Matteo Colombo

Synthesis and Biological Evaluation of 9-Oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile Analogues as Potential Inhibitors of Deubiquitinating Enzymes

High‐throughput screening highlighted 9‐oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile (1) as an active inhibitor of ubiquitin‐specific proteases (USPs), a family of hydrolytic enzymes involved in the removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1, led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.

DE‐loop mutations affect β2 microglobulin stability, oligomerization, and the low‐pH unfolded form

β2 microglobulin (β2m) is the light chain of class‐I major histocompatibility complex (MHC‐I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of β2m aggregation, several β2m mutants have been previously studied. Among these, three single‐residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect β2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild‐type (w.t.) β2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray‐ionization mass spectrometry (ESI‐MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE‐loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, β2m DE‐loop mutations display long‐range effects, affecting stability and structural properties of the native protein and its low‐pH intermediate. The evidence presented here hints to a crucial role played by the DE‐loop in determining the overall properties of native and partially folded β2m.

Synthesis of new bicyclic lactam peptidomimetics by ring-closing metathesis reactions

Abstract An efficient and versatile synthetic method for the preparation of new fused bicyclic lactams 3a and 3b is described. The spirane cyclopentane nucleus was easily installed by diallylation of the pyroglutamate derivative 18 followed by ring-closing metathesis (RCM). A more practical and stereoselective method for the allylation of the α-methoxy carbamate 21 , involving the use of InCl3 as a Lewis acid, was developed. In the crucial coupling reaction of the diastereomeric mixture of cis- and trans-pirrolidine derivatives 5a and 5b with N-Cbz vinyl phenylalanine only the cis isomer was found to react. An RCM reaction on the dipeptides 25a and 25b followed by catalytic hydrogenation, gave the final epimeric bicyclic lactams 3a and 3b . The same synthetic sequence on the model compound 7 , lacking the spiro cyclopentane nucleus, is also reported.

DE loop mutations affect beta2-microglobulin stability and amyloid aggregation

Beta2-microglobulin (beta2m) is the light chain component of class I major histocompatibility complex (MHC-I). beta2m is an intrinsically amyloidogenic protein that can assemble into amyloid fibrils in vitro and in vivo. Several recent reports suggested that the polypeptide loop comprised between beta-strands D and E of beta2m is important for protein stability and for the protein propensity to aggregate as amyloid fibrils. In particular, the roles of Trp60 for MHC-I assembly and beta2m stability have been highlighted by showing that the beta2m Trp60-->Gly mutant is more stable and less prone to aggregation than the wild type protein. To further analyse such properties, the Trp60-->Cys and Asp59-->Pro beta2m mutants have been expressed, purified, and their crystal structures determined. The stability to thermal denaturation and propensity to fibrillar aggregation have also been analysed. The experimental evidences gathered on the two mutants reinforce the hypothesis that conformational strain in the DE loop can affect beta2m stability and amyloid aggregation properties.

Practical stereoselective synthesis of conformationally constrained unnatural proline-based amino acids and peptidomimetics

Abstract A practical synthetic scheme was developed to prepare both the cis- and trans-fused stereoisomers of N-Boc- l -octahydroindole-2-carboxylic acid ( l -Oic) methyl ester. Key event of the synthetic sequence was the ring-closing metathesis of a suitable diallylated proline derivative. This is the first reported practical synthesis of the trans-fused isomer. Functionalization of the octahydroindole nucleus by electrochemical oxidation followed by acid-catalysed allylation paved the way for the preparation of reverse-turn dipeptide mimics.

Evaluation of tubulin polymerization and chronic inhibition of proteasome as citotoxicity mechanisms in bortezomib-induced peripheral neuropathy.

Bortezomib (BTZ) is the first proteasome inhibitor entered in clinical practice. Peripheral neuropathy is likely to be a class side effect of these drugs, although its severity is largely variable, and it deserves to be further investigated, since the mechanisms of BTZ-induced peripheral neurotoxicity (BiPN) are still unknown. In our study, we investigated in vivo and in vitro possible pathogenic events relevant to BiPN using a well-established rat model, with particular reference to the extent of proteasome inhibition and the effects on α-tubulin polymerization in sciatic nerves and dorsal root ganglia specimens obtained from animals treated with chronic regimens at a dose of 0.2 mg/kg intravenously. The same assessments were also performed after a single injection. Moreover, these studies were replicated in vitro using embryonic DRG neurons exposed to 100 nM BTZ and adult DRG neurons exposed to 10–50 nM BTZ for 24 h and 48 h. A significant increase in the polymerized fraction of α-tubulin and prolonged proteasome inhibition were observed after the chronic BTZ treatment in vivo. Recovery to physiological levels was observed after a 4-week follow-up post-treatment period. Proteasome inhibition and increased α-tubulin polymerization were also observed following BTZ treatment of both embryonic and adult DRG neurons in vitro. Our in vivo results suggest that proteasome inhibition and alteration of tubulin dynamics contribute to BiPN. The in vitro systems here described reliably replicate the in vivo results, and might therefore be used for further mechanistic studies on the effects of proteasome inhibitors on neurons.

The interaction between the measles virus nucleoprotein and the Interferon Regulator Factor 3 relies on a specific cellular environment

BackgroundThe genome of measles virus consists of a non-segmented single-stranded RNA molecule of negative polarity, which is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. The N protein possesses an intrinsically disordered C-terminal domain (aa 401–525, NTAIL) that is exposed at the surface of the viral nucleopcapsid. Thanks to its flexible nature, NTAIL interacts with several viral and cellular partners. Among these latter, the Interferon Regulator Factor 3 (IRF-3) has been reported to interact with N, with the interaction having been mapped to the regulatory domain of IRF-3 and to NTAIL. This interaction was described to lead to the phosphorylation-dependent activation of IRF-3, and to the ensuing activation of the pro-immune cytokine RANTES gene.ResultsAfter confirming the reciprocal ability of IRF-3 and N to be co-immunoprecipitated in 293T cells, we thoroughly investigated the NTAIL-IRF-3 interaction using a recombinant, monomeric form of the regulatory domain of IRF-3. Using a large panel of spectroscopic approaches, including circular dichroism, fluorescence spectroscopy, nuclear magnetic resonance and electron paramagnetic resonance spectroscopy, we failed to detect any direct interaction between IRF-3 and either full-length N or NTAIL under conditions where these latter interact with the C-terminal X domain of the viral phosphoprotein. Furthermore, such interaction was neither detected in E. coli nor in a yeast two hybrid assay.ConclusionAltogether, these data support the requirement for a specific cellular environment, such as that provided by 293T human cells, for the NTAIL-IRF-3 interaction to occur. This dependence from a specific cellular context likely reflects the requirement for a human or mammalian cellular co-factor.

Synthesis of spiroazabicycloalkane amino acid scaffolds as reverse-turn inducer dipeptide mimics

Abstract A practical approach to the synthesis of a conformationally constrained spiroazabicycloalkane aminoacid scaffold as a reverse-turn inducer dipeptide mimic is described.

Synthesis of substituted conformationally constrained 6,5- and 7,5-fused bicyclic lactams as dipeptide mimics

Using a convenient and practical route we report the preparation of a series of rigid surrogates of amino acids and dipeptides for application within constrained peptide analogues, and for employment as input for combinatorial science. These substituted 2-oxo-1-azabicycloalkane amino acids have the potential of replicating the backbone geometry and side-chain function of dipeptide residues like serine, lysine, glutamate, and related amino acids.

D-strand perturbation and amyloid propensity in beta-2 microglobulin

Proteins hosting main β‐sheets adopt specific strategies to avoid intermolecular interactions leading to aggregation and amyloid deposition. Human beta‐2 microglobulin (β2m) displays a typical immunoglobulin fold and is known to be amyloidogenic in vivo. Upon severe kidney deficiency, β2m accumulates in the bloodstream, triggering, over the years, pathological deposition of large amyloid aggregates in joints and bones. A β‐bulge observed on the edge D β‐strand of some β2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation. Conversely, a straight D‐strand, observed in different crystal structures of monomeric β2m, could promote amyloid aggregation. More recently, the different conformations observed for the β2m D‐strand have been interpreted as the result of intrinsic flexibility, rather than being assigned to a functional protective role against aggregation. To shed light on such contrasting picture, the mutation Asp53→Pro was engineered in β2m, aiming to impair the formation of a regular/straight D‐strand. Such a mutant was characterized structurally and biophysically by CD, X‐ray crystallography and MS, in addition to an assessment of its amyloid aggregation trends in vitro. The results reported in the present study highlight the conformational plasticity of the edge D‐strand, and show that even perturbing the D‐strand structure through a Pro residue has only marginal effects on protecting β2m from amyloid aggregation in vitro.