A comparative QSAR study on carbonic anhydrase and matrix metalloproteinase inhibition by sulfonylated amino acid hydroxamates

A quantitative structure-activity relationship (QSAR) study is made on the inhibition of a few isozymes of carbonic anhydrase (CA) and some matrix metalloproteinases (MMPs), both zinc containing families of enzymes, by sulfonylated amino acid hydroxamates. For both enzymes, the inhibition potency of the hydroxamates is found to be well correlated with Kier's first-order valence molecular connectivity index 1chi(v) of the molecule and electrotopological state indices of some atoms. From the results, it is suggested that while hydroxamate-CA binding may involve mostly polar interactions, hydroxamate-MMP and hydroxamate-ChC (ChC: Clostridium histolyticum collagenase, another zinc enzyme related to MMPs) bindings may involve some hydrophobic interactions. Both MMPs and ChC also possess some electronic sites of exactly opposite nature to the corresponding sites in CAs. A group such as C6F5 present in the sulfonyl moiety is shown to be advantageous in both CA and MMP (also ChC) inhibitions, which is supposed to be due to the interaction of this group with Zn2+ ion present in the catalytic site of both families of enzymes.     

A quantitative structure-activity relationship study on some matrix metalloproteinase and collagenase inhibitors

A quantitative structure-activity relationship (QSAR) study is made on some hydroxamic acid-based inhibitors of matrix metalloproteinases (MMPs) and a bacterial collagenase, namely Clostridium histolyticum collagenase (ChC), that also belongs to an MMP family, M-31, using Kier's valence molecular connectivity index (1)chi(v) of the substituents and electrotopological state (E-state) indices of some atoms. The results indicate that out of the four MMPs (MMP-1, MMP-2, MMP-8, and MMP-9) studied, MMP-2 and MMP-9 can be structurally quite similar, but widely differing from MMP-1 and MMP-8 and ChC. For MMP-2 and MMP-9, the inhibition activity of compounds is shown to depend on both (1)chi(v )and E-state indices, while for MMP-1 and MMP-8 it is shown to depend only on E-state indices and for ChC only on (1)chi(v). However, in all the cases, an aromatic group like C(6)F(5) or 3-CF(3)-C(6)H(4) attached to SO(2) moiety in the compounds is indicated to be equally beneficial, due to probably the involvement of fluorine atom(s) in charge-charge interactions with the Zn(2+) ion of the enzymes or in the formation of the hydrogen bonds with some sites of the receptors.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.     

Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors.     

Recent advances in the discovery of zinc-binding motifs for the development of carbonic anhydrase inhibitors

Abstract

In addition to the sulfonamides and their isosteres, recently novel carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) which act by binding to the metal ion from the active site were discovered. Based on the X-ray crystal structure of the CA II–trithiocarbonate adduct, dithiocarbamates, xanthates and thioxanthates were shown to potently inhibit α- and β-CAs. The hydroxamates constitute another class of recently studied CAIs both against mammalian and protozoan enzymes. Another chemotype for which CA inhibitory properties were recently reported is the salicylaldoxime scaffold. X-ray crystal structures were reported for CA II complexed with dithiocarbamates and hydroxamates, whereas the xanthates and salicylaldoximes were investigated by kinetic measurements and docking studies. The dithiocarbamates and the xanthates showed potent antiglaucoma activity in animal models of the disease whereas some hydroxamates inhibited the growth of Trypanosoma cruzii probably by inhibiting the protozoan CA.     

A quantitative structure-activity relationship study on some series of anthranilic acid-based matrix metalloproteinase inhibitors

A quantitative structure-activity relationship (QSAR) study has been made on four different series of anthranilic acid-based matrix metalloproteinase (MMP) inhibitors, in which two substituted aryl rings, one bearing the hydroxamic acid moiety that binds with the zinc atom of MMPs, are joined through a bridge group of sulfonamide. The QSAR results indicate that the sulfonamide group plays a very important role in the inhibition activity of the inhibitors and that the effectiveness of this sulfonamide group can be increased by the presence at the aryl rings or at the sulfonamide nitrogen itself of nitrogen-containing or some such substituents that can increase the electronic character of the sulfonamide group. The hydrophobic character of the molecules is not found to be of any advantage; rather in most of the cases it is shown to have detrimental effect, suggesting that MMPs provide little opportunity to the inhibitors to have a any hydrophobic interactions with them. On the other hand, polarizability of the molecules has been found to be conducive to activity in some cases. Thus the inhibition mechanism seems to predominantly involve the electronic interactions between the inhibitors and the enzymes.     

Dual carbonic anhydrase/matrix metalloproteinase inhibitors incorporating bisphosphonic acid moieties targeting bone tumors

A set of bisphosphonate matrix metalloproteinase (MMP) inhibitors was investigated for inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, some of which are overexpressed in hypoxic tumors. Some of the bisphosphonate revealed to be very potent inhibitors (in the low nanomolar range) of the cytosolic isoform CA II and the membrane-bound CA IX, XII and XIV isozymes, a feature useful for considering them as interesting compounds for bone resorption inhibition applications. We suggest here that it is possible to develop dual enzyme inhibitors bearing bisphosphonate moieties that may target both MMPs and CAs, two families of enzymes involved in tumor formation, growth, and metastasis.     

How many carbonic anhydrase inhibition mechanisms exist?

Six genetic families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) were described to date. Inhibition of CAs has pharmacologic applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents. New classes of CA inhibitors (CAIs) were described in the last decade with enzyme inhibition mechanisms differing considerably from the classical inhibitors of the sulfonamide or anion type. Five different CA inhibition mechanisms are known: (i) the zinc binders coordinate to the catalytically crucial Zn(II) ion from the enzyme active site, with the metal in tetrahedral or trigonal bipyramidal geometries. Sulfonamides and their isosters, most anions, dithiocarbamates and their isosters, carboxylates, and hydroxamates bind in this way; (ii) inhibitors that anchor to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, 2-thioxocoumarins, sulfocoumarins); (iii) inhibitors which occlude the entrance to the active site cavity (coumarins and their isosters), this binding site coinciding with that where CA activators bind; (iv) compounds which bind out of the active site cavity (a carboxylic acid derivative was seen to inhibit CA in this manner), and (v) compounds for which the inhibition mechanism is not known, among which the secondary/tertiary sulfonamides as well as imatinib/nilotinib are the most investigated examples. As CAIs are used clinically in many pathologies, with a sulfonamide inhibitor (SLC-0111) in Phase I clinical trials for the management of metastatic solid tumors, this review updates the recent findings in the field which may be useful for a structure-based drug design approach of more selective/potent modulators of the activity of these enzymes.     

Carbonic anhydrase and matrix metalloproteinase inhibitors. Inhibition of human tumor-associated isozymes IX and cytosolic isozyme I and II with sulfonylated hydroxamates

A series of sulfonylated hydroxamates were synthesized and evaluated as dual inhibitors of both human carbonic anhydrases (hCAs) and matrix metalloproteinases (MMPs), two metalloenzyme families involved in carcinogenesis and tumor invasion processes. The new derivatives were tested on three CA isozymes, the cytosolic isozymes I and II, and the transmembrane, tumor-associated isozyme IX, and also on human gelatinases (MMP-2 and MMP-9). Some of the new derivatives proved to be potent and selective inhibitors of CA II, but only compounds 3b and 6b, devoid of the arylsulfonyl moiety, proved to have a better inhibitory activity on hCA IX than on hCA I and II, in the micromolar range.     

A quantitative structure-activity relationship study of hydroxamate matrix metalloproteinase inhibitors derived from functionalized 4-aminoprolines

A quantitative structure-activity relationship (QSAR) study has been made on the inhibitions of some matrix metalloproteinases (MMPs) by functionalized 4-aminoproline based hydroxamates. Attempts have been made to correlate the inhibition potencies of these hydroxamates with Kier's first-order valence molecular connectivity index ((1)chi(v)) of substituents and electrotopological state (E-state) indices of some atoms. The correlations obtained for the inhibitions of all the enzymes studied, i.e. MMP-1, MMP-2, MMP-3, MMP-7, and MMP-13, were not so uniform, but suggested that in almost all the cases the substituents at the amide nitrogen may be conducive to the activity, though the whole amide group may be sterically unfavourable. Similarly, in most of the cases, the substituens at the phenyl moiety have been found to be beneficial to the inhibition potency and in many cases an electronic role of SO(2) group of the sulfonylphenyl moiety has been indicated.     

Protease Inhibitors: Synthesis of Bacterial Collagenase and Matrix Metalloproteinase Inhibitors Incorporating Succinyl Hydroxamate and Iminodiacetic Acid Hydroxamate Moieties

A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.     

Structural basis for potent slow binding inhibition of human matrix metalloproteinase-2 (MMP-2)

The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.     

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.     

Matrilysin: Expression, purification, and characterization

The expression vector pGEX-2T under the control of the IPTG-inducibletac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin,HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.     

Carbonic Anhydrase Inhibitors: Aromatic Sulfonamides and Disulfonamides Act as Efficient Tumor Growth Inhibitors

Abstract

Aromatic/heterocyclic sulfonamides generally act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we report the unexpected finding that potent aromatic sulfonamide inhibitors of CA, possessing inhibition constants in the range of 10^?-⁸-^?10^?-⁹ M (against all the isozymes), also act as efficient in vitro tumor cell growth inhibitors, with GI₅₀ (molarity of inhibitor producing a 50% inhibition of tumor cell growth) values of 10 nM-35 μM against several leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. The investigated compounds were sulfanilyl-sulfanilamide-, 4-thioureido-benzenesulfonamide- and benzene-1.3-disulfonamide-derivatives. The mechanism of antitumor action with these sulfonamides is unknown, but it might involve either inhibition of several CA isozymes (such as CA IX, CA XII, CA XIV) predominantly present in tumor cells, a reduced provision of bicarbonate for the nucleotide synthesis (mediated by carbamoyl phosphate synthetase II), the acidification of the intracellular milieu as a consequence of CA inhibition or uncoupling of mitochondria and potent CA V inhibition among others. A combination of several such mechanisms is also plausible. Optimization of such derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents/therapies.     

A quantitative structure-activity relationship study on Clostridium histolyticum collagenase inhibitors: roles of electrotopological state indices

A quantitative structure-activity relationship (QSAR) study has been made on eight different series of Clostridium histolyticum collegenase (ChC) inhibitors. These series are comprised of four different groups of sulfonylated amino acids and their corresponding hydroxamates. In each series, the inhibition potency of the compounds has been found to be significantly correlated with the electrotopological state (E-state) indices of nitrogen and sulfur atoms of the sulfonylated amino group in the molecules, showing the importance of the electronic characterstics of these atoms in controlling the inhibition potency of the compounds.     

MMPs as therapeutic targets--still a viable option?

Matrix metalloproteinases (MMPs) appear to be ideal drug targets--they are disease-associated, extracellular enzymes with a dependence on zinc for activity. This apparently straightforward target, however, is much more complex than initially realized. Although disease associated, the roles for particular enzymes may be healing rather than harmful making broad-spectrum inhibition unwise; targeting the catalytic zinc with specificity is difficult, since other related proteases as well as non-related proteins can be affected by some chelating groups. While the failure of early-generation MMP inhibitors dampened enthusiasm for this type of drug, there has recently been a wealth of studies examining the basic biology of MMPs which will greatly inform new drug trials in this field.     

Inhibition of poly(A) polymerase by aminoglycosides

Aminoglycosides are potent inhibitors of bacterial growth and are used clinically as antibiotics. However, their usage has declined in recent years due to the emergence of resistance and severe toxic side effects. Here we show that human poly(A) polymerase gamma (PAPgamma) is inhibited by aminoglycosides. The inhibition was pH dependent and could be released by Mg(II) ions in a competitive manner suggesting that electrostatic interactions are important for inhibition and that the binding sites for aminoglycosides overlap with Mg(II) ion binding sites. Kinetic analysis revealed that aminoglycosides of the neomycin and kanamycin families behaved as mixed non-competitive inhibitors for the PAPgamma substrates oligoA15 and ATP. Interestingly, sisomicin and 5-epi-sisomycin showed a competitive mechanism of inhibition for the oligoA15 whereas they inhibited the ATP substrate mixed non-competitive. This implies that different aminoglycosides bind in different ways to a common binding pocket and suggests that the binding sites for related aminoglycosides are not overlapping even if they may share molecular determinants. Our study emphasizes the possibility that aminoglycoside toxicity could be due to interference with housekeeping enzymes involved in breaking and forming phosphodiester bonds.