Target mutation-driven drug discovery

In the era of precision medicine, genetics is becoming a key factor for the choice of therapies. For decades, it has been intuitively thought that variability in drug response relies, at least partly, on gene variants that may affect the pharmacokinetics and pharmacodynamics of drugs. The most considerable evidences regard polymorphisms in genes encoding enzymes and carriers involved in the ADME processes. Nowadays, more attention should be paid to genetic variations in drug targets, especially in cancer and rare diseases

Clinical, Molecular, and Functional Characterization of CLCN1 Mutations in Three Families with Recessive Myotonia Congenita

Myotonia congenita (MC) is an inherited muscle disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. Both recessive (Becker's disease) or dominant (Thomsen's disease) MC are caused by mutations in the CLCN1 gene encoding the voltage-dependent chloride ClC-1 channel, which is quite exclusively expressed in skeletal muscle. More than 200 CLCN1 mutations have been associated with MC. We provide herein a detailed clinical, molecular, and functional evaluation of four patients with recessive MC belonging to three different families. Four CLCN1 variants were identified, three of which have never been characterized. The c.244A>G (p.T82A) and c.1357C>T (p.R453W) variants were each associated in compound heterozygosity with c.568GG>TC (p.G190S), for which pathogenicity is already known. The new c.809G>T (p.G270V) variant was found in the homozygous state. Patch-clamp studies of ClC-1 mutants expressed in tsA201 cells confirmed the pathogenicity of p.G270V, which greatly shifts the voltage dependence of channel activation toward positive potentials. Conversely, the mechanisms by which p.T82A and p.R453W cause the disease remained elusive, as the mutated channels behave similarly to WT. The results also suggest that p.G190S does not exert dominant-negative effects on other mutated ClC-1 subunits. Moreover, we performed a RT-PCR quantification of selected ion channels transcripts in muscle biopsies of two patients. The results suggest gene expression alteration of sodium and potassium channel subunits in myotonic muscles; if confirmed, such analysis may pave the way toward a better understanding of disease phenotype and a possible identification of new therapeutic options

Demographic and clinical determinants of neck pain in idiopathic cervical dystonia.

Cervical dystonia is associated with neck pain in a significant proportion of cases, but the mechanisms underlying pain are largely unknown. In this exploratory study, we compared demographic and clinical variables in cervical dystonia patients with and without neck pain from the Italian Dystonia Registry. Univariable and multivariable logistic regression analysis indicated a higher frequency of sensory trick and a lower educational level among patients with pain

Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in gating-defective or expression-defective mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 mu M NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations

Spread of muscle spasms in hemifacial spasm

Hemifacial spasm (HFS) is a clinical condition characterized by involuntary contractions in facial muscles. The aim of the study was to investigate, systematically in 178 patients with HFS, the frequency of spread from the site of origin to other facial muscles. Patients enrolled underwent a complete neurological examination and a face-to-face interview. Spread of the spasm to other facial muscles was considered to be present in those patients whose spasms onset in a single site and involved both upper and lower facial muscles at the time of examination. We also collected information about gender, age, age at HFS onset, symptom duration, muscles involved by the spasm at the time of onset, and spread of spasm to other facial muscles. Spread of spasms to the other facial muscles of the same side of the face was present in 93.4% of patients with HFS, and latency of spread was related to disease duration and age at onset. In patients with HFS, spread of muscle spasms represents the natural history of HFS

Synthesis and Biological Evaluation of Novel Cinnamic Acid-Based Antimicrobials

The main antimicrobial resistance (AMR) nosocomial strains (ESKAPE pathogens such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are the most widespread bacteria in cutaneous infections. In this work we report the synthesis, in silico skin permeability prediction, antimicrobial, antibiofilm, and wound healing properties of novel cinnamic acid-based antimicrobials (DM1–11) as novel antibacterial drugs for the treatment of ESKAPE-related skin infections. Antimicrobial and wound healing scratch assays were performed to evaluate the antibacterial properties of DM1–11. In silico skin permeability capabilities of DM1–11 were evaluated using Swiss-ADME online database. Cytotoxicity assays were performed on keratinocytes and fibroblasts. DM2, bearing a catechol group on the aromatic ring of the cinnamic portion of the molecule, possesses a significant antibacterial activity against S. aureus (MIC range 16–64 mg/L) and contrasts the biofilm-mediated S. epidermidis infection at low concentrations. Wound healing assays showed that wound closure in 48 h was observed in DM2-treated keratinocytes with a better healing pattern at all the used concentrations (0.1, 1.0, and 10 µM). A potential good skin permeation for DM2, that could guarantee its effectiveness at the target site, was also observed. Cytotoxicity studies revealed that DM2 may be a safe compound for topical use. Taking together all these data confirm that DM2 could represent a safe wound-healing topical agent for the treatment of skin wound infections caused by two of main Gram-positive bacteria belonging to ESKAPE microorganisms

A novel KCNA1 mutation in a patient with paroxysmal ataxia, myokymia, painful contractures and metabolic dysfunctions

Episodic ataxia type 1 (EA1) is a human dominant neurological syndrome characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles that can be triggered by emotional stress and fatigue. This rare disease is caused by missense mutations in the KCNA1 gene coding for the neuronal voltage gated potassium channel Kv1.1, which contributes to nerve cell excitability in the cerebellum, hippocampus, cortex and peripheral nervous system. We identified a novel KCNA1 mutation, E283K, in an Italian proband presenting with paroxysmal ataxia and myokymia aggravated by painful contractures and metabolic dysfunctions. The E283K mutation is located in the S3â\u80\u93S4 extracellular linker belonging to the voltage sensor domain of Kv channels. In order to test whether the E283K mutation affects Kv1.1 biophysical properties we transfected HEK293 cells with WT or mutant cDNAs alone or in a 1:1 combination, and recorded relative potassium currents in the whole-cell configuration of patch-clamp. Mutant E283K channels display voltage-dependent activation shifted by 10 mV toward positive potentials and kinetics of activation slowed by ~ 2 fold compared to WT channels. Potassium currents resulting from heteromeric WT/E283K channels show voltage-dependent gating and kinetics of activation intermediate between WT and mutant homomeric channels. Based on homology modeling studies of the mutant E283K, we propose a molecular explanation for the reduced voltage sensitivity and slow channel opening. Overall, our results suggest that the replacement of a negatively charged residue with a positively charged lysine at position 283 in Kv1.1 causes a drop of potassium current that likely accounts for EA-1 symptoms in the heterozygous carrier