Discovery of KRB-456, a KRAS G12D Switch-I/II Allosteric Pocket Binder That Inhibits the Growth of Pancreatic Cancer Patient-derived Tumors.

Currently, there are no clinically approved drugs that directly thwart mutant KRAS G12D, a major driver of human cancer. Here, we report on the discovery of a small molecule, KRB-456, that binds KRAS G12D and inhibits the growth of pancreatic cancer patient-derived tumors. Protein nuclear magnetic resonance studies revealed that KRB-456 binds the GDP-bound and GCP-bound conformation of KRAS G12D by forming interactions with a dynamic allosteric binding pocket within the switch-I/II region. Isothermal titration calorimetry demonstrated that KRB-456 binds potently to KRAS G12D with 1.5-, 2-, and 6-fold higher affinity than to KRAS G12V, KRAS wild-type, and KRAS G12C, respectively. KRB-456 potently inhibits the binding of KRAS G12D to the RAS-binding domain (RBD) of RAF1 as demonstrated by GST-RBD pulldown and AlphaScreen assays. Treatment of KRAS G12D-harboring human pancreatic cancer cells with KRB-456 suppresses the cellular levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1. Importantly, KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer whose tumors harbor KRAS G12D and KRAS G12V and who relapsed after chemotherapy and radiotherapy. These results warrant further development of KRB-456 for pancreatic cancer. SIGNIFICANCE: There are no clinically approved drugs directly abrogating mutant KRAS G12D. Here, we discovered a small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D. KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer. This discovery warrants further advanced preclinical and clinical studies in pancreatic cancer.

ICT Enabled Disease Diagnosis, Treatment and Management-A Holistic Cost-Effective Approach Through Data Management and Analysis in UAE and India.

This concept paper addresses specific challenges identified in the UN 2030 Agenda Sustainable Development Goals (SDG) as well as the National Health Policy of India (NHP-India) and the Ministry of Health Policy of UAE (MHP-UAE). This policy calls for a digital health technology ecosystem. SDG Goal 1 and its related objectives are conceptualized which serves as the foundation for Virtual Consultations, Tele-pharmacy, Virtual Storage, and Virtual Community (VCom). SDG Goals 2 and 3 are conceptualized as Data Management & Analytical (DMA) Architecture. Individual researchers and health care professionals in India and the UAE can use DMA to uncover and harness PHC and POC data into practical insights. In addition, the DMA would provide a set of core tools for cross-network initiatives, allowing researchers and other users to compare their data with DMA data. In rural, urban, and remote populations of the UAE and India, the concept augments the PHC system with ICT-based interventions. The ICT-based interventions may improve patient health outcomes. The open and flexible design allows users to access various digital materials. Extendable data/metadata format, scalable architecture for petabyte-scale federated discovery. The modular DMA is designed using existing technology and resources. Public health functions include population health assessment, policy development, and monitoring policy implementation. PHC and POC periodically conduct syndromic surveillance to identify population risk patterns. In addition, the PHC and POC deploy medical and non-medical preventive measures to prevent disease outbreaks. To assess the impact of social and economic factors on health, epidemiologists must first understand diseases. Improved health due to compliance with holistic disease treatment plans and access to scientific health information.

Autonomous Tool for Monitoring Multi-Morbidity Health Conditions in UAE and India.

Multi-morbidity is the presence of two or more long-term health conditions, including defined physical or mental health conditions, such as diabetes or schizophrenia. One of the regular and critical health cases is an elderly person with a multi-morbid health condition and special complications who lives alone. These patients are typically not familiar with advanced Information and Communications Technology (ICT), but they are comfortable using smart devices such as wearable watches and mobile phones. The use of ICT improves medical quality, promotes patient security and data security, lowers operational and administrative costs, and gives the people in charge to make informed decisions. Additionally, the use of ICT in healthcare practices greatly reduces human errors, enhances clinical outcomes, ramps up care coordination, boosts practice efficiencies, and helps in collecting data over time. The proposed research concept provides a natural technique to implement preventive health care innovative solutions since several health sensors are embedded in devices that autonomously monitor the patients' health conditions in real-time. This enhances the elder's limited ability to predict and respond to critical health situations. Autonomous monitoring can alert doctors and patients themselves of unexpected health conditions. Real-time monitoring, modeling, and predicting health conditions can trigger swift responses by doctors and health officials in case of emergencies. This study will use data science to stimulate discoveries and breakthroughs in the United Arab Emirates (UAE) and India, which will then be reproduced in other world areas to create major gains in health for people, communities, and populations.

Assembly mechanism of early Hsp90-Cdc37-kinase complexes.

Molecular chaperones have an essential role for the maintenance of a balanced protein homeostasis. Here, we investigate how protein kinases are recruited and loaded to the Hsp90-Cdc37 complex, the first step during Hsp90-mediated chaperoning that leads to enhanced client kinase stability and activation. We show that conformational dynamics of all partners is a critical feature of the underlying loading mechanism. The kinome co-chaperone Cdc37 exists primarily in a dynamic extended conformation but samples a low-populated, well-defined compact structure. Exchange between these two states is maintained in an assembled Hsp90-Cdc37 complex and is necessary for substrate loading. Breathing motions at the N-lobe of a free kinase domain partially expose the kinase segment trapped in the Hsp90 dimer downstream in the cycle. Thus, client dynamics poise for chaperone dependence. Hsp90 is not directly involved during loading, and Cdc37 is assigned the task of sensing clients by stabilizing the preexisting partially unfolded client state.

G6036A substitution in mitochondrial COX I gene compromises cytochrome c oxidase activity in thiamine responsive Leigh syndrome patients.

Cytochrome c oxidase (COX) deficiency is known to be associated with Leigh syndrome (LS), however there are limited studies on genetic screening of mitochondrial (mt) DNA encoding COX genes as well as the functional validation of identified variants. In our previous studies, we cared for total 165 LS patients and analyzed the nucleotide variations across entire mt genome. We observed a high level of genetic heterogeneity in these patients. We identified various reported and novel variation across entire genome including COX genes. In our present study we have further studied and functionally validated the selected novel nucleotide variant of COX I and COX II gene using different in-silico tools and trans mitochondrial cybrid based assays. As a result of our study, G6036A (G45S) variant of COX I gene, reduced the COX activity in both spectrophotometric as well as In-gel BN-PAGE assays. FACS analysis also revealed this variant to affect the mitochondrial membrane potential in the respective cybrids. Interestingly most of our in-silico studies indicated that this variant might affect the secondary structure and confirmation of COX I protein. Thus we report the first missense mutation in the COX I gene of LS patients and justify its pathogenic role in these patients by different assays. Variant A7746G (N54K) in COX II gene was also predicted to affect the secondary structure as well as stability of COX II protein. Though, the effect of this variant was not significant, however it will be interesting to investigate its significance by other assays in future.

Mechanism of proton transfer in class A ß-lactamase catalysis and inhibition by avibactam.

Gram-negative bacteria expressing class A ß-lactamases pose a serious health threat due to their ability to inactivate all ß-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A ß-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved ß-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A ß-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl-enzyme complexes with ß-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKa to be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.

Genetic heterogeneity of mitochondrial genome in thiamine deficient Leigh syndrome patients.

In our previously published study, we cared for 165 thiamine deficient Leigh syndrome (LS) patients who presented in acute life threatening conditions with severe neurological abnormalities. However the molecular basis for this atypical phenotype was not explored. This study is an effort to undermine the possible molecular defects in mitochondria of those patients and put-forth an explanation towards this clinical presentation. Protein coding genes of mitochondrial (mt) DNA were sequenced in total 165 LS patients and 94 age matched controls. To understand their pathogenic significance, nucleotide variations were also studied using various in-silico tools. Histochemical and electron microscopic analysis was also done in tissue samples obtained from 23 patients. We observed a very high level of genetic heterogeneity across the mt DNA of all these patients. In the concordance of published literature we also observed a large number of variations in ND5 gene (hot spot for LS). We also observed a total 13 nucleotide variations across COX genes, which is otherwise not common in LS. As per in-silico analysis, many of these variations were suggested to be pathogenic. Histochemical and electron microscopic studies also suggested the defects in the mitochondria of these patients. As these patients were thiamine deficient, hence we propose that genetic defects and thiamine deficiency may together severely affect the ATP levelof these patients, leading to acute and life threatening clinical presentation. Present study has opened up many avenues for further research towards understanding the genetic basis and possible role of thiamine deficiency in LS patients.

Design and Structure Determination of a Composite Zinc Finger Containing a Nonpeptide Foldamer Helical Domain.

A number of foldamer backbones have been described as useful mimics of protein secondary structure elements, enabling for example the design of synthetic oligomers with the ability to engage specific protein surfaces. Synthetic folded backbones can also be used to create artificial proteins in which a folded peptide segment (e.g., an α-helix, a loop) is replaced by its unnatural counterpart, with the expectation that the resulting molecule would maintain its ability to fold while manifesting new exploitable features. The similarities in screw sense, pitch, and polarity between peptide α-helices and oligourea 2.5-helices suggest that a tertiary structure could be retained when swapping the two backbones in a protein sequence. In the present work, we move a step toward the creation of such composite proteins by replacing the 10-residue long original α-helical segment in the Cys2His2 zinc finger 3 of transcription factor Egr1 (also known as Zif268) by an oligourea sequence bearing two appropriately spaced imidazole side chains for zinc coordination. We show by spectroscopic techniques and mass spectrometry analysis under native conditions that the ability of the peptide/oligourea hybrid to coordinate the zinc ion is not affected by the foldamer replacement. Moreover, detailed NMR analysis provides evidence that the engineered zinc finger motif adopts a folded structure in which the native ß-sheet arrangement of the peptide region and global arrangement of DNA-binding side chains are preserved. Titration in the presence of the Egr1 target DNA sequence supports binding to GC bases as reported for the wild-type zinc finger.

Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation.

During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner.

1H, 13C, and 15N chemical shift assignments of a G-quadruplex forming sequence within the KRAS proto-oncogene promoter region.

Single stranded guanine rich DNA (or RNA) sequences adopt noncanonical secondary structures called G-quadruplexes (G4). Functionally, quadruplexes control gene transcription and regulate activities such as replication, gene recombination or alternative splicing. Hence they are potential targets for cancer, neuronal, and viral related diseases. KRAS is one of the most mutated oncogenes in the genome of cancer cells and contains a nuclease hypersensitive element (NHE) sequence capable of forming G-quadruplexes via its six runs of guanines. In our work, we are interested in the NMR structure of the major G4 scaffold formed in the KRAS NHE region with a mutated sequence of 22 residues. Here, we report 1H, 13C and 15N chemical shift assignments the G4 formed within KRAS22RT sequence.

Antibiotics in dental implants: A review of literature.

The routine use of antibiotics in oral implant treatment seems to be widespread. The pre- or post-operative use of antibiotics in conjunction with implant surgery and its correlation with failure and success rates are poorly documented in the literature. The debate regarding overprescription of antibiotics raises the need for a critical evaluation of proper antibiotic coverage in association with implant treatment. The benefits of prophylactic antibiotics are well-recognized in dentistry. However, their routine use in the placement of endosseous dental implants remains controversial. The purpose of this review is to know the efficacy of antibiotic prophylaxis in implant dentistry.

Implementation and Operational Research: High Loss to Follow-up Among Children on Pre-ART Care Under National AIDS Program in Madurai, South India.

BACKGROUND: Information on the follow-up of HIV-infected children enrolled into preantiretroviral therapy (Pre-ART) care under routine program settings is limited in India. Knowledge on the magnitude of loss to follow-up (LFU) and its reasons will help programs to retain children in HIV care. We aimed to assess the proportion of LFU among children in Pre-ART care and its associated factors. METHODS: In this retrospective cohort study, we reviewed the records of all HIV-infected children (aged <15 years) registered from 2005 to 2012 at an ART center, Madurai, South India. LFU during Pre-ART care was defined as having not visited the ART center within a year of registration. RESULTS: Of 426 children enrolled in Pre-ART care, 211 (49%) were females and 301 (71%) were in the 5- to 14-year age group. At 1 year of registration, 348 (82%) were lost to follow-up. Of 348, 81 returned to care after 1 year of enrollment, whereas 267 (63% of all children) were permanently lost to follow-up. The proportion of LFU remained high from 2005 to 2012. WHO staging, CD4 count, and opportunistic infection were the significant factors associated with lost to follow-up on multivariate analysis. CONCLUSIONS: LFU was alarmingly high indicating poor clinical and programmatic monitoring among HIV-infected children enrolled in Pre-ART care. A system for active tracing of those missing a clinic appointment intensified supervision, and monitoring along with qualitative research is urgently needed. This will help to understand the exact reasons for LFU based on which effective interventions may be planned for reducing such losses.

Effect of oral prophylactic instrumentation on the surface texture of all metal restorative materials.

INTRODUCTION: In the inaccessible areas on the crown the removal of calculus and stains by hand and ultrasonic instrumentation is the method for cleaning to preserve and increase the longevity of the restoration. However, when oral prophylaxis is performed on restorative crowns, it may produce some surface alterations and may favour plaque accumulation. STATEMENT OF PROBLEM: Many patients may have restored their teeth with artificial crowns and would come to the dental office for oral prophylaxis. If a routine oral prophylaxis is followed, its effect on the restorative materials and the plaque accumulation can be studied. MATERIALS AND METHODS: A total of 15 disc shaped wax patterns were invested and casted for cast titanium (Group A) and the remaining 15 disk shaped for nickel-chromium (Group B). The obtained castings were finished and polished. All the specimens were subjected to hand and ultrasonic scaling for 15 s. Profilometer and scanning electron microscopic was used to analyze and evaluate the surface roughness. Specimens of each group were embedded on the anterior lingual aspects of the removable lower retention plates. 5 volunteers were asked to wear it in the mouth for 24 h for 7 days. After 7 days, the specimens were stained with plaque disclosing solutions and the photomicrographs were taken by the optical stereomicroscope and the plaque accumulations were assessed in percentage. RESULTS: The difference in average surface roughness (µm) of the polished test specimens was maximum for ultrasonic scaling than hand scaling and maximum for Group A than Group B. Plaque accumulation in percentage on the treated specimens was found to be nonsignificant but, mean plaque accumulation was maximum on ultrasonic scaling surface than hand scaling and maximum for Group A than Group B. Surface roughness was found to be statistically significant after hand scaling (F = 9.377, P = 0.000) and ultrasonic scaling (F = 5.373, P = 0.0000) by Student t-test. CONCLUSION: The Surface roughness and plaque accumulation on the specimens were more for Group A than Group B and maximum produced by ultrasonic scaling than hand scaling.

Novel heteroaryl phosphonicdiamides PTPs inhibitors as anti-hyperglycemic agents.

BACKGROUND: Chronic and oral administration of benzylamine improves glucose tolerance. Picolylamine is a selective functional antagonist of the human adenosine A2B receptor. Phosphonic diamide derivatives enhance the cellular permeability and in turn their biological activities. METHODS: A series of heteroaryl phosphonicdiamide derivatives were designed as therapeutics to control and manage type2 diabetes. Initially defined Lipinski parameters encouraged them as safer drugs. Molecular docking of these compounds against Protein tyrosine phosphatase (PTP), the potential therapeutic target of type 2 diabetes, revealed their potential binding ability explaining their anti-diabetic activity in terms of PTP inhibition. Human intestinal absorption, Caco-2 cell permeability, MDCK cell permeability, BBB penetration, skin permeability and plasma protein binding abilities of the title compounds were calculated by PreADMET server. A convenient method has been developed for the synthesis of title compounds through the formation of 1-ethoxy-N,N'-bis(4-fluorobenzyl/pyridin-3-ylmethyl)phosphinediamine by the reaction of 4-fluorobenzylamine/ 3-picolylamine with ethyldichlorophosphite, subsequently reacted with heteroaryl halides using lanthanum(III) chloride as a catalyst. RESULTS: All the compounds exhibited significant in vitro anti-oxidant activity and in vivo evaluation in streptozotocin induced diabetic rat models revealed that the normal glycemic levels were observed on 12(th) day by 9a and 20(th) day by 5b, 5c, 9e and 9f. The remaining compounds also exhibited normal glycemic levels by 25(th) day. CONCLUSION: The results from molecular modeling, in vitro and in vivo studies are suggesting them as safer and effective therapeutic agents against type2 diabetes. Graphical Abstract Development of PTPs inhibitors.

DAO-9 (2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazole) exhibits p53 induced apoptogenesis through caspase-3 mediated endonuclease activity in murine carcinoma.

One of the main strategies to inhibit the tumor growth is to promote the biochemical events leading to DNA degradation, which would eventually culminate in apoptosis. We have earlier reported that the 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazole(DAO-9) possessed anti-cancer activity. To address the exact molecular mechanism underlying anti-cancer property, present study focused on evaluating the anti-tumor effect of the DAO-9 on murine ascites carcinoma cells using various in vivo and in vitro assays. The in vivo assays implicated a strong regression in tumor growth of ascites carcinoma after treatment which is due to apoptogenic efficacy as assessed through structural morphology of EAC cells by Giemsa, Acridine orange, Annexin V staining and FACS analysis. Nucleosomal DNA fragmentation induced by DAO-9 is due to activation of caspase-3 mediated DNAse as verified by endonuclease assays and immunoblot analysis. The caspase-3 activation mechanism is by induction of intrinsic cascade signaling molecules, such as p53, Bax, Bad and cytochrome c (cyt c) expression as verified by western blot. The results concluded that the tumor inhibiting activity of DAO-9 is due to activation of the apoptotic signaling cascade, which could be translated into targeted anti-cancer drug in the near future.

Selective separation, detection of zotepine and mass spectral characterization of degradants by LC-MS/MS/QTOF / 药物分析学报

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C18 column (250 mm ~ 4.6 mm i.d., 5 mm) with a mobile phase containing a gradient mixture of solvents, A (0.05%trifluoroacetic acid (TFA), pH ? 3.0) and B (acetonitrile). The eluted compounds were monitored at 254 nm;the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of 41.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC-MS/MS and their fragmentation pathways were proposed.

Rigid nasal endoscopy in the diagnosis and treatment of epistaxis.

BACKGROUND AND OBJECTIVES: Epistaxis is one of the common symptoms encountered in the Otorhinolaryngology department. Many times the cause for epistaxis is not found on anterior and posterior rhinoscopy. The present study was undertaken to assess the role of rigid nasal endoscope in the diagnosis and treatment of epistaxis, where normal anterior and posterior rhinoscopy did not reveal any specific finding. METHODS: Fifty patients with epistaxis were studied using rigid nasal endoscope under local anaesthesia. Patients who were above 15 years with nasal bleeding and who were willing for rigid nasal endoscopy were included in the study. Patients less than 15 years were not included in the study because nasal endoscopy was difficult in them under local anaesthesia. Only those patients in whom, the cause for epistaxis could not be made out on anterior and posterior rhinoscopy were chosen for the study, this was done in order to remove the bias for nasal endoscopy. RESULTS: The use of the nasal endoscope allowed diagnosis of bleeding points and treating them directly. Epistaxis was more in male patients especially in the 3rd and after the 5th decade. On endoscopic examination,the bleeding points were identified as coming from the crevices of the lateral nasal wall, posterior spur on the septum, posterior deviation of the septum with ulcer, congested polyps, enlarged and congested adenoids, scabs or crusts in the crevices of the lateral nasal wall and angiofibroma. Endoscope also helps in the treatment of epistaxis, which includes endoscopic selective nasal packing using gelfoam, endoscopic cautery or diathermy and endoscopic polypectomy. Other patients with adenoids, scabs and crusts and angiofibroma were managed on their merits. INTERPRETATION AND CONCLUSION: Nasal endoscopy helps not only in the localisation of the bleeding point but also in the treatment of those bleeding areas that are situated in the posterior and lateral part of the nose.

Improving the chemical shift dispersion of multidimensional NMR spectra of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) have recently attracted the attention of the scientific community challenging the well accepted structure-function paradigm. In the characterization of the dynamic features of proteins nuclear magnetic resonance spectroscopy (NMR) is a strategic tool of investigation. However the peculiar properties of IDPs, with the lack of a unique 3D structure and their high flexibility, have a strong impact on NMR observables (low chemical shift dispersion, efficient solvent exchange broadening) and thus on the quality of NMR spectra. Key aspects to be considered in the design of new NMR experiments optimized for the study of IDPs are discussed. A new experiment, based on direct detection of (13)C(α), is proposed.