Giovanni Cercignani

N-O-isopropyl sulfonamido-based hydroxamates: design, synthesis and biological evaluation of selective matrix metalloproteinase-13 inhibitors as potential therapeutic agents for osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a key enzyme implicated in the degradation of the extracellular matrix in osteoarthritis (OA). For this reason, MMP-13 synthetic inhibitors are being sought as potential therapeutic agents to prevent cartilage degradation and to halt the progression of OA. Herein, we report the synthesis and in vitro evaluation of a new series of selective MMP-13 inhibitors possessing an arylsulfonamidic scaffold. Among these potential inhibitors, a very promising compound was discovered exhibiting nanomolar activity for MMP-13 and was highly selective for this enzyme compared to MMP-1, -14, and TACE. This compound acted as a slow-binding inhibitor of MMP-13 and was demonstrated to be effective in an in vitro collagen assay and in a model of cartilage degradation. Furthermore, a docking study was conducted for this compound in order to investigate its binding interactions with MMP-13 and the reasons for its selectivity toward MMP-13 versus other MMPs.

Design, synthesis, biological evaluation, and NMR studies of a new series of arylsulfones as selective and potent matrix metalloproteinase-12 inhibitors.

Overexpression of macrophage elastase (MMP-12), a member of the matrix metalloproteinases family, can be linked to tissue remodeling and degradation in some inflammatory processes, such as chronic obstructive pulmonary disease (COPD), emphysema, rheumatoid arthritis (RA), and atherosclerosis. On this basis, MMP-12 can be considered an attractive target for studying selective inhibitors that are useful in the development of new therapies for COPD and other inflammatory diseases. We report herein the design, synthesis, and in vitro evaluation of a new series of compounds, possessing an arylsulfonyl scaffold, for their potential as selective inhibitors of MMP-12. The best compound in the series showed an IC50 value of 0.2 nM, with good selectivity over MMP-1 and MMP-14. A docking study was carried out on this compound in order to investigate its binding interactions with MMP-12, and NMR studies on the complex with the MMP-12 catalytic domain were able to validate the proposed binding mode.

A unified theory of enzyme kinetics based upon the systematic analysis of the variations of kcat, KM, and kcat/KM and the relevant ΔG0≠ values—possible implications in chemotherapy and biotechnology

To elucidate the kinetic properties of critical enzymatic situations that have previously escaped classification, we performed a systematic analysis of all the possible variations of the kinetic constants kcat, KM, and ksp 5 kcat/KM, encompassing all aspects of enzymology. The equation gives a total of thirteen theoretically possible cases, comprising the reference case plus 12 different sets of variations, which can be divided into six principal cases and six specular ones. The six relevant cases are examined individually in the context of each of the main chapters of enzymology, i.e. as regards mechanism of action, specificity of substrate and isoenzyme, reversible and irreversible inhibition, and mutation of residues (enzyme evolution and enzyme engineering). Some critical cases where ksp does not hold as a specificity index are classified for the first time. Interestingly, the six possible cases correspond to the five known cases of reversible inhibition (competitive, non-competitive, incompetitive, mixed competitive/non-competitive, and mixed incompetitive/non-competitive) plus an additional case of biphasic nature (activation-inhibition), which is crucial for a full understanding of specificity and which leads us to propose some modification to the definition of enzyme specificity. The systematic approach to enzymology outlined herein could find practical applications in various sectors of biotechnology, including chemotherapy.

Potent Arylsulfonamide Inhibitors of Tumor Necrosis Factor-α Converting Enzyme Able to Reduce Activated Leukocyte Cell Adhesion Molecule Shedding in Cancer Cell Models

Activated leukocyte cell adhesion molecule (ALCAM) plays a relevant role in tumor biology and progression. Our previous studies showed that ALCAM is expressed at the surface of epithelial ovarian cancer (EOC) cells and is released in a soluble form by ADAM-17-mediated shedding. This process is relevant to EOC cell motility and invasiveness, which is reduced by nonspecific inhibitors of ADAM-17. For this reason, ADAM-17 may represent a new useful target in anticancer therapy. Herein, we report the synthesis and biological evaluation of new ADAM-17 inhibitors containing an arylsulfonamidic scaffold. Among the new potential inhibitors, two very promising compounds 17 and 18 were discovered, with a nanomolar activity for ADAM-17 isolated enzyme. These compounds proved to be also the most potent in inhibiting soluble ALCAM release in cancer cells, showing a nanomolar activity on A2774 and SKOV3 cell lines.

Effects of sequential stimuli on Halobacterium salinarium photobehavior.

We analyzed the motor photoresponses of Halobacterium salinarium to different test stimuli applied after a first photophobic response produced by a step-down of red-orange light (prestimulus). We observed that pulses given with a suitable delay after the prestimulus produced unusual responses. Pulses of blue, green, or red-orange light, each eliciting no response when applied alone, produced a secondary photophobic response when applied several seconds after the prestimulus; the same occurred with a negative blue pulse (rapid shut-off and turning on of a blue light). Conversely, no secondary photophobic response was observed when the test stimulus was a step (a step-up for red-orange light, a step-down for blue light) of the same wavelength and intensity. When the delay was varied, different results were obtained with different wavelengths; red-orange pulses were typically effective in producing a secondary photophobic response, even with a delay of 2 s, whereas the response to a blue pulse was suppressed when the test stimulus was applied within 5 s after the prestimulus. The secondary photophobic response to pulses was abolished by reducing the intensity of the prestimulus without affecting the primary photophobic response. These results, some of which were previously reported in the literature as inverse effects, must be produced by a facilitating mechanism depending on the prestimulus itself, the occurrence of reversals being per se ineffective. The fact that red-orange test stimuli are facilitated even at the shortest delay, whereas those of different wavelengths become effective only after several seconds, suggests that the putative mechanism of the facilitating effect is specific for different signaling pathways.

New trends in photobiology: Photobehaviour of Halobacterium halobium: proposed models for signal transduction and motor switching

Abstract The swimming behaviour of Halobacterium halobium is influenced by variations in light intensity and wavelength. These stimuli are sensed through two retinal-containing proteins: sensory rhodopsin I and II (SR-I and SR-II), each displaying distinct spectra and photocycles. Light as an energy source is used by H. halobium through two other retinal-containing membrane proteins, acting as photon-driven ionic pumps, bacteriorhodopsin and halorhodopsin. In their ground states, SR-I and SR-II have an absorption maximum at 587 and 487 nm respectively. They have been shown to function as photoreceptors that signal, by a chemical transduction chain, to the motor switch controlling the direction of rotation of a flagellar bundle at the cell pole. Changes from clockwise to counterclockwise rotation or vice versa produce reversals in the swimming direction of H. halobium . Phototaxis occurs because the time interval between reversals is altered by response to light stimuli. SR-I acts as a receptor for attractant (red—orange) light stimuli and displays photochromic properties: when absorption in this wavelength range increases, reversals become less frequent and the pigment is transformed into a blue-absorbing species (SR 373 ). This intermediate of the SR-I photocycle appears to act both as a signalling state for the reversal-suppressing transduction chain and as a receptor for repellent (blue) light stimuli; these are effective in eliciting more frequent reversals when delivered against a red—orange background. SR-II, also called phoborhodopsin, only senses blue—green light (repellent) stimuli, producing an increased frequency of reversals. This sensor—effector system has been subjected to various types of analysis and constitutes the second best known example of this kind in prokaryotes, the first being chemotaxis in enterobacteria. Differences and similarities between these two natural models are described. Several mathematical models have been proposed to interpret the mechanisms underlying taxis phenomena. These are typically kinetic schemes for the transduction chain, which are partly hypothetical in H. halobium , due to the limited amount of convincing data on the nature of the (chemical) signal reaching the motor switch. Basically, two types of model have been suggested to account for the time interval distribution of reversals in the swimming behaviour of H. halobium : in the first (of which two variants have been proposed), the stochastic properties of the putative transduction chain signalling to the motor switch are involved; in the second, the hypothesis that an endogeneous oscillator is responsible for the motor switch control is maintained. Structural and biochemical data relevant for a model treatment of the processes occurring at the transduction chain are reported. The available behavioural data in H. halobium are also reported and discussed in detail, comparing the experimental approaches and the different interpretations. The implications of the proposed kinetic models are discussed.

Kinetic characterization of 4,4′-biphenylsulfonamides as selective non-zinc binding MMP inhibitors

Abstract We describe the characterisation of a series of 4,4′-biphenylsulfonamides as selective inhibitors of matrix metalloproteases MMP-2 and -13, two enzymes involved in cell invasion and angiogenesis. Double-inhibitor studies in the presence of acetohydroxamic acid show that these molecules do not bind the catalytic zinc. Moreover, two of the characterised inhibitors (11 and 19) act as non-competitive inhibitors, whereas the para-methyl ester derivative 13 behaves as a competitive inhibitor. This finding suggests that this class of molecules binds to a catalytic subsite, possibly the S1′-pocket. Moreover, since these compounds also act as inhibitors of carbonic anhydrases (CAs), another family of enzymes involved in cell invasion, they could be potentially useful as CA/MMP dual target inhibitors with increased efficacy as anticancer agents.

Synthesis, inhibitory activity towards human leukocyte elastase and molecular modelling studies of 1-carbamoyl-4-methyleneaminoxyazetidinones.

Some monocyclic beta-lactam derivatives of type 3 (MAOAs) in which the leaving group (LG) on the C(4) is a methyleneaminoxy moiety, were synthesised and tested in vitro and in vivo for their inhibitory activity towards human leukocyte elastase (HLE). Some compounds showed an appreciable in vitro inhibitory activity against this enzyme. Effects on the anti-HLE activity due to the nature of the substituents R and R(1) present on their LG were observed and rationalised by means of molecular modelling techniques. The results of in vivo pharmacological tests indicated that MAOAs, while showing an inhibitory activity on the haemorrhage induced by HLE, did not exhibit any effects due to the R and R(1) substituents.

A comment on the sensitivity of fish to low electric fields.

The sensitivity of sharks, skates, rays, and similar animals to extremely low electric fields is a popular topic in the nonlinear analysis community. It has been considered often in general editorial comments (Tsong, 1994; Glanz, 1996) and very recently in the “New and Notable” section of Biophysical Journal (Moss, 1997). Indeed, the sensitivity of some fish to electric fields appears to be astounding. Behavioral evaluation of the lowest field perceived by rays, found earlier to be 10 nV/cm in water (Kalmijn, 1982), has recently been reduced to 1–2 nV/cm (Kalmijn, 1997).

Photoresponses of Halobacterium salinarum to repetitive pulse stimuli.

Halobacterium salinarum cells from 3-day-old cultures have been stimulated with different patterns of repetitive pulse stimuli. A short train of 0.6-s orange light pulses with a 4-s period resulted in reversal peaks of increasing intensity. The reverse occurred when blue light pulses were delivered as a finite train: with a 3-s period, the response declined in sequence from the first to the last pulse. To evaluate the response of the system under steady-state conditions of stimulation, continuous trains of pulses were also applied; whereas blue light always produced a sharply peaked response immediately after each pulse, orange pulses resulted in a declining peak of reversals that lasted until the subsequent pulse. An attempt to account for these results in terms of current excitation/adaptation models shows that additional mechanisms appear to be at work in this transduction chain.