Discovery of potent antimycobacterial agents targeting lumazine synthase (RibH) of Mycobacterium tuberculosis.

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, as a potential drug target against Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We employ a high-throughput molecular docking approach to screen ~ 600,000 compounds targeting RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 compounds that exhibit potent antimycobacterial activity. These compounds also reduce intracellular burden of M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these three compounds enhance the bactericidal effect of first-line anti-TB drugs, isoniazid and rifampicin. Corroborating with the in silico predicted high docking scores along with favourable ADME and toxicity profiles, all three compounds demonstrate binding affinity towards purified lumazine synthase enzyme in vitro, in addition these compounds exhibit riboflavin displacement in an in vitro assay with purified lumazine synthase indicative of specificity of these compounds to the active site. Further, treatment of M. tb with these compounds indicate reduced production of flavin adenine dinucleotide (FAD), the ultimate end product of the riboflavin biosynthetic pathway suggesting the action of these drugs on riboflavin biosynthesis. These compounds also show acceptable safety profile in mammalian cells, with a high selective index. Hence, our study validates RibH as an important drug target against M. tb and identifies potent antimycobacterial agents.

Development and clinical evaluation of a dried urine spot method for detection of morphine among opioid users.

BACKGROUND: In recent years, drug testing in body fluids has gained popularity for validating self-reported drug use. The storage and transportation of urine specimens is a major concern for remote areas where the facilities for performing drug abuse testing are lacking. AIMS AND OBJECTIVES: The aim of the present study was to develop an efficient method for testing opiate in dried urine spots (DUS) and to evaluate its clinical applicability. MATERIALS AND METHODS: The methodology involved optimization of conditions for extraction, recovery, short-, and long-term stability (room temperature, 4°C,-20°C) for detection of opiate from dried urine spots. Further, the extraction efficiency from dried urine spots was compared with the conventional drug testing methodology. The screening was done by using enzyme-linked immunosorbent assay technique, and confirmation was achieved by gas chromatography equipped with nitrogen phosphorus detector. RESULTS: Deionized water was found to be a suitable extracting solvent compare to bi-carbonate buffer (pH 9.2) and saline. Primary screening was achieved by 2 punches taken from a 20-µl (diameter 1.3 cm) spotted urine samples, whereas confirmation was achieved by 2 complete circles each of 20 µl sample volume. The recovery was found to be 99.41% in water. No sign of significant degradation was seen among all storage conditions. CONCLUSIONS: In the current study, DUS has achieved the same level of precision and reproducibility as that of standard methods used for drug testing in urine. Hence, the DUS sampling appears to have potential to detect opiate among drug users in a clinical setting.

Evaluation of Dried Urine Spot Method to Screen Cotinine among Tobacco Dependents: An Exploratory Study.

BACKGROUND AND OBJECTIVES: Assessment of cotinine, a metabolite of nicotine in body fluids, is an important approach for validating the self-report among tobacco users. Adaptation of assays on dried urine spots (DUSs) has advantages of ease of collection, transportation, minimal invasiveness, and requirement of small volume. The aim of the present study was to develop an efficient method for testing cotinine in DUSs and evaluating its clinical applicability. METHODS: This involved optimization of conditions for detection, recovery, and stability of cotinine from dried urine, spotted on filter paper. Enzyme-linked immunosorbent assay was used for screening, whereas confirmation was done by gas chromatography. For clinical applicability, urine samples of tobacco users were tested. RESULTS AND INTERPRETATION: Water was found to be a suitable extracting solvent as compared to carbonate-bicarbonate buffer (pH 9.2) and saline. Screening was achieved by two punches taken from a 20 µl (diameter 1.3 cm) spotted urine samples, and confirmation was achieved by five complete circles each of 20 µl sample volume. The recovery was found to be 97% in water. Limit of detection for the method was found to be 100 ng/ml. No signs of significant degradation were found under all storage conditions. All the urine samples of tobacco users were found to be positive by a conventional method as well as DUSs, and the method proved to be efficient. CONCLUSIONS: DUS samples are a useful alternative for biological monitoring of recent nicotine use, especially in developing countries where sample logistics could be an important concern.

Dried urine spots for detection of benzodiazepines.

BACKGROUND AND AIM: Benzodiazepines (BZD) are widely prescribed to substance users. However, the nonmedical use of prescription BZD often leads to abuse and dependence. Therefore, it is important to detect BZD among substance users seeking treatment. The aim of the present study was to develop an efficient method for testing BZD on dried urine spot (DUS) and evaluating its clinical applicability. METHODS: This involved optimization of conditions for the detection, recovery, and stability of BZD from dried urine, spotted on filter paper. Enzyme linked immuno-sorbent assay was used for screening whereas confirmation was done by gas chromatography. For clinical applicability, urine samples of BZD users were tested. RESULTS: The recovery was found to be 99.7% in de-ionized water from 20 µl spotted urine samples. Limit of detection, inter-day and intra-day CV were found to be 100 ng/ml, 4.22% and 3.83%, respectively. BZD were found stable in DUS for 3 weeks at room temperature, and for 3 months at 4°C and -20°C. All the urine samples of benzodiazepine users were found positive by conventional method as well as the DUS method. CONCLUSION: DUS method proved to be efficient for BZD testing with advantages of ease of collection, transportation, minimal invasiveness and small sample volume. It offers a useful alternative for BZD testing especially in developing countries where logistics of sample collection and transportation could be an important concern.

Laboratory approach for diagnosis of toluene-based inhalant abuse in a clinical setting.

The steady increase of inhalant abuse is a great challenge for analytical toxicologists. This review describes an overview of inhalant abuse including the extent of the problem, types of products abused, modes of administration, pharmacology and effects of inhalants, the role of laboratory, interpretation of laboratory results and clinical considerations. Regular laboratory screening for inhalant abuse as well as other substance abuse and health risk behaviors must be a part of standard clinical care.

Laboratory profiles of treatment-seeking subjects with concurrent dependence on cannabis and other substances: a comparative study.

BACKGROUND: Cannabis is one of the most widely used illicit drugs in India and worldwide. It is considered to have a minimal effect on physical health. OBJECTIVES: The aim of this study was to compare the laboratory profiles of treatment-seeking patients who were cannabis dependent, and drug users who concurrently use other substances, with non-users. MATERIALS AND METHODS: Medical records of patients, whose urine was tested for the detection of cannabis within the last year, were considered for the study. The inclusion criteria for the study group were; co-morbid diagnosis of cannabis dependence according to DSM-IV TR criteria, positive urine drug screen for cannabis, and at least one biochemical or hematological examination report during the treatment period. The subjects who underwent all of the above mentioned tests, but who were negative for any psychoactive substance with no past or current history of substance use, were placed in the control group. RESULTS: A total of 51 subjects fulfilled the inclusion criteria for the study group and 30 subjects were considered as controls. There was no significant difference found between the demographic profiles of the subject and control groups. The mean duration of cannabis use in the patients was 9.53 ± 8.06 years. Serum levels of; bilirubin, SGOT (serum glutamic oxaloacetic transaminase), SGPT (serum glutamic pyruvic transaminase), total protein, alkaline phosphatase, ESR, and eosinophil counts, were raised in; 13.7%, 15.6%, 33.3%, 17.6%, 37.2%, 75% and 5.8% of subjects, respectively. The relative monocyte count was lower than normal in 92% of cases. Physical complaints were reported in 98% of subjects. The two groups showed significant differences in serum alkaline phosphatase [t (79) = 6.5, P ≤ 0.01], TLC [t (79) = 2.36, P = 0.03] and hemoglobin levels [t (79) = 5.50, P ≤ 0.01]. CONCLUSIONS: Abnormal laboratory parameters were observed in patients with cannabis dependence. The study emphasizes the need for regular physical examinations and laboratory investigations for cannabis users.