Structure‐Based Design of a Eukaryote‐Selective Antiprotozoal Fluorinated Aminoglycoside

Aminoglycosides (AG) are antibiotics that lower the accuracy of protein synthesis by targeting a highly conserved RNA helix of the ribosomal A‐site. The discovery of AGs that selectively target the eukaryotic ribosome, but lack activity in prokaryotes, are promising as antiprotozoals for the treatment of neglected tropical diseases, and as therapies to read‐through point‐mutation genetic diseases. However, a single nucleobase change A1408G in the eukaryotic A‐site leads to negligible affinity for most AGs. Herein we report the synthesis of 6′‐fluorosisomicin, the first 6′‐fluorinated aminoglycoside, which specifically interacts with the protozoal cytoplasmic rRNA A‐site, but not the bacterial A‐site, as evidenced by X‐ray co‐crystal structures. The respective dispositions of 6′‐fluorosisomicin within the bacterial and protozoal A‐sites reveal that the fluorine atom acts only as a hydrogen‐bond acceptor to favorably interact with G1408 of the protozoal A‐site. Unlike aminoglycosides containing a 6′‐ammonium group, 6′‐fluorosisomicin cannot participate in the hydrogen‐bonding pattern that characterizes stable pseudo‐base‐pairs with A1408 of the bacterial A‐sites. Based on these structural observations it may be possible to shift the biological activity of aminoglycosides to act preferentially as antiprotozoal agents. These findings expand the repertoire of small molecules targeting the eukaryotic ribosome and demonstrate the usefulness of fluorine as a design element.     

Structure‐Based and in silico Design of Hsp90 Inhibitors

The molecular chaperone Hsp90 is responsible for activation and stabilization of several oncoproteins in cancer cells, and has emerged as an important target in cancer treatment because of this pivotal role. In recent years, interests have arisen around structure‐based design of small molecules aimed at inhibiting the chaperone activity of Hsp90. In this review, we illustrate the recent advances in structure‐based and in silico strategies aimed at discovering and optimizing Hsp90 inhibitors.     

Truncation and Optimisation of Peptide Inhibitors of Cyclin‐Dependent Kinase 2‐Cyclin A Through Structure‐Guided Design

Peptides that inhibit cyclin‐dependent kinase 2 by blocking the macromolecular substrate recruitment site of cyclin A were simplified, for example, by replacement of dipeptide units with β‐amino acids. The smallest inhibitor retaining activity was a tripeptide, whose binding mode was confirmed by X‐ray crystallography. This result suggests that nonpeptidic cyclin groove inhibitors may be feasible therapeutic agents.

     

Structure‐Based Design of New KSP‐Eg5 Inhibitors Assisted by a Targeted Multicomponent Reaction

An integrated multidisciplinary approach that combined structure‐based drug design, multicomponent reaction synthetic approaches and functional characterization in enzymatic and cell assays led to the discovery of new kinesin spindle protein (KSP) inhibitors with antiproliferative activity. A focused library of new benzimidazoles obtained by a Ugi+Boc removal/cyclization reaction sequence generated low‐micromolar‐range KSP inhibitors as promising anticancer prototypes. The design and functional studies of the new chemotypes were assessed by computational modeling and molecular biology techniques. The most active compounds— 20 (IC₅₀=1.49 μM , EC₅₀=3.63 μM ) and 22 (IC₅₀=1.37 μM , EC₅₀=6.90 μM )—were synthesized with high efficiency by taking advantage of the multicomponent reactions.     

Sugar‐Based Arylsulfonamide Carboxylates as Selective and Water‐Soluble Matrix Metalloproteinase‐12 Inhibitors

Matrix metalloproteinase‐12 (MMP‐12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a β‐N‐acetyl‐d ‐glucosamine moiety, were designed and synthesized as MMP‐12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP‐12 inhibitor with improved water solubility, compound 3 [(R)‐2‐(N‐(2‐(3‐(2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl)thioureido)ethyl)biphenyl‐4‐ylsulfonamido)‐3‐methylbutanoic acid], was identified.     

Structure‐Based Optimization of Aldose Reductase Inhibitors Originating from Virtual Screening

Virtual screening discovered two prospective hits as potential leads for aldose reductase inhibition. Based on their crystal structures with the enzyme, a systematic optimization has been performed to reveal a first structure–activity relationship. A central thiophen moiety and a terminal nitro group exhibit the best binding properties.

     

Synthesis and Quantitative Structure–Activity Relationships of Selective BCRP Inhibitors

The breast cancer resistance protein (BCRP/ABCG2) is a member of the ABC transporter superfamily. This protein has a number of physiological functions, including protection of the human body from xenobiotics. The overexpression of BCRP in certain tumor cell lines causes cross‐resistance against various drugs used in chemotherapeutic treatment. In a previous work we showed that a new class of compounds derived from XR9576 (tariquidar) selectively inhibits BCRP. In this work we synthesized more members of this class, with modification on the second and third aromatic rings. The inhibitory activities against BCRP and P‐gp were assayed using a Hoechst 33342 assay for BCRP and a calcein AM assay for P‐gp. Finally, quantitative structure–activity relationships for both aromatic rings were established. The results obtained show the importance of the electron density on the third aromatic ring, influenced by substituents, pointing to interactions with aromatic residues of the protein binding site. In the second aromatic ring the activity of compounds is influenced by the steric volume of the substituents.     

Design,Synthesis, and Biological Evaluation of Tetrahydro‐β‐carboline Derivatives as Selective Sub‐Nanomolar Gelatinase Inhibitors

Targeting matrix metalloproteinases (MMPs) is a pursued strategy for treating several pathological conditions, such as multiple sclerosis and cancer. Herein, a series of novel tetrahydro‐β‐carboline derivatives with outstanding inhibitory activity toward MMPs are present. In particular, compounds 9 f , 9 g , 9 h and 9 i show sub‐nanomolar IC₅₀ values. Interestingly, compounds 9 g and 9 i also provide remarkable selectivity toward gelatinases; IC₅₀=0.15 nm for both toward MMP‐2 and IC₅₀=0.63 and 0.58 nm , respectively, toward MMP‐9. Molecular docking simulations, performed by employing quantum mechanics based partial charges, shed light on the rationale behind binding involving specific interactions with key residues of S1′ and S3′ domains. Taken together, these studies indicate that tetrahydro‐β‐carboline represents a promising scaffold for the design of novel inhibitors able to target MMPs and selectively bias gelatinases, over the desirable range of the pharmacokinetics spectrum.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

Identification of HDAC6‐Selective Inhibitors of Low Cancer Cell Cytotoxicity

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform‐selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first‐generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell‐cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole‐based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1–3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.     

Structure–Activity Relationship of Tumor‐Selective 5‐Substituted 2‐Amino‐3‐carboxymethylthiophene Derivatives

Methyl‐2‐amino‐5‐[2‐(4‐methoxyphenethyl)]thiophene‐3‐carboxylate ( 8 c ) is the prototype of a well‐defined class of tumor‐selective agents. Compound 8 c preferentially inhibited the proliferation of a number of tumor cell lines including many human T‐lymphoma/leukemia cells, but also several prostate, renal, central nervous system and liver tumor cell types. Instead, a broad variety of other tumor cell lines including B‐lymphomas and HeLa cells were not affected. The tumor selectivity (TS; selectivity index or preferential suppression of CEM lymphoma (IC₅₀=0.90 μM ) versus HeLa tumor cell carcinoma (IC₅₀=39 μM )) amounted up to ～43 for 8 c . At higher concentrations, the compound proved cytotoxic rather than cytostatic. The antiproliferative potency and selectivity of 8 c could be preserved by replacing the ethyl linker between the 2‐amino‐3‐carboxymethylthiophene and the substituted aryl by a thioalkyl but not by an oxyalkyl nor an aminoalkyl. Among >50 novel 8 c derivatives, the 5‐(4‐ethyl‐ and 4‐isopropylarylmethylthio)thiophene analogues, methyl‐2‐amino‐5‐((4‐ethylphenylthio)methyl)thiophene‐3‐carboxylate ( 13 m ) and methyl‐2‐amino‐5‐((4‐isopropylphenylthio)methyl)thiophene‐3‐carboxylate ( 13 n ), were more potent (IC₅₀: 0.3–0.4 μM ) and selective (TS: 100–144) anti‐T‐lymphoma/leukemia agents than the prototype compound.     

The Development of Selective Inhibitors of NagZ: Increased Susceptibility of Gram‐Negative Bacteria to β‐Lactams

The increasing incidence of inducible chromosomal AmpC β‐lactamases within the clinic is a growing concern because these enzymes deactivate a broad range of even the most recently developed β‐lactam antibiotics. As a result, new strategies are needed to block the action of this antibiotic resistance enzyme. Presented here is a strategy to combat the action of inducible AmpC by inhibiting the β‐glucosaminidase NagZ, which is an enzyme involved in regulating the induction of AmpC expression. A divergent route facilitating the rapid synthesis of a series of N‐acyl analogues of 2‐acetamido‐2‐deoxynojirimycin is reported here. Among these compounds are potent NagZ inhibitors that are selective against functionally related human enzymes. These compounds reduce minimum inhibitory concentration values for β‐lactams against a clinically relevant Gram‐negative bacterium bearing inducible chromosomal AmpC β‐lactamase, Pseudomonas aeruginosa. The structure of a NagZ–inhibitor complex provides insight into the molecular basis for inhibition by these compounds.     

High‐Throughput Virtual Screening Using Quantum Mechanical Probes: Discovery of Selective Kinase Inhibitors

A procedure based on semi‐empirical quantum mechanical (QM) calculations of interaction energy is proposed for the rapid screening of compound poses generated by high‐throughput docking. Small molecules (consisting of 2–10 atoms and termed “probes”) are overlapped with polar groups in the binding site of the protein target. The interaction energy values between each compound pose and the probes, calculated by a semi‐empirical Hamiltonian, are used as filters. The QM probe method does not require fixed partial charges and takes into account polarization and charge‐transfer effects which are not captured by conventional force fields. The procedure is applied to screen ～100 million poses (of 2.7 million commercially available compounds) obtained by high‐throughput docking in the ATP binding site of the tyrosine kinase erythropoietin‐producing human hepatocellular carcinoma receptor B4 (EphB4). Three QM probes on the hinge region and one at the entrance pocket are employed to select for binding affinity, while a QM probe on the side chain of the so‐called gatekeeper residue (a hypervariable residue in the kinome) is used to enforce selectivity. The poses with favorable interactions with the five QM probes are filtered further for hydrophobic matching and low ligand strain. In this way, a single‐digit micromolar inhibitor of EphB4 with a relatively good selectivity profile is identified in a multimillion‐compound library upon experimental tests of only 23 molecules.     

Structure–Activity Relationships of Benzenesulfonamide‐Based Inhibitors towards Carbonic Anhydrase Isoform Specificity

Carbonic anhydrases (CAs) are implicated in a wide range of diseases, including the upregulation of isoforms CA IX and XII in many aggressive cancers. However, effective inhibition of disease‐implicated CAs should minimally affect the ubiquitously expressed isoforms, including CA I and II, to improve directed distribution of the inhibitors to the cancer‐associated isoforms and reduce side effects. Four benzenesulfonamide‐based inhibitors were synthesized by using the tail approach and displayed nanomolar affinities for several CA isoforms. The crystal structures of the inhibitors bound to a CA IX mimic and CA II are presented. Further in silico modeling was performed with the inhibitors docked into CA I and XII to identify residues that contributed to or hindered their binding interactions. These structural studies demonstrated that active‐site residues lining the hydrophobic pocket, especially positions 92 and 131, dictate the positional binding and affinity of inhibitors, whereas the tail groups modulate CA isoform specificity. Geometry optimizations were performed on each ligand in the crystal structures and showed that the energetic penalties of the inhibitor conformations were negligible compared to the gains from active‐site interactions. These studies further our understanding of obtaining isoform specificity when designing small molecule CA inhibitors.     

Structure‐Based Design of a Monosaccharide Ligand Targeting Galectin‐8

Galectin‐8 is a β‐galactoside‐recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3‐O‐[1‐carboxyethyl]‐β‐d ‐galactopyranoside (compound 6 ) as a ligand to target the N‐terminal domain of galectin‐8 (galectin‐8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3′‐O‐sialylated lactose (3′‐SiaLac), with galectin‐8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin‐8N for 6 was 32.8 μm , whereas no significant affinity was detected for the C‐terminal domain of galectin‐8 (galectin‐8C). The crystal structure of the galectin‐8N– 6 complex validated the predicted binding conformation and revealed the exact protein–ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin‐8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide‐based scaffold as a binder of galectin‐8.