Identification of novel inhibitors targeting TIRAP interactions with BTK and PKCδ in inflammation through an in silico approach.

Advanced computational tools focusing on protein-protein interaction (PPI) based drug development is a powerful platform to accelerate the therapeutic development of small lead molecules and repurposed drugs. Toll/interleukin-1 receptor (TIR) domain-containing adapter protein (TIRAP) and its interactions with other proteins in macrophages signalling are crucial components of severe or persistent inflammation. TIRAP activation through Bruton's tyrosine kinase (BTK) and Protein Kinase C delta (PKCδ) is essential for downstream inflammatory signalling. We created homology-based structural models of BTK and PKCδ in MODELLER 9.24. TIRAP interactions with BTK and PKCδ in its non-phosphorylated and phosphorylated states were determined by multiple docking tools including HADDOCK 2.4, pyDockWEB and ClusPro 2.0. Food and Drug Administration (FDA)-approved drugs were virtually screened through Discovery Studio LibDock and Autodock Vina tools to target the common TIR domain residues of TIRAP, which interact with both BTK and PKC at the identified interfacial sites of the complexes. Four FDA-approved drugs were identified and found to have stable interactions over a range of 100 ns MD simulation timescales. These drugs block the interactions of both kinases with TIRAP in silico. Hence, these drugs have the potential to dampen downstream inflammatory signalling and inflammation-mediated disease.

ACE inhibitors, angiotensin receptor blockers and endothelial injury in COVID-19.

BACKGROUND: COVID-19 is caused by the coronavirus SARS-CoV-2, which uses angiotensin-converting enzyme 2 (ACE-2) as a receptor for cellular entry. It is theorized that ACE inhibitors (ACE-Is) or angiotensin receptor blockers (ARBs) may increase vulnerability to SARS-CoV-2 by upregulating ACE-2 expression, but ACE-I/ARB discontinuation is associated with clinical deterioration. OBJECTIVE: To determine whether ACE-I and ARB use is associated with acute kidney injury (AKI), macrovascular thrombosis and in-hospital mortality. METHODS: A retrospective, single-centre study of 558 hospital inpatients with confirmed COVID-19 admitted from 1 March to 30 April 2020, followed up until 24 May 2020. AKI and macrovascular thrombosis were primary end-points, and in-hospital mortality was a secondary end-point. RESULTS: AKI occurred in 126 (23.1%) patients, 34 (6.1%) developed macrovascular thrombi, and 200 (35.9%) died. Overlap propensity score-weighted analysis showed no significant effect of ACE-I/ARB use on the risk of occurrence of the specified end-points. On exploratory analysis, severe chronic kidney disease (CKD) increases odds of macrovascular thrombi (OR: 8.237, 95% CI: 1.689-40.181, P = 0.009). The risk of AKI increased with advancing age (OR: 1.028, 95% CI: 1.011-1.044, P = 0.001) and diabetes (OR: 1.675, 95% CI: 1.065-2.633, P = 0.025). Immunosuppression was associated with lower risk of AKI (OR: 0.160, 95% CI: 0.029-0.886, P = 0.036). Advancing age, dependence on care, male gender and eGFR < 60 mL min-1 /1.73 m2 increased odds of in-hospital mortality. CONCLUSION: We did not identify an association between ACE-I/ARB use and AKI, macrovascular thrombi or mortality. This supports the recommendations of the European and American Societies of Cardiology that ACE-Is and ARBs should not be discontinued during the COVID-19 pandemic.

Identifying the inhibition of TIR proteins involved in TLR signalling as an anti-inflammatory strategy.

Toll/IL1 receptor (TIR) adaptor proteins continue to be an integral part of Toll-like receptors' (TLR) signalling involved in inflammation. Signalling is likely to be initiated by these TIR adaptors when they are recruited to a TIR-TIR interface formed by TLR dimerization. Among these, myeloid differentiation factor-88 (MyD88), MyD88 adapter-like protein (Mal), TIR domain-containing adaptor protein inducing interferon-ß (TRIF) and TRIF-related adaptor molecule (TRAM) play pivotal roles at many steps in the signalling events leading to inflammation. The presence of the conserved BB loop residues in the TIR domain of all these important adaptor proteins make them possible targets for inhibition by synthetic compounds. We have designed compounds based on an already known MyD88 TIR dimerization inhibitor, T6167923, which binds well not only to the original target but also to the TIR domains of Mal, TRIF and TRAM. The designed inhibitors are based on modifications of the bromophenyl-sulphonyl-thiophenyl-piperazine-carboxamide series of compounds. We have further suggested modifications in these high-affinity compounds for efficient absorption inside the body. Further, a pharmacophore model highlighting important structural interaction features has been developed. The screened compounds are better in binding to the TIR proteins then the parent compound and hence are good starting points for multi-TIR inhibition.

Probing PARP1-inhibitor complexes for the development of novel inhibitors.

Poly (ADP-ribose) polymerase 1 (PARP1) is the most important member of the PARP family which has been shown to have a direct involvement in the development of cancer. A strategy to rationalize the structure based drug discovery of PARP1 inhibitors has been discussed. So far studies regarding varied scaffold PARP1 inhibitors have been done, however the current study focus on how the available data from potent PARP1 inhibitors could be combined and utilized for developing a robust model for the development of novel inhibitors. Through detailed analyses of PARP1-inhibitor binding, a pharmacophore model has been developed followed by a virtual screen of potential inhibitors. The resulting high-affinity binding hits following the defined pharmacophore model and making the critical interactions were selected as final potential leads. Hence, using the approaches of pharmacophore design, docking based virtual screening and conformation alignment, we have identified important leads which satisfy all parameters of the screening process. The developed pharmacophore model as well as the strategy is very straightforward for screening novel inhibitors and could thus be used as a prototype for PARP1 structure based drug discovery.

Biochemical characterization of dipeptidylcarboxypeptidase of Leishmania donovani.

The incidence of parasitic infection, leishmaniasis, has been steadily increasing worldwide. Since, the existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance, there is an urgent need for development of potent, mechanism-based anti-parasitic agents. The peptidases of protozoan parasites are becoming increasingly important for their role in parasite survival and pathogenecity. Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4 mM and Vmax, 1.173 µmole/ml/min. The enzyme was more sensitive to 1,10 phenanthroline than EDTA and was 80% inhibited in presence of NaCl. Among various protease inhibitors, pepstatin was found as potent inhibitor of LdDCP.

Homology modeling and docking studies of Comamonas testosteroni B-356 biphenyl-2,3-dioxygenase involved in degradation of polychlorinated biphenyls.

Biphenyl dioxygenase is a microbial enzyme which catalyzes the stereospecific dioxygenation of aromatic rings of biphenyl congeners leading to their degradation. Hence, it has attracted the attention of researchers due to its ability to oxidize chlorinated biphenyls, which are one of the serious environmental contaminants. In the present study, the three-dimensional model of alpha-subunit of biphenyl dioxygenase (BphA) from Comamonas testosteroni B-356 has been constructed. The resulting model was further validated and used for docking studies with a class of chlorinated biphenyls such as biphenyl,3,3'-dichlorobiphenyl and 4,4'-dichlorobiphenyl. The kinetic parameters of these biphenyl compounds were well matched with the docking results in terms of conformational and distance constraints. The binding properties of these biphenyl compounds along with identification of critical active site residues could be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

The efficacy of disease modifying anti-rheumatic drugs in rheumatoid arthritis in local patients of Karachi.

The primary objective of the study is to assess the efficacy of the 'Disease Modifying AntiRheumatic Drugs (DMARDs) on the disease activity in Rheumatoid Arthritis (RA) in the local patients of Karachi. The secondary objective is to evaluate whether the combination of two concurrent DMARDs (Combination Therapy) is superior to a single DMARD (Mono-therapy). This is an open labeled retrospective case series. One hundred and five consecutive patients fulfilling 1987 ACR criteria for the diagnosis of RA were initially selected from the case notes of out patients department. Sixty nine patients fulfilled the inclusion criteria and were finally recruited for analysis. Details of the Tender Joint Count (TJC), Swolen Joint Count (SJC), Patient Global Assessment (PGA) and ESR were obtained at six weeks, three months, six months and one year. Out of the 69 patients studied 48 were in the mono-therapy group and 21 in the combination therapy group. Methotrexate (MTX) was the most commonly used single DMARD (75%) as well as the most frequent component of the combination groups (85%). The TJC, SJC and PGA analyses of all patients show that DMARDs are effective agents for clinically controlling RA activity. The speed of their beneficial effect is slow and unlike analgesics and NSAIDS, may take up to six weeks to start working. The 6 week responses showed 32.49% improvement in TJC, 33.19% improvement in SJC and 59% better responses in PGA. This response continued to show further improvement and at six months when TJC improved by 63.41%, SJC by 53.21% and PGA with 81% better responses. After 6 months the response reached a plateau but nevertheless maintained until 1 year with improvements in TJC by 66.23%, SJC by 56.48% and PGA with 88.23% better responses. The changes in ESR did not go parallel with the other three outcome measures. The mean baseline ESR of 56 reduced to 44 at 6 weeks but rose again gradually to 54 at 1 year. The sub-group analysis did not show the overall superiority of combination therapy over mono-therapy. DMARDs are effective in controlling disease activity in RA. Their effect starts slowly over 6 week and may take up to 6 months to show full benefits. The beneficial effect was maintained for at least 1 year. Sub-group analysis did not show any advantage of combination therapy over mono-therapy in this series of patients. Methotrexote being the most frequently used DMARDs in both groups and being most cost effective agent seems to be the most useful drug in RA in the developing world.

Quantitation of free amino acids in biological samples by high-performance liquid chromatography. Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis.

An automatic on-line high-performance liquid chromatographic method based on a precolumn derivatization with o-phthalaldehyde has been developed to quantitate levels of free amino acids in cerebrospinal fluid (CSF) and plasma samples from 12 patients with multiple sclerosis (MS) and 12 controls. The analytical method gave reproducible results with relative standard deviations of 0.5-3% for all amino acids. The separation of 24 amino acids was performed on a reversed-phase C18 column, using two solvents and a multiple-step gradient. Each chromatographic experiment was completed within 40 min. The results showed higher levels of Glu, Gln, Gly and Ala and lower levels of Met, Val, Phe and Lys in plasma of MS patients. In CSF, increased levels of Gln, Arg, Ser and Tyr and decreased levels of Asp, Glu, Met, gamma-aminobutyric acid and Phe were found in MS patients, whereas the levels of other amino acids remained more or the less same in both groups.

Signature size in the psychiatric diagnosis: a significant clinical sign?

To test the hypothesis that patients' signatures may have a useful potential in making psychiatric diagnoses, the authors conducted a correlation study between signature sizes and psychiatric diagnoses. 252 medical records at St. Louis State Hospital in Missouri, USA, were randomized for the measurement of the signature sizes and assessment of DSM-III diagnoses. Analysis of variance and pair-wise comparison show that the signature size in the manic group is significantly larger than those of any other categories of psychiatric diagnoses (p less than 0.05), and that the signature size of organic mental disorder is significantly larger than those of the normal group (p less than 0.05). The authors suggest that further studies are needed to develop the clinical significance, if any, for interpreting patients' signatures.