Investigation of flap flexibility of β-secretase using molecular dynamic simulations

Flap motif and its dynamics were extensively reported in aspartate proteases, e.g. HIV proteases and plasmepsins. Herein, we report the first account of flap dynamics amongst different conformations of β-secretase using molecular dynamics simulation. Various parameters were proposed and a selected few were picked which could appropriately describe the flap motion. Three systems were studied, namely Free (BACE_Free) and two ligand-bound conformations, which belonged to space groups P6₁22 (BACE_Bound1) and C222₁ (BACE_Bound2), respectively and four parameters (distance between the flaps tip residue, Thr72 and Ser325, d₁; dihedral angle, ? (Thr72-Asp32-Asp228-Ser325); TriCα angles, θ₁ (Thr72-Asp32-Ser325), and θ₂ (Thr72-Asp228-Ser325)) were proposed to understand the change in dynamics of flap domain and the extent of flap opening and closing. Analysis of, θ₂, d₁, θ₁ and ? confirmed that the BACE_Free adopted semi-open, open and closed conformations with slight twisting during flap opening. However, BACE_Bound1 (P6122) showed an adaptation to open conformation due to lack of hydrogen bond interaction between the ligand and flap tip residue. A slight flap twisting, ? (lateral twisting) was observed for BACE_Bound1 during flap opening which correlates with the opening of BACE_Free. Contradictory to the BACE_Bound1, the BACE_Bound2 locked the flap in a closed conformation throughout the simulation due to formation of a stable hydrogen bond interaction between the flap tip residue and ligand. Analyses of all three systems highlight that d₁, θ₂ and ? can be precisely used to describe the extent of flap opening and closing concurrently with snapshots along the molecular dynamics trajectory across several conformations of β-secretase.     

Discovery of potent iminoheterocycle BACE1 inhibitors

The synthesis of a series of iminoheterocycles and their structure–activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aβ₄₀ levels upon subcutaneous and oral administration to rats.     

Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1-calcium interaction

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the β-secretase, BACE1, is the first step in the production of the Aβ peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca²⁺ binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca²⁺. Isothermal titration calorimetry was then used to characterize the Ca²⁺-BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (k_d = 2.0 × μM) between Ca²⁺ and BACE1 and that the binding process was exothermic (ΔH = − 3.5 kcal/mol). We also could demonstrate that low concentrations of Ca²⁺ (μM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca²⁺ and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca²⁺.     

The role of tyrosine 71 in modulating the flap conformations of BACE1

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is a potential target for treating Alzheimer's disease. BACE1's binding site is partially covered by a flexible loop on its N-terminal domain, known as the "flap," which has been found in several conformations in crystal structures of BACE1 and other aspartyl proteases. The side chain of the invariant residue Tyr71 on the flap adopts several rotameric orientations, leading to our hypothesis that the orientation of this residue dictates the movement and conformations available to the flap. We investigated this hypothesis by performing 220 ns of molecular dynamics simulations of bound and unbound wild-type BACE1 as well as the unbound Y71A mutant. Our findings indicate that the flap exhibits various degrees of mobility and adopts different conformations depending on the Tyr71 orientation. Surprisingly, the "self-inhibited" form is stable in our simulations, making it a reasonable target for drug design. The alanine mutant, lacking a large side chain at position 71, displays significant differences in flap dynamics from wild type, freely sampling very open and closed conformations. Our simulations show that Tyr71, in addition to its previously determined functions in catalysis and substrate binding, has the important role of modulating flap conformations in BACE1.     

Mapping of interaction domains mediating binding between BACE1 and RTN/Nogo proteins

BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the beta-secretase site. Membrane bound reticulon (RTN) family proteins interact with BACE1 and negatively modulate BACE1 activity through preventing access of BACE1 to its cellular APP substrate. Here, we focused our study on RTN3 and further show that a C-terminal QID triplet conserved among mammalian RTN members is required for the binding of RTN to BACE1. Although RTN3 can form homo- or heterodimers in cells, BACE1 mainly binds to the RTN monomer and disruption of the QID triplet does not interfere with the dimerization. Correspondingly, the C-terminal region of BACE1 is required for the binding of BACE1 to RTNs. Furthermore, we show that the negative modulation of BACE1 by RTN3 relies on the binding of RTN3 to BACE1. The knowledge from this study may potentially guide discovery of small molecules that can mimic the effect of RTN3 on the inhibition of BACE1 activity.     

Identification of a cis-acting element involved in the regulation of BACE1 mRNA alternative splicing

β-site APP cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step during proteolysis of the β-amyloid precursor protein, a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, and cis -acting elements important for its regulation have not been identified. We constructed and compared several BACE1 minigenes and found that BACE1 sequence from exon 3 through exon 5 was required for minigenes to undergo correct splicing. Minigene splicing was validated by showing specific splicing inhibition upon splice site mutation. Furthermore, we showed that mutation of the minigene at a predicted exonic splicing enhancer in exon 4 of BACE1 increased exon 4 skipping. Therefore, we have for the first time found evidence of a regulatory site involved in BACE1 alternative splicing, and these data indicate that minor sequence changes can dramatically alter BACE1 alternative splicing.     

Extracellular release of BACE1 holoproteins from human neuronal cells

BACE1 is a membrane-bound aspartyl protease involved in production of the Alzheimer's amyloid beta-protein. The BACE1 ectodomain is partially cleaved to generate soluble BACE1, but the physiological significance of this event is unclear. During our characterization of BACE1 shedding from human neuroblastoma SH-SY5Y cells stably expressing BACE1, we unexpectedly found that detectable amounts of BACE1 holoproteins were released extracellularly along with soluble BACE1. Treatment with the metalloprotease inhibitor, TAPI-1, inhibited BACE1 shedding but increased BACE1 holoprotein release. Soluble and full-length BACE1 were released in parallel, at least partly originating from the plasma membrane. Furthermore, the release of soluble BACE1, but not full-length BACE1, was increased by deletion of the C-terminal dileucine motif, indicating that dysregulated BACE1 sorting affects BACE1 shedding. These findings suggest that the release of BACE1 holoproteins may be a physiologically relevant cellular process.     

Molecular docking based virtual screening of natural compounds as potential BACE1 inhibitors: 3D QSAR pharmacophore mapping and molecular dynamics analysis

Beta-site APP cleaving enzyme1 (BACE1) catalyzes the rate determining step in the generation of Aβ peptide and is widely considered as a potential therapeutic drug target for Alzheimer’s disease (AD). Active site of BACE1 contains catalytic aspartic (Asp) dyad and flap. Asp dyad cleaves the substrate amyloid precursor protein with the help of flap. Currently, there are no marketed drugs available against BACE1 and existing inhibitors are mostly pseudopeptide or synthetic derivatives. There is a need to search for a potent inhibitor with natural scaffold interacting with flap and Asp dyad. This study screens the natural database InterBioScreen, followed by three-dimensional (3D) QSAR pharmacophore modeling, mapping, in silico ADME/T predictions to find the potential BACE1 inhibitors. Further, molecular dynamics of selected inhibitors were performed to observe the dynamic structure of protein after ligand binding. All conformations and the residues of binding region were stable but the flap adopted a closed conformation after binding with the ligand. Bond oligosaccharide interacted with the flap as well as catalytic dyad via hydrogen bond throughout the simulation. This led to stabilize the flap in closed conformation and restricted the entry of substrate. Carbohydrates have been earlier used in the treatment of AD because of their low toxicity, high efficiency, good biocompatibility, and easy permeability through the blood–brain barrier. Our finding will be helpful in identify the potential leads to design novel BACE1 inhibitors for AD therapy.     

Screening a series of sialyltransferases for possible BACE1 substrates

Deposition of amyloid β-peptide (Aβ) and neurofibrillary tangles in the brain are hallmarks of Alzheimer’s disease (AD) pathogenesis. BACE1, a membrane-bound aspartic protease that cleaves amyloid precursor protein (APP) to produce Aβ, has been implicated in triggering the pathogenesis of the disease. We previously reported that BACE1 also cleaved α2,6-sialyltransferase (ST6Gal I) in the Golgi apparatus and induced its secretion from the cell. Since most glycosyltransferases show Golgi localization and many of these are cleaved and secreted from the cell, we hypothesized that other glycosyltransferases may also be BACE1 substrates. Here, we focused on a series of sialyltransferases as candidates for BACE1 substrates. We found that BACE1 cleaved polysialyltransferase ST8Sia IV (PST) in vitro. We further found that BACE1 overexpression in COS cells enhanced the secretion of ST3Gal I, II, III and IV, although these sialyltransferases were not cleaved by BACE1 in vitro. These results suggest that BACE1 expression affects glycosylation not only by directly cleaving glycosyltransferases but also by modifying the secretion of glycosyltransferases via some other mechanisms.     

Flap flexibility amongst plasmepsins I,II, III,IV, and V: Sequence,structural, and molecular dynamics analyses

Herein, for the first time, we comparatively report the opening and closing of apo plasmepsin I – V. Plasmepsins belong the aspartic protease family of enzymes, and are expressed during the various stages of the P. falciparum lifecycle, the species responsible for the most lethal and virulent malaria to infect humans. Plasmepsin I, II, IV and HAP degrade hemoglobin from infected red blood cells, whereas plasmepsin V transport proteins crucial to the survival of the malaria parasite across the endoplasmic reticulum. Flap‐structures covering the active site of aspartic proteases (such as HIV protease) are crucial to the conformational flexibility and dynamics of the protein, and ultimately control the binding landscape. The flap‐structure in plasmepsins is made up of a flip tip in the N‐terminal lying perpendicular to the active site, adjacent to the flexible loop region in the C‐terminal. Using molecular dynamics, we propose three parameters to better describe the opening and closing of the flap‐structure in apo plasmepsins. Namely, the distance, d₁, between the flap tip and the flexible region; the dihedral angle, ?, to account for the twisting motion; and the TriCα angle, θ₁. Simulations have shown that as the flap‐structure twists, the flap and flexible region move apart opening the active site, or move toward each other closing the active site. The data from our study indicate that of all the plasmepsins investigated in the present study, Plm IV and V display the highest conformational flexibility and are more dynamic structures versus Plm I, II, and HAP. Proteins 2015; 83:1693–1705. © 2015 Wiley Periodicals, Inc.     

Physiological and behavioral effects of amphetamine in BACE1−/− mice

β‐Site APP‐cleaving Enzyme 1 (BACE1) is a protease that has been linked to schizophrenia, a severe mental illness that is potentially characterized by enhanced dopamine (DA) release in the striatum. Here, we used acute amphetamine administration to stimulate neuronal activity and investigated the neurophysiological and locomotor‐activity response in BACE1‐deficient (BACE1^?/?) mice. We measured locomotor activity at baseline and after treatment with amphetamine (3.2 and 10 mg/kg). While baseline locomotor activity did not vary between groups, BACE1^?/? mice exhibited reduced sensitivity to the locomotor‐enhancing effects of amphetamine. Using high‐performance liquid chromatography (HPLC) to measure DA and DA metabolites in the striatum, we found no significant differences in BACE1^?/? compared with wild‐type mice. To determine if DA neuron excitability is altered in BACE1^?/? mice, we performed patch‐clamp electrophysiology in putative DA neurons from brain slices that contained the substantia nigra. Pacemaker firing rate was slightly increased in slices from BACE1^?/? mice. We next measured G protein‐coupled potassium currents produced by activation of D2 autoreceptors, which strongly inhibit firing of these neurons. The maximal amplitude and decay times of D2 autoreceptor currents were not altered in BACE1^?/? mice, indicating no change in D2 autoreceptor‐sensitivity and DA transporter‐mediated reuptake. However, amphetamine (30 µm )‐induced potassium currents produced by efflux of DA were enhanced in BACE1^?/? mice, perhaps indicating increased vesicular DA content in the midbrain. This suggests a plausible mechanism to explain the decreased sensitivity to amphetamine‐induced locomotion, and provides evidence that decreased availability of BACE1 can produce persistent adaptations in the dopaminergic system.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

β-Site Amyloid Precursor Protein Cleaving Enzyme 1(BACE1) Regulates Notch Signaling by Controlling the Cleavage of Jagged 1 (Jag1) and Jagged 2 (Jag2) Proteins

BACE1 is a type I transmembrane aspartyl protease that cleaves amyloid precursor protein at the β-secretase site to initiate the release of β-amyloid peptide. As a secretase, BACE1 also cleaves additional membrane-bound molecules by exerting various cellular functions. In this study, we showed that BACE1 can effectively shed the membrane-anchored signaling molecule Jagged 1 (Jag1). We also mapped the cleavage sites of Jag1 by ADAM10 and ADAM17. Although Jag1 shares a high degree of homology with Jag2 in the ectodomain region, BACE1 fails to cleave Jag2 effectively, indicating a selective cleavage of Jag1. Abolished cleavage of Jag1 in BACE1-null mice leads to enhanced astrogenesis and, concomitantly, reduced neurogenesis. This characterization provides biochemical evidence that the Jag1-Notch pathway is under the control of BACE1 activity.     

BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (⁴⁹⁵DDISLL⁵⁰⁰) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules.     

Evaluation of an Allosteric BACE Inhibitor Peptide to Identify Mimetics that Can Interact with the Loop F Region of the Enzyme and Prevent APP Cleavage

The aspartyl protease BACE1 (BACE) has emerged as an appealing target for reduction of amyloid-β in Alzheimer's disease. The clinical fate of active-site BACE inhibitors may depend on potential side effects related to enzyme and substrate selectivity. One strategy to reduce this risk is through development of allosteric inhibitors that interact with and modulate the Loop F region unique to BACE1. Previously, a BACE-inhibiting antibody (Ab) was shown by co-crystallization to bind and induce conformational changes of Loop F, resulting in backbone perturbations at the distal S6 and S7 subsites, preventing proper binding of a long APP-like substrate to BACE and inhibiting its cleavage. In an effort to discover small Loop F-interacting molecules that mimic the Ab inhibition, we evaluated a peptide series with a YPYF(I/L)P(L/Y) motif that was reported to bind a BACE exosite. Our studies show that the most potent inhibitor from this series, peptide 65007, has a similar substrate cleavage profile to the Ab and reduces sAPPβ levels in cell models and primary neurons. As our modeling indicates, it interacts with the Loop F region causing a conformational shift of the BACE protein backbone near the distal subsites. The peptide-bound enzyme adopts a conformation that closely overlays with the crystal structure (PDB: 3R1G) from Ab binding. Importantly, peptide 65007 appears to be BACE substrate and enzyme selective, showing little inhibition of NRG1, PSGL1, CHL1, or Cat D. Thus, peptide 65007 is a promising lead for discovery of Loop F-interacting small-molecule mimetics as allosteric inhibitors of BACE.     

New substituted aminopyrimidine derivatives as BACE1 inhibitors: in silico design,synthesis and biological assays

We report in this work new substituted aminopyrimidine derivatives acting as inhibitors of the catalytic site of BACE1. These compounds were obtained from a molecular modeling study. The theoretical and experimental study reported here was carried out in several steps: docking analysis, Molecular Dynamics (MD) simulations, Quantum Theory Atom in Molecules (QTAIM) calculations, synthesis and bioassays and has allowed us to propose some compounds of this series as new inhibitors of the catalytic site of BACE1. The QTAIM study has allowed us to obtain an excellent correlation between the electronic densities and the experimental data of IC₅₀. Also, using combined techniques (MD simulations and QTAIM calculations) enabled us to describe in detail the molecular interactions that stabilize the different L-R complexes. In addition, our results allowed us to determine what portion of these compounds should be changed in order to increase their affinity with the BACE1. Another interesting result is that a sort of synergism was observed when the effects of these new catalytic site inhibitors were combined with Ac-Tyr5-Pro6-Tyr7-Asp8-Ile9-Pro10-Leu11-NH₂, which we have recently reported as a modulator of BACE1 acting on its exosite.     

Characterization of a mouse model overexpressing beta‐site APP‐cleaving enzyme 2 reveals a new role for BACE2

BACE2 is homologous to BACE1, a β‐secretase that is involved in the amyloidogenic pathway of amyloid precursor protein (APP), and maps to the Down syndrome critical region of chromosome 21. Alzheimer disease neuropathology is common in Down syndrome patients at relatively early ages, and it has thus been speculated that BACE2 co‐overexpression with APP would promote the early neurodegenerative phenotype. However, the in vivo function of BACE2 has not yet been elucidated. The aim of the present work has been to analyse the impact of in vivo BACE2 overexpression using a transgenic mouse model. Our results suggest that BACE2 is not involved in the amyloidogenic pathway, cognitive dysfunction or cholinergic degeneration. However, TgBACE2 animals showed increased anxiety‐like behaviour along with increased numbers of noradrenergic neurones in locus coeruleus, thus suggesting an unexpected role of BACE2 overexpression.     

Hydroxyethylamine-based inhibitors of BACE1: P1–P3 macrocyclization can improve potency,selectivity, and cell activity

We describe a systematic study of how macrocyclization in the P₁–P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APP_SW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.     

Label‐free quantitative analysis of the membrane proteome of Bace1 protease knock‐out zebrafish brains

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β‐secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter‐aided sample preparation, strong‐anion exchange fractionation, and label‐free quantification. We used bace1‐deficient zebrafish and quantified differences in protein levels between wild‐type and bace1 ?/? zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild‐type and bace1 ?/? zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty‐four membrane proteins accumulated in the bace1 ?/? brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican‐1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock‐out zebrafish tissue and demonstrates that combining gene knock‐out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.     

Sulfonamido-derivatives of unsubstituted carbazoles as BACE1 inhibitors

A novel series of variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylsulfonamides has been synthesized and assayed for β-Secretase (BACE1) inhibitory activity. BACE1 is a widely recognized drug target for the prevention and treatment of Alzheimer's Disease (AD). The introduction of benzyl substituents on the nitrogen atom of the arylsulfonamide moiety has so far led to the best results, with three derivatives showing IC₅₀ values ranging from 1.6 to 1.9?μM. Therefore, a significant improvement over the previously reported series of N-carboxamides (displaying IC₅₀’s?≥?2.5?μM) has been achieved, thus suggesting an active role of the sulfonamido-portion in the inhibition process. Preliminary molecular modeling studies have been carried out to rationalize the observed structure-activity relationships.