sham-treated guinea pigs. Our data provide insights that tetanus immunization generates antibodies which induce heterologous chlamydial immunity and promote protection beyond the intended target pathogen.

Chemo- and biocatalytic esterification of marchantin A and cytotoxic activity of ester derivatives.

Chemical and biocatalytic synthesis of seven previously undescribed marchantin A ester derivatives has been presented. Chemical synthesis afforded three peresterified bisbibenzyl products (TE1-TE3), while enzymatic method, using lipase, produced regioselective monoester derivatives (ME1-ME4). The antiproliferative activities of all prepared derivatives of marchantin A were tested on MRC-5 healthy human lung fibroblast, A549 human lung cancer, and MDA-MB-231 human breast cancer cell lines. All tested esters were less cytotoxic in comparison to marchantin A, but they also exhibited lower cytotoxicity against healthy cells. Monoesters displayed higher cytotoxic activities than the corresponding peresterified products, presumably due to the presence of free catechol group. Monohexanoyl ester ME3 displayed the same IC50 like marchantin A against MDA-MB-231 cells, but the selectivity was higher. In this way, regioselective enzymatic monoesterification enhanced selectivity of marchantin A. ME3 was also the most active among all derivatives against lung cancer cells A549 with the slightly lower activity and selectivity in comparison to marchantin A.

N-Benzyl Derivatives of Long-Chained 4-Amino-7-chloro-quionolines as Inhibitors of Pyocyanin Production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a leading cause of nosocomial infections that are becoming increasingly difficult to treat due to the occurrence of antibiotic resistant strains. Since P. aeruginosa virulence is controlled through quorum sensing, small molecule treatments inhibiting quorum sensing signaling pathways provide a promising therapeutic option. Consequently, we synthesized a series of N-octaneamino-4-aminoquinoline derivatives to optimize this chemotype's antivirulence activity against P. aeruginosa via inhibition of pyocyanin production. The most potent derivative, which possesses a benzofuran substituent, provided effective inhibition of pyocyanin production (IC50 = 12 µM), biofilm formation (BFIC50 = 50 µM), and motility. Experimentally, the compound's activity is achieved through competitive inhibition of PqsR, and structure-activity data were rationalized using molecular docking studies.

Computational Analysis of Non-covalent Interactions in Phycocyanin Subunit Interfaces.

Protein-protein interactions are an important phenomenon in biological processes and functions. We used the manually curated non-redundant dataset of 118 phycocyanin interfaces to gain additional insight into this phenomenon using a robust inter-atomic non-covalent interaction analyzing tool PPCheck. Our observations indicate that there is a relatively high composition of hydrophobic residues at the interfaces. Most of the interface residues are clustered at the middle of the range which we call "standard-size" interfaces. Furthermore, the multiple interaction patterns founded in the present study indicate that more than half of the residues involved in these interactions participate in multiple and water-bridged hydrogen bonds. Thus, hydrogen bonds contribute maximally towards the stability of protein-protein complexes. The analysis shows that hydrogen bond energies contribute to about 88 % to the total energy and it also increases with interface size. Van der Waals (vdW) energy contributes to 9.3 %±1.7 % on average in these complexes. Moreover, there is about 1.9 %±1.5 % contribution by electrostatic energy. Nevertheless, the role by vdW and electrostatic energy could not be ignored in interface binding. Results show that the total binding energy is more for large phycocyanin interfaces. The normalized energy per residue was less than -16 kJ mol-1 , while most of them have energy in the range from -6 to -14 kJ mol-1 . The non-covalent interacting residues in these proteins were found to be highly conserved. Obtained results might contribute to the understanding of structural stability of this class of evolutionary essential proteins with increased practical application and future designs of novel protein-bioactive compound interactions.

Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity.

This paper discusses antioxidative and biological activities of 25 novel amidino substituted benzamides with a variety of heteroaromatic nuclei attached to the benzamide moiety and with a variable number of methoxy or hydroxy substituents. Targeted compounds, bearing either amidino or 2-imidazolinyl substituent, were obtained in the Pinner reaction from cyano precursors. 3D-QSAR models were generated to predict antioxidative activity of the 25 novel aromatic and heteroaromatic benzamide derivatives. The compounds were tested for antioxidative activity using in vitro spectrophotometric assays. Direct validation of 3D-QSAR approach for predicting activities of novel benzamide derivatives was carried out by comparing experimental and computationally predicted antioxidative activity. Experimentally determined activities for all novel compounds were found to be within a standard deviation of error of the models. Following this, structure-activity relationships among the synthesized compounds are discussed. Furthermore, antiproliferative activity in vitro against HeLa cells as well as antibacterial and antifungal activity was tested to confirm the other biological activities of the prepared compounds.

Bis-guanylhydrazones as efficient anti-Candida compounds through DNA interaction.

Candida spp. are leading causes of opportunistic mycoses, including life-threatening hospital-borne infections, and novel antifungals, preferably aiming targets that have not been used before, are constantly needed. Hydrazone- and guanidine-containing molecules have shown a wide range of biological activities, including recently described excellent antifungal properties. In this study, four bis-guanylhydrazone derivatives (BG1-4) were generated following a previously developed synthetic route. Anti-Candida (two C. albicans, C. glabrata, and C. parapsilosis) minimal inhibitory concentrations (MICs) of bis-guanylhydrazones were between 2 and 15.6 µg/mL. They were also effective against preformed 48-h-old C. albicans biofilms. In vitro DNA interaction, circular dichroism, and molecular docking analysis showed the great ability of these compounds to bind fungal DNA. Competition with DNA-binding stain, exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane, and activation of metacaspases were shown for BG3. This pro-apoptotic effect of BG3 was only partially due to the accumulation of reactive oxygen species in C. albicans, as only twofold MIC and higher concentrations of BG3 caused depolarization of mitochondrial membrane which was accompanied by the decrease of the activity of fungal mitochondrial dehydrogenases, while the activity of oxidative stress response enzymes glutathione reductase and catalase was not significantly affected. BG3 showed synergistic activity with amphotericin B with a fractional inhibitory concentration index of 0.5. It also exerted low cytotoxicity and the ability to inhibit epithelial cell (TR146) invasion and damage by virulent C. albicans SC5314. With further developments, BG3 may further progress in the antifungal pipeline as a DNA-targeting agent.

Anion-π interactions in active centers of superoxide dismutases.

We investigated 1060 possible anion-π interactions in a data set of 41 superoxide dismutase active centers. Our observations indicate that majority of the aromatic residues are capable to form anion-π interactions, mainly by long-range contacts, and that there is preference of Trp over other aromatic residues in these interactions. Furthermore, 68% of total predicted interactions in the dataset are multiple anion-π interactions. Anion-π interactions are distance and orientation dependent. We analyzed the energy contribution resulting from anion-π interactions using ab initio calculations. The results showed that, while most of their interaction energies lay in the range from -0 to -4kcalmol-1, those energies can be up to -9kcalmol-1 and about 34% of interactions were found to be repulsive. Majority of the suggested anion-π interacting residues in ternary complexes are metal-assisted. Stabilization centers for these proteins showed that all the six residues found in predicted anion-π interactions are important in locating one or more of such centers. The anion-π interacting residues in these proteins were found to be highly conserved. We hope that these studies might contribute useful information regarding structural stability and its interaction in future designs of novel metalloproteins.

Human serum albumin binding of certain antimalarials.

Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37°C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

Long-Chain 4-Aminoquinolines as Quorum Sensing Inhibitors in Serratia marcescens and Pseudomonas aeruginosa.

Antibiotic resistance has become a serious global threat to public health; therefore, improved strategies and structurally novel antimicrobials are urgently needed to combat infectious diseases. Here we report a new type of highly potent 4-aminoquinoline derivatives as quorum sensing inhibitors in Serratia marcescens and Pseudomonas aeruginosa, exhibiting weak bactericidal activities (minimum inhibitory concentration (MIC) > 400 µM). Through detailed structure-activity study, we have identified 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines (5 and 10) as biofilm formation inhibitors with 50% biofilm inhibition at 69 µM and 63 µM in S. marcescens and P. aeruginosa, respectively. These two compounds, 5 and 10, are the first quinoline derivatives with anti-biofilm formation activity reported in S. marcescens. Quantitative structure-activity relationship (QSAR) analysis identified structural descriptors such as Wiener indices, hyper-distance-path index (HDPI), mean topological charge (MTC), topological charge index (TCI), and log D(o/w)exp as the most influential in biofilm inhibition in this bacterial species. Derivative 10 is one of the most potent quinoline type inhibitors of pyocyanin production described so far (IC50 = 2.5 µM). While we have demonstrated that 5 and 10 act as Pseudomonas quinolone system (PQS) antagonists, the mechanism of inhibition of S. marcescens biofilm formation with these compounds remains open since signaling similar to P. aeruginosa PQS system has not yet been described in Serratia and activity of these compounds on acylhomoserine lactone (AHL) signaling has not been detected. Our data show that 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines present the promising scaffolds for developing antivirulence and anti-biofilm formation agents against multidrug-resistant bacterial species.

α-Glucosidase inhibitory activity and cytotoxic effects of some cyclic urea and carbamate derivatives.

The inhibitory activities of selected cyclic urea and carbamate derivatives (1-13) toward α-glucosidase (α-Gls) in in vitro assay were examined in this study. All examined compounds showed higher inhibitory activity (IC50) against α-Gls compared to standard antidiabetic drug acarbose. The most potent was benzyl (3,4,5-trimethoxyphenyl)carbamate (12) with IC50 = 49.85 ± 0.10 µM. In vitro cytotoxicity of the investigated compounds was tested on three human cancer cell lines HeLa, A549 and MDA-MB-453 using MTT assay. The best antitumour activity was achieved with compound 2 (trans-5-phenethyl-1-phenylhexahydro-1H-imidazo[4,5-c]pyridin-2(3H)-one) against MDA-MB-453 human breast cancer cell line (IC50 = 83.41 ± 1.60 µM). Cyclic ureas and carbamates showed promising anti-α-glucosidase activity and should be further tested as potential antidiabetic drugs. The PLS model of preliminary QSAR study indicated that, in planing the future synthesis of more potent compounds, the newly designed should have the substituents capable of polar interactions with receptor sites in various positions, while avoiding the increase of their lipophilicity.

Anion-π interactions in complexes of proteins and halogen-containing amino acids.

We analyzed the potential influence of anion-π interactions on the stability of complexes of proteins and halogen-containing non-natural amino acids. Anion-π interactions are distance and orientation dependent and our ab initio calculations showed that their energy can be lower than -8 kcal mol(-1), while most of their interaction energies lie in the range from -1 to -4 kcal mol(-1). About 20 % of these interactions were found to be repulsive. We have observed that Tyr has the highest occurrence among the aromatic residues involved in anion-π interactions, while His made the least contribution. Furthermore, our study showed that 67 % of total interactions in the dataset are multiple anion-π interactions. Most of the amino acid residues involved in anion-π interactions tend to be buried in the solvent-excluded environment. The majority of the anion-π interacting residues are located in regions with helical secondary structure. Analysis of stabilization centers for these complexes showed that all of the six residues capable of anion-π interactions are important in locating one or more of such centers. We found that anion-π interacting residues are sometimes involved in simultaneous interactions with halogens as well. With all that in mind, we can conclude that the anion-π interactions can show significant influence on molecular organization and on the structural stability of the complexes of proteins and halogen-containing non-natural amino acids. Their influence should not be neglected in supramolecular chemistry and crystal engineering fields as well.

Quantitative structure retention/activity relationships of biologically relevant 4-amino-7-chloroquinoline based compounds.

The chromatographic behaviour of series of 4-amino-7-chloroquinoline (4,7-ACQ) based compounds was studied by reversed-phase thin-layer chromatography (RPTLC) with binary mobile phases containing water and the organic modifiers, DMSO or acetone. The lipophilicity of the studied compounds was determined by extrapolation of retention parameters RM to pure water content in mobile phase. In order to obtain some basic insight into the chromatographic behaviour and structural features of investigated compounds, PCA was performed on both chromatographic data (RM values) and calculated 2D and 3D structural descriptors. Both QSRR and QSAR models were built by means of the partial least squares (PLS) statistical method. It was found that descriptors which encode hydrophobic (dispersive) interactions have positive influence on retention, while influence of descriptors encoding polar interactions was negative. According to the obtained PLS model for inhibition of botulinum neurotoxin serotype A light chain, hydrophobic interactions influence positively on the mechanism of action of the investigated 4,7-ACQ, while polar interactions are less favoured. Contrary, the results of PLS modelling of activity against Plasmodium falciparum strains (W2, D6 and TM91C235) indicate that higher polarity of 4,7-ACQ contribute to their higher antimalarial activity.

Reinvestigating Old Pharmacophores: Are 4-Aminoquinolines and Tetraoxanes Potential Two-Stage Antimalarials?

The syntheses and antiplasmodial activities of various substituted aminoquinolines coupled to an adamantane carrier are described. The compounds exhibited pronounced in vitro and in vivo activity against Plasmodium berghei in the Thompson test. Tethering a fluorine atom to the aminoquinoline C(3) position afforded fluoroaminoquinolines that act as intrahepatocytic parasite inhibitors, with compound 25 having an IC50 = 0.31 µM and reducing the liver load in mice by up to 92% at 80 mg/kg dose. Screening our peroxides as inhibitors of liver stage infection revealed that the tetraoxane pharmacophore itself is also an excellent liver stage P. berghei inhibitor (78: IC50 = 0.33 µM). Up to 91% reduction of the parasite liver load in mice was achieved at 100 mg/kg. Examination of tetraoxane 78 against the transgenic 3D7 strain expressing luciferase under a gametocyte-specific promoter revealed its activity against stage IV-V Plasmodium falciparum gametocytes (IC50 = 1.16 ± 0.37 µM). To the best of our knowledge, compounds 25 and 78 are the first examples of either an 4-aminoquinoline or a tetraoxane liver stage inhibitors.

Is increased susceptibility to Balkan endemic nephropathy in carriers of common GSTA1 (*A/*B) polymorphism linked with the catalytic role of GSTA1 in ochratoxin a biotransformation? Serbian case control study and in silico analysis.

Although recent data suggest aristolochic acid as a putative cause of Balkan endemic nephropathy (BEN), evidence also exists in favor of ochratoxin A (OTA) exposure as risk factor for the disease. The potential role of xenobiotic metabolizing enzymes, such as the glutathione transferases (GSTs), in OTA biotransformation is based on OTA glutathione adducts (OTHQ-SG and OTB-SG) in blood and urine of BEN patients. We aimed to analyze the association between common GSTA1, GSTM1, GSTT1, and GSTP1 polymorphisms and BEN susceptibility, and thereafter performed an in silico simulation of particular GST enzymes potentially involved in OTA transformations. GSTA1, GSTM1, GSTT1 and GSTP1 genotypes were determined in 207 BEN patients and 138 non-BEN healthy individuals from endemic regions by polymerase chain reaction (PCR). Molecular modeling in silico was performed for GSTA1 protein. Among the GST polymorphisms tested, only GSTA1 was significantly associated with a higher risk of BEN. Namely, carriers of the GSTA1*B gene variant, associated with lower transcriptional activation, were at a 1.6-fold higher BEN risk than those carrying the homozygous GSTA1*A/*A genotype (OR = 1.6; p = 0.037). In in silico modeling, we found four structures, two OTB-SG and two OTHQ-SG, bound in a GSTA1 monomer. We found that GSTA1 polymorphism was associated with increased risk of BEN, and suggested, according to the in silico simulation, that GSTA1-1 might be involved in catalyzing the formation of OTHQ-SG and OTB-SG conjugates.

Second generation steroidal 4-aminoquinolines are potent, dual-target inhibitors of the botulinum neurotoxin serotype A metalloprotease and P. falciparum malaria.

Significantly more potent second generation 4-amino-7-chloroquinoline (4,7-ACQ) based inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain were synthesized. Introducing an amino group at the C(3) position of the cholate component markedly increased potency (IC50 values for such derivatives ranged from 0.81 to 2.27 µM). Two additional subclasses were prepared: bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives; both classes provided inhibitors with nanomolar-range potencies (e.g., the Ki of compound 67 is 0.10 µM). During BoNT/A challenge using primary neurons, select derivatives protected SNAP-25 by up to 89%. Docking simulations were performed to rationalize the compounds' in vitro potencies. In addition to specific residue contacts, coordination of the enzyme's catalytic zinc and expulsion of the enzyme's catalytic water were a consistent theme. With respect to antimalarial activity, the compounds provided better IC90 activities against chloroquine resistant (CQR) malaria than CQ, and seven compounds were more active than mefloquine against CQR strain W2.