RP-HPLC separation of interconvertible rotamers of a 5-tetrahydroisoquinolin-6-yl-pyridin-3-yl acetic acid derivative and confirmation by VT NMR and DFT study.

Due to emergence of drug resistance and drug tolerability, there is urgent need for discovery of new chemical entity for the treatment of HIV infection. As a part of in-house small molecule drug discovery program for HIV infection, sodium-2-(tert-butoxy)- 2-(5-(2-(2-chloro-6-methylbenzyl)- 1,2,3,4-tetrahydroisoquinolin-6-yl)- 4-(4,4-dimethylpiperidin-1-yl)- 2,6-dimethylpyridin-3-yl) acetate (SCMTDDA) was prepared as an intermediate for the synthesis of an API, designed as a HIV-1 integrase inhibitor. Initially, the final compound was isolated as a mixture of rotamers. In the current study, we have developed a simple and efficient achiral, reversed phase (RP) HPLC method to separate the interconvertible rotamers of SCMTDDA. The effect of several parameters, including stationary phase, buffer, modifiers and column temperature, were optimized for the chromatographic separation and it was observed that best separation was achieved on a SunFire C18 column using TFA/acetonitrile (ACN) - methanol (MeOH) (1:1 v/v) as the mobile phase at 10 0C. The chromatographic observations were complemented with variable-temperature NMR and energy barrier calculations using density functional theory (DFT).

Prochlorperazine enhances radiosensitivity of non-small cell lung carcinoma by stabilizing GDP-bound mutant KRAS conformation.

Lung cancer is considered as leading cancer with the highest mortality. The KRAS-oncogenic mutations are dominant in lung carcinoma leading to poor prognosis and radioresistance, which is a major impediment to radiotherapy. Thus, KRAS mutant inhibitors that synergistically sensitize tumours to radiation are urgently needed. In pursuance of the search for a novel radiosensitizer, high-throughput screening of FDA-approved drugs was performed at active site of K-Ras. Prochlorperazine (PCZ), an antipsychotic drug, showed good binding affinity with KRAS-mutant proteins. PCZ binds to the GTP-binding pocket of KRAS-mutant protein and inhibits its constitutive activation by stabilizing the GDP-bound conformation of K-Ras mutants by 9 kcal/mol compared to WT. PCZ alongwith radiation decreased the clonogenic survival of KRAS-mutant NSCLC but not KRAS-WT cells. The combination treatment activates p-ATM, p53, and p21 proteins, leading to cell cycle arrest. PCZ with increasing radiation caused a linear increase in γH2AX foci, suggesting enhanced DSBs-associated apoptosis in radioresistant A549 cells. Pharmacokinetics study showed Cmax = 526 ng/ml at 30min, 4.6h half-life in plasma, and highest accumulation in tumours. PCZ and 10Gy irradiation synergistically radiosensitize mice xenografts via downregulation of Ras/Raf/MEK/ERK pathway. Our efforts have led to the discovery of PCZ as a lead compound. In preclinical analyses, treatment with PCZ alone and in combination with radiation led to regression of KRAS-G12S tumours.

Optimization of Nicotinamides as Potent and Selective IRAK4 Inhibitors with Efficacy in a Murine Model of Psoriasis.

IRAK4 is an attractive therapeutic target for the treatment of inflammatory conditions. Structure guided optimization of a nicotinamide series of inhibitors has been expanded to explore the IRAK4 front pocket. This has resulted in the identification of compounds such as 12 with improved potency and selectivity. Additionally 12 demonstrated activity in a pharmacokinetics/pharmacodynamics (PK/PD) model. Further optimization efforts led to the identification of the highly kinome selective 21, which demonstrated a robust PD effect and efficacy in a TLR7 driven model of murine psoriasis.

Electrophilicity of pyridazine-3-carbonitrile, pyrimidine-2-carbonitrile, and pyridine-carbonitrile derivatives: a chemical model to describe the formation of thiazoline derivatives in human liver microsomes.

Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure-property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)pyridazine-3-carbonitrile, 6-p-tolylpyridazine-3-carbonitrile], pyrimidines [5-p-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)pyrimidine-2-carbonitrile], and pyridines [5-p-tolylpicolinonitrile and 5-(4-fluorophenyl)picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and N-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and N-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by in silico modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts.

Structure-guided discovery of 1,3,5 tri-substituted benzenes as potent and selective matriptase inhibitors exhibiting in vivo antitumor efficacy.

Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.

Discovery of Pyridyl Bis(oxy)dibenzimidamide Derivatives as Selective Matriptase Inhibitors.

Matriptase belongs to trypsin-like serine proteases involved in matrix remodeling/degradation, growth regulation, survival, motility, and cell morphogenesis. Herein, we report a structure-based approach, which led to the discovery of sulfonamide and amide derivatives of pyridyl bis(oxy)benzamidine as potent and selective matriptase inhibitors. Co-crystal structures of selected compounds in complex with matriptase supported compound designing. Additionally, WaterMap analyses indicated the possibility of occupying a distinct pocket within the catalytic domain, exploration of which resulted in >100-fold improvement in potency. Co-crystal structure of 10 with matriptase revealed critical interactions leading to potent target inhibition and selectivity against other serine proteases.

Identification of domains and domain interface residues in multidomain proteins from graph spectral method.

We present a novel method for the identification of structural domains and domain interface residues in proteins by graph spectral method. This method converts the three-dimensional structure of the protein into a graph by using atomic coordinates from the PDB file. Domain definitions are obtained by constructing either a protein backbone graph or a protein side-chain graph. The graph is constructed based on the interactions between amino acid residues in the three-dimensional structure of the proteins. The spectral parameters of such a graph contain information regarding the domains and subdomains in the protein structure. This is based on the fact that the interactions among amino acids are higher within a domain than across domains. This is evident in the spectra of the protein backbone and the side-chain graphs, thus differentiating the structural domains from one another. Further, residues that occur at the interface of two domains can also be easily identified from the spectra. This method is simple, elegant, and robust. Moreover, a single numeric computation yields both the domain definitions and the interface residues.