Low Nanomolar Inhibitors Research Articles

7-Aryl-triazolyl-substituted sulfocoumarins are potent, selective inhibitors of the tumor-associated carbonic anhydrase IX and XII

Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the tumor-associated ones hCA IX and XII.

Inhibition of carbonic anhydrase isoforms I, II, IV, VII and XII with carboxylates and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds

We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthranilic acids and trimellitic anhydride chloride, followed by reaction with primary amines and were tested for the inhibition of five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and XII, some of which are involved in serious pathologies (CA II, IV and XII in glaucoma; CA VII in epilepsy; CA XII in some solid tumors). The carboxylic acids were generally poor inhibitors of isoforms hCA I, II and IV but were highly effective, low nanomolar inhibitors of hCA VII and XII. The sulfonamides inhibited all isoforms significantly, and some of them were sub-nanomolar hCA VII inhibitors, although their isoform selectivity was lower compared to the carboxylates. This study proves that carboxylic acids incorporating a phthalic anhydride/phthalimide based scaffold may lead to isoform-selective inhibitors by applying the tail approach, mostly used up until now for obtaining sulfonamide, sulfamide and sulfamate CA inhibitors.

Anion and sulfonamide inhibition studies of an α-carbonic anhydrase from the Antarctic hemoglobinless fish Chionodraco hamatus.

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from the Antarctic hemoglobinless fish Chionodraco hamatus (icefish). The new enzyme, denominated ChaCA, has a good catalytic activity for the physiologic CO2 hydration to bicarbonate reaction, similar to that of the low activity human isoform hCA I, with a kcat of 5.3×10(5) s(-1), and a kcat/Km of 3.7×10(7) M(-1) s(-1). The enzyme was inhibited in the submillimolar range by most inorganic anions (cyanate, thiocyanate, cyanide, bicarbonate, halides), whereas sulfamide, sulfamate, phenylboronic/phenylarsonic acids were micromolar inhibitors, with KIs in the range of 9-77 μM. Many clinically used drugs, such as acetazolamide, methazolamide, dorzolamide, brinzolamide, topiramate and benzolamide were low nanomolar inhibitors, with KIs in the range of 39.1-77.6 nM. As the physiology of CO2/bicarbonate transport or the Root effect in this Antarctic fish are poorly understood at this moment, such inhibition data may give a more detailed insight in the role that CAs play in these phenomena, by the use of inhibitors described here as physiologic tools.

Discovery, Development, and SAR of Aminothiazoles as LIMK Inhibitors with Cellular Anti-Invasive Properties.

As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.

From ribavirin to NAD analogues and back to ribavirin in search for anticancer agents

Abstract Ribavirin, a broad-spectrum antiviral agent is used in the clinic alone or in combination with other antivirals and/or interferons. Numerous structural analogues of ribavirin have been developed, among them tiazofurin, which is inactive against viruses but is a potent anticancer drug. Tiazofurin was found to inhibit nicotinamide adenine dinucleotide (NAD)-dependent inosine monophosphate dehydrogenase (IMPDH) after metabolic conversion into tiazofurin adenine dinucleotide (TAD), which binds well but could not serve as IMPDH cofactor. TAD showed high selectivity against human IMPDH vs. other cellular dehydrogenases. Mycophenolic acid (MPA) was even more specific, binding at the cofactor-binding domain of IMPDH. Ribavirin adenine dinucleotide, however, did not show any significant inhibition at the enzymatic level. We synthesized numerous NAD analogues in which natural nicotinamide riboside was replaced by tiazofurin, MPA moiety, or benzamide riboside, and the adenosine moiety as well as the pyrophosphate linker were broadly modified. Some of these compounds were found to be low nanomolar inhibitors of the enzyme and sub-micromolar inhibitors of cancer cell line proliferation. The best were as potent as tyrosine kinase inhibitor gleevec heralded as a ‘magic bullet’ against chronic myelogenous leukemia. In recent years, ribavirin was rediscovered as a potential anticancer agent against number of tumors including leukemia. It was clearly established that its antitumor activity is related to the inhibition of an oncogene, the eukaryotic translation initiation factor (eIF4E).

Preclinical Pharmacokinetics and In Vitro Metabolism of Asunaprevir (BMS-650032), a Potent Hepatitis C Virus NS3 Protease Inhibitor

Asunaprevir (ASV; BMS-650032), a low nanomolar inhibitor of the hepatitis C virus (HCV) NS3 protease, is currently under development, in combination with other direct-acting antiviral (DAA) agents for the treatment of chronic HCV infection. Extensive nonclinical and pharmacokinetic studies have been conducted to characterize the ADME properties of ASV. ASV has a moderate to high clearance in preclinical species. In vitro reaction phenotyping studies demonstrated that the oxidative metabolism of ASV is primarily mediated via CYP3A4; however, studies in bile-duct cannulated rats and dogs suggest that biliary elimination may contribute to overall ASV clearance. ASV is shown to have hepatotropic disposition in all preclinical species tested (liver to plasma ratios >40). The translation of in vitro replicon potency to clinical viral load decline for a previous lead BMS-605339 was leveraged to predict a human dose of 2 mg BID for ASV. Clinical drug-drug interaction (DDI) studies have shown that at therapeutically relevant concentrations of ASV the potential for a DDI is minimal. The need for an interferon free treatment combined with ASV's initial clinical trial data support development of ASV as part of a fixed dose combination for the treatment of patients chronically infected with HCV genotype 1.

Reducing GBA2 Activity Ameliorates Neuropathology in Niemann-Pick Type C Mice.

The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two β-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1 -/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1 -/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1 -/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1 -/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.

Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

A set of heteroaryl-N-carbonylbenzenesulfonamides has been designed, synthesized, and screened as inhibitors of human carbonic anhydrases (hCAs). The new sulfonamide derivatives were tested against hCA I, hCA II, hCA VII, hCA IX, and hCA XII isoforms using acetazolamide (AAZ, 1) and topiramate (TPM, 2) as reference compounds. Six compounds were low nanomolar inhibitors of tumor-associated hCA IX isoform (Ki values < 10 nM); among them we identified three arylsulfonamides showing unexpected inefficacy over brain distributed hCA VII isoform (hCA IX/hCA VII selectivity ratio > 1500 for compound 5c). Thus, these compounds can offer the opportunity to highlight the interactions preventing the inhibition of hCA VII mainly expressed in central nervous system. Thereby, we used structural and computational techniques to study in depth the interaction with hCAs. In an effort to confirm the inhibitory action we determined crystal structures of five selected heteroaryl-N-carbonylbenzenesulfonamides (4a, 4b, 4e, 5c, and 5e) in complex with hCA II. Moreover, to explore the lack of inhibitory effects of selected compounds (e.g.4b and 5c) we also performed docking studies into hCA VII catalytic site.

Structure-Guided Design of Novel l-Cysteine Derivatives as Potent KSP Inhibitors.

Kinesin spindle protein (KSP), known as Hs Eg5, a member of the kinesin-5 family, plays an important role in the formation and maintenance of the bipolar spindle. We previously reported S-trityl-l-cysteine derivatives as selective KSP inhibitors. Here, we report further optimizations using docking modeling in the L5 allosteric binding site, which led to the discovery of several high affinity derivatives with two fused phenyl rings in the trityl group giving low nanomolar range KSP ATPase inhibition. The representative derivatives potently inhibited cell growth of HCT116 cells in correlation with KSP inhibitory activities and significantly suppressed tumor growth in the xenograft model in vivo.

Synthesis of sulfonamides incorporating piperazinyl-ureido moieties and their carbonic anhydrase I, II, IX and XII inhibitory activity.

By using SLC-0111 (4-fluorophenylureido-benzenesulfonamide), a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in Phase I clinical trials as an antitumor agent as lead molecule, a series of benzenesulfonamide derivatives incorporating ureido moieties was synthesized. The new compounds contain a 4-N-substituted piperazine fragment in which the ureido linker has been included, and were tested as inhibitors of the cytosolic human (h) hCA I and II isoforms, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. Depending on the substitution pattern at the piperazine ring, low nanomolar inhibitors were detected against all four isoforms, making the new class of sulfonamides of interest for various pharmacologic applications.

Synthesis and carbonic anhydrase I, II, IX and XII inhibitory activity of sulfamates incorporating piperazinyl-ureido moieties

A series of sulfamates were synthesized using as lead compound SLC-0111, a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in Phase I clinical trials. The new derivatives incorporated ureido moieties as spacers between the benzene sulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor, but the urea moieties were part of a substituted piperazine ring system. The derivatives (and some of their phenol precursors) were tested for the inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). Generally hCA I was not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 1.0–94.4nM), IX (KIs in the range of 0.91–36.9nM), and XII (KIs in the range of 1.0–84.5nM). The best substitution fragments at the piperazine ring included the following moieties: 3-methylphenyl, 2,3-dimethylphenyl, 4-methoxyphenyl, 6-arylpyrimidine-2-yl.

Discovery of dihydroisoquinolinone derivatives as novel inhibitors of the p53-MDM2 interaction with a distinct binding mode.

Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogs. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors.

Synthesis of Schiff base derivatives of 4-(2-aminoethyl)-benzenesulfonamide with inhibitory activity against carbonic anhydrase isoforms I, II, IX and XII.

Schiff base derivatives were obtained by reaction of 4-(2-aminoethyl)benzenesulfonamide with aromatic aldehydes. The corresponding secondary amine derivatives were also prepared by reduction of the imine compounds with NaBH4. These derivatives were investigated as inhibitors of four human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic isozymes hCA I and II, as well as, the transmembrane, tumor-associated hCA IX and XII. Some of the newly synthesised compounds showed effective inhibitory activities against these CA isozymes. Many low nanomolar inhibitors were detected against all isoforms among the secondary amines whereas the Schiff bases were by far less active compared to the corresponding reduced derivatives among all investigated isoforms.

The β-carbonic anhydrase from the malaria mosquito Anopheles gambiae is highly inhibited by sulfonamides

A β-carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from Anopheles gambiae, the mosquito species mainly involved in the transmission of malaria. The new enzyme, AgaCA, showed a significant catalytic activity for the physiologic reaction, CO2 hydration to bicarbonate and protons, with a kcat of 7.2×105s−1 and kcat/Km of 5.6×107M−1s−1, being thus similar to parasite β-CAs which were discovered earlier as drug targets for antifungal or anti-protozoan agents. An inhibition study of AgaCA with a panel of aromatic, aliphatic and heterocyclic sulfonamides allowed us to identify several low nanomolar inhibitors of the enzyme. Benzolamide and aminobenzolamide showed inhibition constants of 6.8–9.8nM, whereas a structurally related aromatic derivative, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide was the strongest inhibitor with a KI of 6.1nM. As β-CAs are not present in mammals, including humans, finding effective and selective A. gambiae CA inhibitors may lead to alternative procedures for controlling malaria by impairing the growth of its transmission vector, the mosquito.

The design, synthesis and biological evaluation of conformationally restricted 4-substituted-2,6-dimethylfuro[2,3-d]pyrimidines as multi-targeted receptor tyrosine kinase and microtubule inhibitors as potential antitumor agents

A series of eleven conformationally restricted, 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines was designed to explore the bioactive conformation required for dual inhibition of microtubule assembly and receptor tyrosine kinases (RTKs), and their biological activities are reported. All three rotatable single bonds in the lead compound 1 were sequentially restricted to address the role of each in SAR for microtubule and RTK inhibitory effects. Compounds 2, 3, 7 and 10 showed microtubule depolymerizing activity comparable to or better than the lead 1, some with nanomolar EC50 values. While compound 8 had no effect on microtubules, 8 and 10 both showed potent RTK inhibition with nanomolar IC50s. These compounds confirm that the bioactive conformation for RTK inhibition is different from that for tubulin inhibition. The tetrahydroquinoline analog 10 showed the most potent dual tubulin and RTK inhibitory activities (low nanomolar inhibition of EGFR, VEGFR2 and PDGFR-β). Compound 10 has highly potent activity against many NCI cancer cell lines, including several chemo-resistant cell lines, and could serve as a lead for further preclinical studies.

Nanomolar Inhibitors of the Transcription Factor STAT5b with High Selectivity over STAT5a

Src homology 2 (SH2) domains play a central role in signal transduction. Although many SH2 domains have been validated as drug targets, their structural similarity makes development of specific inhibitors difficult. The cancer-relevant transcription factors STAT5a and STAT5b are particularly challenging small-molecule targets because their SH2 domains are 93 % identical on the amino acid level. Here we present the natural product-inspired development of the low-nanomolar inhibitor Stafib-1, as the first small molecule which inhibits the STAT5b SH2 domain (Ki=44 nM) with more than 50-fold selectivity over STAT5a. The binding site of the core moiety of Stafib-1 was validated by functional analysis of point mutants. A prodrug of Stafib-1 was shown to inhibit STAT5b with high selectivity over STAT5a in tumor cells. Stafib-1 provides the first demonstration that naturally occurring SH2 domains with more than 90 % sequence identity can be selectively targeted with small organic molecules.

Swapped-domain constructs of the glycoprotein-41 ectodomain are potent inhibitors of HIV infection.

The conformational rearrangement of N- and C-heptad repeats (NHR, CHR) of the HIV-1 glycoprotein-41 (gp41) ectodomain into a trimer of hairpins triggers virus-cell fusion by bringing together membrane-spanning N- and C-terminal domains. Peptides derived from the NHR and CHR inhibit fusion by targeting a prehairpin intermediate state of gp41. Typically, peptides derived from the CHR are low nanomolar inhibitors, whereas peptides derived from the NHR are low micromolar inhibitors. Here, we describe the inhibitory activity of swapped-domain gp41 mimics of the form CHR-loop-NHR, which were designed to form reverse hairpin trimers exposing NHR grooves. We observed low nanomolar inhibition of HIV fusion in constructs that possessed the following properties: an extended NHR C-terminus, an exposed conserved hydrophobic pocket on the NHR, high helical content, and trimer stability. Low nanomolar activity was independent of CHR length. CD studies in membrane mimetic dodecylphosphocholine micelles suggested that bioactivity could be related to the ability of the inhibitors to interact with a membrane-associated prehairpin intermediate. The swapped-domain design resolves the problem of unstable and weakly active NHR peptides and suggests a different mechanism of action from that of CHR peptides in inhibition of HIV-1 fusion.

Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

A unified approach toward the rational design of selective low nanomolar human neutrophil elastase inhibitors

A computer-aided campaign boosted the discovery of potent human neutrophil elastase (HNE) inhibitors.

A class of 4-sulfamoylphenyl-ω-aminoalkyl ethers with effective carbonic anhydrase inhibitory action and antiglaucoma effects.

We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The structure–activity relationship was drawn for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms, whereas several isoform-selective were also identified. X-ray crystal structures of two new sulfonamides bound to the physiologically dominant CA II isoform showed the tails of these derivatives bound within the hydrophobic half of the enzyme active site through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate salt) showed effective IOP lowering properties in an animal model of glaucoma. Several fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a–c) or tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII.