Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

PURPOSE: Coffin-Siris and Nicolaides-Baraitser syndromes are recognizable neurodevelopmental disorders caused by germline variants in BAF complex subunits. The SMARCC2 BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort. METHODS: Clinical symptoms for 41 novel and 24 previously published affected individuals were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlations, molecular data were standardized and grouped into non-truncating and likely gene-disrupting (LGD) variants. Missense variant protein expression and BAF-subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays. RESULTS: Neurodevelopmental delay with intellectual disability, muscular hypotonia, and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, whereas non-truncating variants were mostly de novo and presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms. In vitro testing showed decreased protein expression for N-terminal missense variants similar to LGD. CONCLUSION: This study improved SMARCC2 variant classification and identified discernible SMARCC2-associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

Molecular characteristics of several drugs evaluated from solvent/water partition measurements: Solvation parameters and intramolecular hydrogen bond indicator.

A wide set of well-known drugs, most of them included in the Abraham´s reference database, covering a wide variety of chemical structures and therapeutical functionalities were chosen in order to determine some molecular properties from solvent/water partition measurements. Partition data from aqueous solutions and four different solvents (n-dodecane, toluene, chloroform and n-octanol) were measured and reported. From them, Abraham´s molecular descriptors of selected compounds (A, B and S, accounting for hydrogen bond donor, hydrogen bond acceptor and dipolarity/polaritzability, respectively) were estimated. A and B values derived from the experimental measurements strongly agree with the tabulated ones showing the suitability of the used procedure to achieve reliable values for new molecules. However, obtained S values differ from those previously reported for several compounds. Moreover, values for a new indicator of the propensity to form intramolecular hydrogen bonds (Δlog Poct-tol) were estimated from the experimental data and also calculated according to both, the Abraham´s model and the molecular structures (SMD). The quality of both series of calculated descriptors was evaluated by contrast with the experimental values and satisfactory results were obtained in both instances. Thus, the Abraham´s way is useful when molecular descriptors are available but very good estimations can be achieved by SMD, which only requires the drug´s molecular structure.

BDV Syndrome: An Emerging Syndrome With Profound Obesity and Neurodevelopmental Delay Resembling Prader-Willi Syndrome.

CONTEXT: CPE encodes carboxypeptidase E, an enzyme that converts proneuropeptides and propeptide hormones to bioactive forms. It is widely expressed in the endocrine and central nervous system. To date, 4 individuals from 2 families with core clinical features including morbid obesity, neurodevelopmental delay, and hypogonadotropic hypogonadism, harboring biallelic loss-of-function (LoF) CPE variants, have been reported. OBJECTIVE: We describe 4 affected individuals from 3 unrelated consanguineous families, 2 siblings of Syrian, 1 of Egyptian, and 1 of Pakistani descent, all harboring novel homozygous CPE LoF variants. METHODS: After excluding Prader-Willi syndrome (PWS), exome sequencing was performed in both Syrian siblings. The variants identified in the other 2 individuals were reported as research variants in a large-scale exome study and in the ClinVar database. Computational modeling of all possible missense alterations allowed assessing CPE tolerance to missense variants. RESULTS: All affected individuals were severely obese with neurodevelopmental delay and other endocrine anomalies. Three individuals from 2 families shared the same CPE homozygous truncating variant c.361C > T, p.(Arg121*), while the fourth carried the c.994del, p.(Ser333Alafs*22) variant. Comparison of clinical features with previously described cases and standardization according to the Human Phenotype Ontology terms indicated a recognizable clinical phenotype, which we termed Blakemore-Durmaz-Vasileiou (BDV) syndrome. Computational analysis indicated high conservation of CPE domains and intolerance to missense changes. CONCLUSION: Biallelic truncating CPE variants are associated with BDV syndrome, a clinically recognizable monogenic recessive syndrome with childhood-onset obesity, neurodevelopmental delay, hypogonadotropic hypogonadism, and hypothyroidism. BDV syndrome resembles PWS. Our findings suggest missense variants may also be clinically relevant.

Ionizable Drug Self-Associations and the Solubility Dependence on pH: Detection of Aggregates in Saturated Solutions Using Mass Spectrometry (ESI-Q-TOF-MS/MS).

It is widely accepted that solubility-pH profiles of ionizable compounds follow the Henderson-Hasselbalch equation. However, several studies point out that compounds often undergo additional processes in saturated solutions, such as sub-micellar oligomerization, micellar aggregation, or drug-buffer complexation among others, which make the experimental profiles deviate from the behavior predicted by the Henderson-Hasselbalch equation. Often, the presence of additional processes is supported by the analysis of experimental data through solubility computer programs. However, the purpose of this work is to experimentally prove the aggregation phenomena for a series of bases for which deviations from the theoretical profile have been observed. To this end, five monoprotic bases (lidocaine, maprotiline, cyproheptadine, bupivacaine, and mifepristone) susceptible to form ionic aggregates in solution have been selected, and mass spectrometry has been the technique of choice to prove the presence of aggregation. High declustering potentials have been applied to prevent aggregates from forming in the ionization source of the mass spectrometer. In addition, haloperidol has been used as a negative control since according to its profile, it is not suspected to form ionic aggregates. In all instances, except for haloperidol, the analysis of the saturated solutions revealed the presence of mixed-charged dimers (aggregates formed by a neutral molecule and a charged one) and even trimers in the case of mifepristone and bupivacaine. For lidocaine, the most soluble of the compounds, the presence of neutral aggregates was also detected. These experiments support the hypothesis that the simple Henderson-Hasselbalch equation may explain the solubility-pH behavior of certain compounds, but it can be somewhat inaccurate in describing the behavior of many other substances.

Single-cell RNA sequencing of adult mouse testes.

Spermatogenesis is an efficient and complex system of continuous cell differentiation. Previous studies investigating the transcriptomes of different cell populations in the testis relied either on sorting cells, cell depletion, or juvenile animals where not all stages of spermatogenesis have been completed. We present single-cell RNA sequencing (scRNA-Seq) data of 2,500 cells from the testes of two 8-week-old C57Bl/6J mice. Our dataset includes all spermatogenic stages from preleptotene to condensing spermatids as well as individual spermatogonia, Sertoli and Leydig cells. The data capture the full continuity of the meiotic and postmeiotic stages of spermatogenesis, and is thus ideally suited for marker discovery, network inference and similar analyses for which temporal ordering of differentiation processes can be exploited. Furthermore, it can serve as a reference for future studies involving single-cell RNA-Seq in mice where spermatogenesis is perturbed.

Critical comparison of shake-flask, potentiometric and chromatographic methods for lipophilicity evaluation (log Po/w) of neutral, acidic, basic, amphoteric, and zwitterionic drugs.

In the present study three different procedures have been compared for the determination of the lipophilicity of the unionized species (log Po/w) of neutral, acidic, basic, amphoteric, and zwitterionic drugs. Shake-flask, potentiometric and chromatographic approaches have been assayed in a set of 66 representative compounds in different phases of advanced development. An excellent equivalence has been found between log Po/w values obtained by shake-flask and potentiometry, while the chromatographic approach is less accurate but very convenient for screening purposes when a high-throughput is required. In the case of zwitterionic and amphoteric compounds, either for shake-flask and chromatographic methods, the pH has to be accurately selected in order to ensure the compound to be in its neutral form.

Effect of vinylpyrrolidone polymers on the solubility and supersaturation of drugs; a study using the Cheqsol method.

The development of methods to increase the bioavailability of drugs is of great interest, especially for those which are poorly soluble or permeable. One of the strategies to enhance the solubility (which in turn has the potential of increase bioavailability) of drugs is the use of additives in the formulation process, so that the drug can stay supersaturated in biological fluids for a period of time long enough to allow absorption. The use of polymers as pharmaceutical excipients in order to stabilize the supersaturation of drugs is common practice. In this work, the ability of different polymers of vinylpyrrolidone (K-12, K-17, K-25, K-29/32, K-90) and a copolymer of vinylpyrrolidone and vinylacetate (S-630) have been tested for their impact on the supersaturation of drugs. Sixteen drugs of different chemical nature have been selected, and analyzed using the Cheqsol method. The results of the drug alone, and of physical mixtures with the different polymers at several polymer:drug ratios have been compared in terms of supersaturation extent and duration. It has been observed that acidic compounds displayed enhanced solubility in different ways: sometimes the supersaturated state of the drug is maintained for a long time, due to the precipitation of an amorphous solid, as determined by X-ray diffraction studies; on other occasions supersaturation increases but only for a short time, compared to the drug alone, and then the drug precipitates to a crystalline form. Only a few basic drugs displayed enhanced solubility in the presence of PVP polymers, in contrast to acidic compounds.

Molecular interactions between warfarin and human (HSA) or bovine (BSA) serum albumin evaluated by isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE).

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction events for both albumins are detected only by FA/CE.

Lipophilicity of amphoteric and zwitterionic compounds: A comparative study of determination methods.

Common drugs intended for action in plasma (antibacterials, antiallergics, diuretics...) often display both acidic and basic behavior, and some of these amphoteric compounds can appear as zwitterions. In such cases, accurate profiling of lipophilicity vs. pH, which plays a fundamental role in drug pharmacokinetics, might be complex. In the present work two common lipophilicity determination methods based on the drug distribution between 1-octanol and aqueous buffer i.e. phase equilibration (shake-flask) and two-phase titration (potentiometry), were compared with a high-throughput lipophilicity index, the Chromatographic Hydrophobicity Index (CHI). The results were also compared with log Do/w pH-profiles calculated by different algorithms from ACD/Labs. Accurate and similar results were obtained for both octanol-water approaches but, due to the lower determination times and the absence of different ion-pairing buffers, potentiometry was shown to be the most convenient method. CHI vs. pH profiles provide rapid and efficient information, which is very convenient for lipophilicity screening purposes, but may differ slightly from shake-flask and potentiometric results.

Phenothiazines solution complexity - Determination of pKa and solubility-pH profiles exhibiting sub-micellar aggregation at 25 and 37°C.

The ionization constants (pKa) and the pH-dependent solubility (log S-pH) of six phenothiazine derivatives (promazine hydrochloride, chlorpromazine hydrochloride, triflupromazine hydrochloride, fluphenazine dihydrochloride, perphenazine free base, and trifluoperazine dihydrochloride) were determined at 25 and 37°C. The pKa values of these low-soluble surface active molecules were determined by the cosolvent method (n-propanol/water at 37°C and methanol/water at 25°C). The log S-pH profiles were measured at 24h incubation time in 0.15M phosphate buffers. The log S-pH "shape-template" method, which critically depends on accurate pKa values (determined independently of solubility data), was used to propose speciation models, which were subsequently refined by rigorous mass-action weighted regression procedure described recently. Differential scanning calorimetry (DSC), UV-visible spectrophotometry, potentiometric, and high performance liquid chromatography (HPLC) measurements were used to characterize the compounds. The intrinsic solubility (S0) values of the three least-soluble drugs (chlorpromazine·HCl, triflupromazine·HCl, and trifluoperazine·2HCl) at 25°C were 0.5, 1.1, and 2.7µg/mL (resp.). These values increased to 5.5, 9.2, and 8.7µg/mL (resp.) at the physiological temperature. The enthalpies of solution for the latter compounds were exceptionally high positive (endothermic) values (99-152kJ·mol(-1)). Cationic sub-micellar aggregates were evident (from the distortions in the log S-pH profiles) for chlorpromazine, fluphenazine, perphenazine, and trifluoperazine at 25°C. The effects persisted at 37°C for chlorpromazine and trifluoperazine. The solids in suspension were apparently amorphous in cases where the drugs were introduced as the chloride salts.

The Ca(2+)-EDTA chelation as standard reaction to validate Isothermal Titration Calorimeter measurements (ITC).

A study about the suitability of the chelation reaction of Ca(2+)with ethylenediaminetetraacetic acid (EDTA) as a validation standard for Isothermal Titration Calorimeter measurements has been performed exploring the common experimental variables (buffer, pH, ionic strength and temperature). Results obtained in a variety of experimental conditions have been amended according to the side reactions involved in the main process and to the experimental ionic strength and, finally, validated by contrast with the potentiometric reference values. It is demonstrated that the chelation reaction performed in acetate buffer 0.1M and 25°C shows accurate and precise results and it is robust enough to be adopted as a standard calibration process.

High-throughput logPo/w determination from UHPLC measurements: Revisiting the chromatographic hydrophobicity index.

A fast and accurate lipophilicity determination is fundamental in the drug discovery process, as long as it is a relevant property in the absorption, distribution, metabolism, excretion and toxicity (ADMET) of a potential drug substance. In the present work, different models based on chromatographic retention values for a large set of compounds and some of their molecular descriptors (calculated by ACD/Labs or CODESSA programs) have been examined in order to establish reliable equations for logPo/w determination from fast chromatographic hydrophobicity index (CHI) measurements. This appears to be a very interesting high-throughput methodology for screening purposes, since CHI values can be measured by UHPLC in very short runs (<4min) and molecular descriptors can be easily computed from the structure of any compound. The selected final descriptors were Abraham's hydrogen-bond acidity (A) and excess molar refraction (E) from ACD/Labs, and hydrogen-bond acidity HDCA-1/TMSA and HOMO-LUMO polarizability descriptors from CODESSA software. The proposed equations allow an accurate determination of logPo/w with standard errors in the range of 0.4 units.

Binding thermodynamics of paromomycin, neomycin, neomycin-dinucleotide and -diPNA conjugates to bacterial and human rRNA.

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity.

Setup and validation of shake-flask procedures for the determination of partition coefficients (logD) from low drug amounts.

Several procedures based on the shake-flask method and designed to require a minimum amount of drug for octanol-water partition coefficient determination have been established and developed. The procedures have been validated by a 28 substance set with a lipophilicity range from -2.0 to 4.5 (logD7.4). The experimental partition is carried out using aqueous phases buffered with phosphate (pH 7.4) and n-octanol saturated with buffered water and the analysis is performed by liquid chromatography. In order to have accurate results, four procedures and eight different ratios between phase volumes are proposed. Each procedure has been designed and optimized (for partition ratios) for a specific range of drug lipophilicity (low, regular and high lipophilicity) and solubility (high and low aqueous solubility). The procedures have been developed to minimize the measurement in the octanolic phase. Experimental logD7.4 values obtained from different procedures and partition ratios show a standard deviation lower than 0.3 and there is a nice agreement when these values are compared with the reference literature ones.

Prediction of the chromatographic retention of acid-base compounds in pH buffered methanol-water mobile phases in gradient mode by a simplified model.

Retention of ionizable analytes under gradient elution depends on the pH of the mobile phase, the pKa of the analyte and their evolution along the programmed gradient. In previous work, a model depending on two fitting parameters was recommended because of its very favorable relationship between accuracy and required experimental work. It was developed using acetonitrile as the organic modifier and involves pKa modeling by means of equations that take into account the acidic functional group of the compound (carboxylic acid, protonated amine, etc.). In this work, the two-parameter predicting model is tested and validated using methanol as the organic modifier of the mobile phase and several compounds of higher pharmaceutical relevance and structural complexity as testing analytes. The results have been quite good overall, showing that the predicting model is applicable to a wide variety of acid-base compounds using mobile phases prepared with acetonitrile or methanol.

Molecular details of INH-C10 binding to wt KatG and Its S315T mutant.

Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multitherapeutic regimens. Because of the increasing resistance of Mycobacterium tuberculosis to INH, mainly associated with mutations in the katG gene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between wt KatG or its S315T mutant form and either INH or INH-C10, a new acylated INH derivative. MD simulations were used to explore the conformational space of both proteins, and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C10 seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C10 alkyl chain role and helped to rationalize the significantly lower experimental MIC value observed for INH-C10. This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C10 appears to be a very promising lead compound for drug development.

Gradient retention prediction of acid-base analytes in reversed phase liquid chromatography: a simplified approach for acetonitrile-water mobile phases.

In previous work, a two-parameter model to predict chromatographic retention of ionizable analytes in gradient mode was proposed. However, the procedure required some previous experimental work to get a suitable description of the pKa change with the mobile phase composition. In the present study this previous experimental work has been simplified. The analyte pKa values have been calculated through equations whose coefficients vary depending on their functional group. Forced by this new approach, other simplifications regarding the retention of the totally neutral and totally ionized species also had to be performed. After the simplifications were applied, new prediction values were obtained and compared with the previously acquired experimental data. The simplified model gave pretty good predictions while saving a significant amount of time and resources.

Molecular interactions between some non-steroidal anti-inflammatory drugs (NSAID's) and bovine (BSA) or human (HSA) serum albumin estimated by means of isothermal titration calorimetry (ITC) and frontal analysis capillary electrophoresis (FA/CE).

The interactions between some non-steroidal anti-inflammatory drugs, NSAIDs, (naproxen, ibuprofen and flurbiprofen) and bovine (BSA) or human (HSA) serum albumin have been examined by means of two complementary techniques, isothermal titration calorimetry (ITC) and frontal analysis/capillary electrophoresis (FA/CE). It can be concluded that ITC is able to measure with high precision the strongest drug-albumin interactions but the higher order interactions can be better determined by means of FA/CE. Then, the combination of both techniques leads to a complete evaluation of the binding profiles between the selected NSAIDs and both kind of albumin proteins. When BSA is the binding protein, the NSAIDs show a strong primary interaction (binding constants: 1.5 × 10(7), 8 × 10(5) and 2 × 10(6) M(-1) for naproxen, ibuprofen and flurbiprofen, respectively), and also lower affinity interactions of the same order for the three anti-inflammatories (about 1.7 × 10(4) M(-1)). By contrast, when HSA is the binding protein two consecutive interactions can be observed by ITC for naproxen (9 × 10(5) and 7 × 10(4) M(-1)) and flurbiprofen (5 × 10(6) and 6 × 10(4) M(-1)) whereas only one is shown for ibuprofen (9 × 10(5) M(-1)). Measurements by FA/CE show a single interaction for each drug being the ones of naproxen and flurbiprofen the same that those evaluated by ITC as the second interaction events. Then, the ability of both techniques as suitable complementary tools to establish the whole interaction NSAIDs-albumin profile is experimentally demonstrated and allows foreseeing suitable strategies to establish the complete drug-protein binding profile. In addition, for the interactions analyzed by means of ITC, the thermodynamic signature is established and the relative contributions of the enthalpic and entropic terms discussed.

The contribution of the hydrogen bond acidity on the lipophilicity of drugs estimated from chromatographic measurements.

The influence of the hydrogen bond acidity when the 1-octanol/water partition coefficient (log P(o/w)) of drugs is determined from chromatographic measurements was studied in this work. This influence was firstly evaluated by means of the comparison between the Abraham solvation parameter model when it is applied to express the 1-octanol/water partitioning and the chromatographic retention, expressed as the solute polarity p. Then, several hydrogen bond acidity descriptors were compared in order to determine properly the log P(o/w) of drugs. These descriptors were obtained from different software and comprise two-dimensional parameters such as the calculated Abraham hydrogen bond acidity A and three-dimensional descriptors like HDCA-2 from CODESSA program or WO1 and DRDODO descriptors calculated from Volsurf+software. The additional HOMO-LUMO polarizability descriptor should be added when the three-dimensional descriptors are used to complement the chromatographic retention. The models generated using these descriptors were compared studying the correlations between the determined log P(o/w) values and the reference ones. The comparison showed that there was no significant difference between the tested models and any of them was able to determine the log P(o/w) of drugs from a single chromatographic measurement and the correspondent molecular descriptors terms. However, the model that involved the calculated A descriptor was simpler and it is thus recommended for practical uses.

Solubility-pH profiles of some acidic, basic and amphoteric drugs.

The solubility vs. pH profiles of five ionizable drugs of different nature (a monoprotic acid, a monoprotic base, a diprotic base and two amphoteric compounds showing a zwitterionic species each one) have been determined through two different methodologies: the classical shake-flask (S-F) and the potentiometric Cheqsol methods using in both instances the appropriate Henderson-Hasselbalch (H-H) or derived relationships. The results obtained independently from both approaches are consistent. A critical revision about the influence of the electrolyte used as buffering agent in the S-F method on the obtained solubility values is also performed. Thus, some deviations of the experimental points with respect the H-H profiles can be attributed to specific interactions between the buffering electrolyte and the drug due to the hydrotrophic character of citric and lactic acids. In other cases, the observed deviations are independent of the buffers used since they are caused by the formation of new species such as drug aggregates (cefadroxil) or the precipitation of a salt from a cationic species of the analyzed compound (quetiapine).