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Multivalent nanoconstructs, extensively used for enhancing the recognition of biomolecular targets, have been recently exploited for enzyme inhibition showing interesting properties such as improvement of inhibitory potency and selectivity. We review herein the recent results highlighting the potential of multivalent nanoconstructs for the inhibition of different enzymes, and the emerging trends in the generation and identification of multivalent clusters as enzyme inhibitors.

Multifunctional silica nanoparticles decorated with fluorescent and sulfonamide carbonic anhydrase (CA) inhibitors were prepared and investigated as multivalent enzyme inhibitors against the cytosolic isoforms hCA I and II and the transmembrane tumor-associated ones hCA IX and XII. Excellent inhibitory effects were observed with these nanoparticles, with KI values in the low nanomolar range (6.2-0.67 nM) against all tested isozymes. A significant multivalency effect was seen for the inhibition of the monomeric enzymes hCA I and II compared to the dimeric hCA IX and hCA XII isoforms, where no multivalent effect was observed, suggesting that the multivalent binding is occurring through enzyme clustering., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histidinol dehydrogenase (HDH) has been established as a virulence factor for the human pathogen bacterium Brucella suis. Targeting such a virulence factor is a relevant anti-infectious approach as it could decrease the frequency of antibiotic resistance appearance. In this paper, we describe the synthesis of a family of oxo- and thioxo-imidazo[1,5-c]pyrimidines, potential enzyme inhibitors. Beyond their anti-HDH activity, the synthesis approach of these molecules, never described before, is highly original and these oxo- and thioxo- derivatives can improve dramatically the efficiency of the histidine protection pathway for the synthesis of histidine analogues., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

L-Histidinol dehydrogenase from Brucella suis (BsHDH) is an enzyme involved in the histidine biosynthesis pathway which is absent in mammals, thus representing a very interesting target for the development of anti-Brucella agents. In this paper we report the crystallographic structure of a mutated form of BsHDH both in its unbound form and in complex with a nanomolar inhibitor. These studies provide the first structural background for the rational design of potent HDH inhibitors, thus offering new hints for clinical applications., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

A series of nitroimidazoles incorporating sulfonamide/sulfamide/sulfamate moieties were designed and synthesized as radio/chemosensitizing agent targeting the tumor-associated carbonic anhydrase (CA) isoforms IX and XII. Most of the new compounds were nanomolar inhibitors of these isoforms. Crystallographic studies on the complex of hCA II with the lead sulfamide derivative of this series clarified the binding mode of this type of inhibitors in the enzyme active site cavity. Some of the best nitroimidazole CA IX inhibitors showed significant activity in vitro by reducing hypoxia-induced extracellular acidosis in HT-29 and HeLa cell lines. In vivo testing of the lead molecule in the sulfamide series, in cotreatment with doxorubicin, demonstrated a chemosensitization of CA IX containing tumors. Such CA inhibitors, specifically targeting the tumor-associated isoforms, are candidates for novel treatment strategies against hypoxic tumors overexpressing extracellular CA isozymes.

Pazopanib, a new, multi-targeted tyrosine kinase inhibitor (TKI) used clinically for the treatment of several types of tumors, incorporates a primary sulfonamide moiety normally associated with the inhibition of the metallo enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we show that pazopanib and related sulfonamides such as indisulam, acetazolamide or ureido-substituted peptidomimetic benzenesulfonamides are low nanomolar inhibitors of many of the fifteen human isoforms hCA I-XIV. These data indicate that in addition to the TK inhibitory action, pazopanib may exert antitumor/antimetastatic effects also due to the potent inhibition of the tumor-associated, hypoxia-inducible enzymes CA IX and XII.

In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity, catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The inhibition of this virulence factor by substituted benzylic ketones was a proof of concept that disarming bacteria leads to inhibition of intracellular bacterial growth in macrophage infection. This work describes the design, synthesis and evaluation of 19 new potential HDH inhibitors, using a combination of classical approaches and docking studies. The IC(50)-values of these inhibitors on HDH activity were in the nanomolar range, and several of them showed a 70-100% inhibition of Brucella growth in minimal medium. One selected compound yielded a strong inhibitory effect on intracellular replication of B. suis in human macrophages at concentrations as low as 5 μM, with an overall survival of intramacrophagic bacteria reduced by a factor 10(3). Docking studies with two inhibitors showed a good fitting in the catalytic pocket and also interaction with the second lipophilic pocket binding the cofactor NAD(+). Experimental data confirmed competition between inhibitors and NAD(+) at this site. Hence, these inhibitors can be considered as promising tools in the development of novel anti-virulence drugs.

Histidinol dehydrogenase (HDH, EC EC1.1.1.23) catalyses the final step in the biosynthesis of histidine and constitutes an attractive novel target for the development of new agents against the pathogenous, bacteria Brucella suis. A small library of new HDH inhibitors based on the L-histidinylphenylsulfonyl hydrazide scaffold has been synthesized and their inhibitory activity investigated. The obtained results demonstrate that modification of the group between the histidinyl moiety and the phenyl ring constitutes an important structural factor for the design of effective HDH inhibitors.

Brucella suis histidinol dehydrogenase (HDH) can be efficiently targeted by substrate analogues. The growth of this pathogen in minimal medium was inhibited and the multiplication in human macrophages was totally abolished in the presence of the drugs. These effects have been shown to be correlated with the previously described inhibition of Brucella HDH activity.

Brucella spp. is the causative agent of brucellosis (Malta fever), which is the most widespread zoonosis worldwide. The pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic therapy. Moreover, Brucella is considered as a potential bioterrorism agent. Histidinol dehydrogenase (HDH, EC 1.1.1.23) has been shown to be essential for the intramacrophagic replication of this pathogen. It therefore constitutes an original and novel target for the development of anti-Brucella agents. In this work, we cloned and overexpressed the HDH-encoding gene from Brucella suis, purified the protein and evidenced its biological activity. We then investigated the inhibitory effects of a series of substituted benzylic ketones derived from histidine. Most of the compounds reported here inhibited B. suis HDH in the lower nanomolar range and constitute attractive candidates for the development of novel anti-Brucella agents.

N-Hydroxysulfamide is a 2000-fold more potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to sulfamide. It also inhibits other physiologically relevant isoforms, such as the tumor-associated CA IX and XII (K(I)s in the range of 0.865-1.34microM). In order to understand the binding of this inhibitor to the enzyme active site, the X-ray crystal structure of the human hCA II-N-hydroxysulfamide adduct was resolved. The inhibitor coordinates to the active site zinc ion by the ionized primary amino group, participating in an extended network of hydrogen bonds with amino acid residues Thr199, Thr200 and two water molecules. The additional two hydrogen bonds in which N-hydroxysulfamide bound to hCA II is involved as compared to the corresponding adduct of sulfamide may explain its higher affinity for the enzyme, also providing hints for the design of tight-binding CA inhibitors possessing an organic moiety substituting the NH group in the N-hydroxysulfamide structure.

Sulfamide, a quite simple molecule incorporating the sulfonamide functionality, widely used by medicinal chemists for the design of a host of biologically active derivatives with pharmacological applications, may give rise to at least five types of derivatives, by substituting one to four hydrogen atoms present in it, which show specific biological activities. Recently, some of these compounds started to be exploited for the design of many types of therapeutic agents. Among the enzymes for which sulfamide-based inhibitors were designed, are the carbonic anhydrases (CAs), a large number of proteases belonging to the aspartic protease (HIV-1 protease, gamma-secretase), serine protease (elastase, chymase, tryptase, and thrombin among others), and metalloprotease (carboxypeptidase A (CPA) and matrix metalloproteinases (MMP)) families. Some steroid sulfatase (STS) and protein tyrosine phosphatase inhibitors belonging to the sulfamide class of derivatives have also been reported. In all these compounds, many of which show low nanomolar affinity for the target enzymes for which they have been designed, the free or substituted sulfamide moiety plays important roles for the binding of the inhibitor to the active site cavity, either by directly coordinating to a metal ion found in some metalloenzymes (CAs, CPA, STS), usually by means of one of the nitrogen atoms present in the sulfamide motif, or as in the case of the cyclic sulfamides acting as HIV protease inhibitors, interacting with the catalytically critical aspartic acid residues of the active site by means of an oxygen atom belonging to the HN-SO2-NH motif, which substitutes a catalytically essential water molecule. In other cases, the sulfamide moiety is important for inducing desired physico-chemical properties to the drug-like compounds incorporating it, such as enhanced water solubility, better bioavailability, etc., because of the intrinsic properties of this highly polarized moiety when attached to an organic scaffold. This interesting motif is thus of great value for the design of pharmacological agents with a lot of applications., (2006 Wiley Periodicals, Inc.)

A series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfonamides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic alcohols (methanol and ethanol). The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with K(I)s in the range of 75-136nM, hCA II with K(I)s in the range of 13-278nM, and hCA IX with K(I)s in the range of 0.12-549nM. The first hCA IX-selective inhibitors were thus detected, as the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity ratios for CA IX over CA II inhibition in the range of 166-706. Furthermore, some of these compounds have subnanomolar affinity for hCA IX, with K(I)s in the range 0.12-0.34nM. These derivatives are interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumors. Clear renal cell carcinoma, which is the most lethal urologic malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading candidate of such novel therapies.

A series of aliphatic sulfamates and related derivatives incorporating cyclic/polycyclic (steroidal) moieties in their molecules has been synthesized and assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA) and, more precisely, of the cytosolic isozymes CA I and II and the transmembrane, tumor-associated isozyme CA IX. The most potent CA I inhibitor was n-tetradecyl sulfamate and some (substituted)benzyl/phenethyl sulfamates (inhibition constants in the low micromolar range). Against CA II, low nanomolar inhibitors (0.7-3.4 nM) were n-decyl sulfamate and the (substituted)benzyl/phenethyl derivatives mentioned above. Effective CA II inhibition was also observed for the hydroxy/keto derivatives of dehydroepiandrosterone sulfamate. Efficient CA IX inhibitory properties, with inhibition constants in the range of 9-23 nM, were observed for the aliphatic sulfamates C(10)-C(16) (with the most potent inhibitor being the n-dodecyl derivative) and the (substituted)benzyl/phenethyl sulfamates. The inhibition profile of the three investigated isozymes with this type of compound was rather different, allowing us to hope that the preparation of CA IX-selective inhibitors is possible (selectivity ratios toward hCA IX versus hCA II in the range of 5-63 has been observed for some of these compounds, whereas for the clinically used sulfonamides this parameter is in the range of 0.23-0.51). These data are critical for the design of novel antitumor therapies, mainly for hypoxic tumors that overexpress CA IX, which are nonresponsive to radiation or chemotherapy.

A series of sulfamates or bis-sulfamates incorporating aliphatic, aromatic, polycyclic (steroidal), and sugar moieties in their molecules has been synthesized and assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more precisely of the cytosolic isozymes CA I andII, and the transmembrane, tumor-associated isozymes CA IX. Some of these compounds were previously reported to act as inhibitors of steroid sulfatases, among which estrone sulfatase (ES) and dehydroepiandrosterone sulfatase (DHEAS) are the key therapeutic targets for estrogen-dependent tumors. Very potent (nanomolar) inhibitors were detected against the three investigated CA isozymes. Best CA I inhibitors were phenylsulfamate and some of its 4-halogeno derivatives, as well as the aliphatic compound n-octyl sulfamate. Against CA II, low nanomolar inhibitors (1.1-5 nM) were phenylsulfamate and some of its 4-halogeno/nitro derivatives, n-octyl sulfamate, and estradiol 3,17beta-disulfamate among others. All the investigated sulfamates showed efficient CA IX inhibitory properties, with inhibition constants in the range of 18-63 nM. The best CA IX inhibitor detected so far was 4-chlorophenylsulfamate. These data are critical for the design of novel antitumor properties, mainly for hypoxic tumors that overexpress CA IX, which are nonresponsive to radiation or chemotherapy. The antitumor properties of the ES/DHEAS inhibitors in clinical trials may on the other hand also be due to their potent inhibitory properties of CA isozymes involved in tumorigenicity, such as CA II and CA IX.