MmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.
The majority of melanocytic neoplasms can be correctly diagnosed using routine histopathologic analysis. However, a significant minority of tumors have ambiguous histopathologic attributes that overlap between melanocytic nevi and melanoma. Ancillary tests that assist in distinguishing potentially lethal melanomas from benign melanocytic nevi with atypical histopathologic features are available, but still need refining.Most melanomas have chromosomal copy number aberrations, frequently involving multiple chromosomes. With rare exceptions, such anomalies are not found in melanocytic nevi. This difference formed the basis to develop assays that can help distinguish melanoma from nevi by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). FISH can detect chromosomal copy number changes of a limited number of loci within individual cells. By contrast, CGH assesses copy number across the entire genome, but typically is performed on bulk cell populations so that copy number changes in individual cells or subpopulations of cells can go undetected. Both FISH and CGH have been used to provide genomic information in histopathologically ambiguous melanocytic tumors that can assist pathologists make correct diagnoses.
Buschke-Ollendorff syndrome represents an autosomal dominant disorder characterized by connective tissue nevi and osteopoikilosis. Cutaneous lesions may contain either predominantly elastic fibers or predominantly collagen fibers or may show both connective tissue components. The disease results from mutations in LEMD3 (MAN1), which lead to enhanced transforming growth factor-? (TGF-?) signaling and resultant changes in fibroblast function. TGF-? alterations have been implicated in a number of fibrotic disorders, and it is therefore not surprising that a range of cutaneous and skeletal abnormalities have been associated with Buschke-Ollendorff syndrome. Herein, we report a novel association between ossifying fibroma and Buschke-Ollendorff syndrome and discuss how these conditions are likely to be mechanistically linked.
A high-throughput screen (HTS) was performed to identify molecules specifically active against Helicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment of H. pylori infection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, and in vitro validations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes. In vivo validation included testing in the mouse model of H. pylori infection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 ?g/ml and good selectivity for H. pylori compared to a panel of commensal bacteria. HPi1 was also effective in a mouse model of H. pylori infection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specific H. pylori therapeutics.
Previous studies have demonstrated the utility of fluorescence in situ hybridization (FISH) as an ancillary method in the diagnostic workup of histopathologically ambiguous melanocytic neoplasms. A combination of probes targeting 3 loci on chromosome 6 and 1 on 11q has been reported to distinguish unequivocal melanomas and nevi with a sensitivity and specificity of 87% and 96%, respectively. However, information on how FISH should be integrated into routine clinical testing is limited. We report our experience of FISH testing of 804 ambiguous melanocytic lesions performed as part of routine workup at University of California, San Francisco. The main category (47% of all cases) for which FISH testing was requested was Spitz tumors. Other categories included the distinction of possible melanoma from combined nevi (9%), acral or mucosal nevi (9%), Clark/dysplastic nevi (7%), and blue or deep penetrating nevi (6%) and to assess the possibility of nevoid melanoma (4%). Of the ambiguous tumors successfully tested, 88% received a more definitive benign or malignant final diagnosis. Of the 630 cases that tested negative by FISH, the final diagnosis was benign in 489 (78%) cases, ambiguous in 91 cases (14%), and malignant in 50 cases (8%). A positive FISH result was observed in 124 cases, with a final diagnosis of melanoma in 117 (94%). One (1%) FISH-positive case had an equivocal final diagnosis, and 6 (5%) were interpreted, despite the positive FISH result, as melanocytic nevi. We conclude that FISH testing can help reduce the number of equivocal diagnoses in ambiguous melanocytic neoplasms, in particular if FISH testing is positive, and discuss the challenges and limitations of FISH in clinical practice.
The rise of resistant pathogens and chronic infections tolerant to antibiotics presents an unmet need for novel antimicrobial compounds. Identifying broad-spectrum leads is challenging due to the effective penetration barrier of Gram-negative bacteria, formed by an outer membrane restricting amphipathic compounds, and MDR pumps. In chronic infections, pathogens are shielded from the immune system by biofilms or host cells, and dormant persisters tolerant to antibiotics are responsible for recalcitrance to chemotherapy with conventional antibiotics. We reasoned that the dual need for broad-spectrum and sterilizing compounds could be met by developing prodrugs that are activated by bacteria-specific enzymes, and that these generally reactive compounds could kill persisters and accumulate over time due to irreversible binding to targets. We report the development of a screen for prodrugs, based on identifying compounds that non-specifically inhibit reduction of the viability dye Alamar Blue, and then eliminate generally-toxic compound by testing for cytotoxicity. A large pilot of 55,000 compounds against E. coli produced 20 hits, 3 of which were further examined. One compound, ADC111, is an analog of a known nitrofuran prodrug nitrofurantoin, and its activity depends on the presence of activating enzymes nitroreductases. ADC112 is an analog of another known antimicrobial tilbroquinol with unknown mechanism of action, and ADC113 does not belong to an approved class. All three compounds had a good spectrum and showed good to excellent activity against persister cells in biofilm and stationary cultures. These results suggest that screening for overlooked prodrugs may present a viable platform for antimicrobial discovery.
IMPORTANCE Cold panniculitis is a self-limited condition, manifesting as erythematous plaques or nodules after cold exposure, that typically affects infants and children. Recently, a variant involving the lateral thighs of equestrians has been described. Since the original report of this variant, some confusion has arisen in the literature in which the terms equestrian cold panniculitis and equestrian perniosis are both used. Outside of this presentation, cold panniculitis in adults is exceedingly rare. OBSERVATIONS We describe 2 adult patients using ice-pack therapy for chronic back pain who developed erythematous, purpuric plaques at the site of ice-pack application. Histopathologic findings from both patients were similar and showed overlapping features of perniosis and cold panniculitis that closely resembled the pattern seen in cutaneous lupus erythematosus. CONCLUSIONS AND RELEVANCE Ice-pack dermatosis is an uncommon cold-induced process that occurs in adults using long-term ice-pack therapy. The clinical manifestations include erythematous to purpuric plaques with a livedolike appearance and superficial ulceration. The histopathologic features resemble those seen in cutaneous lupus erythematosus with a superficial and deep perivascular and periadnexal dermatitis with increased dermal mucin and a superficial lobular panniculitis.
Human injuries related to stingray attacks include deep puncture wounds, envenomation, and foreign body reactions owing to retained tail fragments. Herein we report a patient who sustained a stingray injury that produced a subcutaneous granulomatous dermatitis and panniculitis with necrobiosis and review the topic of stingray injuries.
Psoriasis is a chronic, inflammatory skin disease caused by a combination of environmental and genetic factors. The Tnip1 gene encodes A20 binding and inhibitor of NF-?B-1 (ABIN-1) protein and is strongly associated with susceptibility to psoriasis in humans. ABIN-1, a widely expressed ubiquitin-binding protein, restricts TNF- and TLR-induced signals. In this study, we report that mice lacking ABIN-1 specifically in dendritic cells (DCs), ABIN-1(fl) CD11c-Cre mice, exhibit perturbed immune homeostasis. ABIN-1-deficient DCs display exaggerated NF-?B and MAPK signaling and produce more IL-23 than do normal cells in response to TLR ligands. Challenge of ABIN-1(fl) CD11c-Cre mice with topical TLR7 ligand leads to greater numbers of Th17 and TCR?? T cells and exacerbated development of psoriaform lesions. These phenotypes are reversed by DC-specific deletion of the TLR adaptor MyD88. These studies link ABIN-1 with IL-23 and IL-17, and they provide cellular and molecular mechanisms by which ABIN-1 regulates susceptibility to psoriasis.
Azoles are among the most successful classes of antifungals. They act by inhibiting ?-14 lanosterol demethylase in the ergosterol biosynthesis pathway. Oropharyngeal candidiasis (OPC) occurs in about 90% of HIV-infected individuals, and 4 to 5% are refractory to current therapies, including azoles, due to the formation of resistant biofilms produced in the course of OPC. We reasoned that compounds affecting a different target may potentiate azoles to produce increased killing and an antibiofilm therapeutic. 2-Adamantanamine (AC17) was identified in a screen for compounds potentiating the action of miconazole against biofilms of Candida albicans. AC17, a close structural analog to the antiviral amantadine, did not affect the viability of C. albicans but caused the normally fungistatic azoles to become fungicidal. Transcriptome analysis of cells treated with AC17 revealed that the ergosterol and filamentation pathways were affected. Indeed, cells exposed to AC17 had decreased ergosterol contents and were unable to invade agar. In vivo, the combination of AC17 and fluconazole produced a significant reduction in fungal tissue burden in a guinea pig model of cutaneous candidiasis, while each treatment alone did not have a significant effect. The combination of fluconazole and AC17 also showed improved efficacy (P value of 0.018) compared to fluconazole alone when fungal lesions were evaluated. AC17 is a promising lead in the search for more effective antifungal therapeutics.
Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (sEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: (1) to increase selectivity for anti-TB activity over human sEH activity, (2) to optimize PK profiles including solubility and (3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead isoxazole, oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub ?g/mL minimum inhibitory concentrations.
Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ~75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.
Melanoma incidence has been rising steadily for decades, whereas mortality rates have remained flat. This type of discordant pattern between incidence and mortality has been linked to diagnostic drift in cancers of the thyroid, breast, and prostate. Ancillary tests, such as fluorescent in situ hybridization (FISH), are now being used to help differentiate melanomas from melanocytic nevi. Multicolor FISH has been shown to distinguish between these 2 with 86.7% sensitivity and 95.4% specificity. To assess the ability of FISH to differentiate melanomas with metastatic or lethal potential from those with an indolent disease course, we performed FISH with probes targeting 6p25, centromere 6, 6q23, and 11q13 on 144 primary melanomas with a minimal tumor thickness of 2 mm and compared the development of metastatic disease and melanoma-specific mortality as well as relapse-free and disease-specific survival between FISH-positive and FISH-negative cases. Of the melanomas, 82% were positive by FISH according to previously defined criteria. The percentage was significantly higher (93%) in cases that developed systemic metastases (n=43) than in patients that did not (77%, n=101). FISH-positive primaries had a significantly increased risk of metastasis or melanoma-related death compared with FISH-negative cases odds ratio 4.11; confidence interval, 1.14-22.7 and odds ratio 7.0, confidence interval 1.03-300.4, respectively. FISH status remained an independent parameter when controlling for known prognostic factors. These data indicate that the group of melanomas diagnosed with routine histopathology that lack aberrations detected by FISH is enriched for melanomas with a more indolent disease course. This suggests that molecular techniques can assist in a more accurate identification of tumors with metastatic potential.
Despite its modest potential benefit, alpha-interferon is one of the most frequently employed therapies for melanoma. With the increasing incidence of melanoma, a parallel increase in interferon use and the associated adverse reactions that accompany interferon therapy should be expected. We present a case of an interferon-induced sarcoidosis-like reaction in a melanoma patient that was initially misinterpreted clinically and radiographically as metastatic melanoma. The etiology of sarcoidosis remains a mystery, but appears to involve Th-1 cytokines such as interferon and interleukin-2. Observance of a sarcoidosis-like reaction induced by interferon therapy lends additional support to the importance of this cytokine in the pathogenesis of sarcoidosis. It is important for pathologists to be aware of this entity when interpreting biopsies from melanoma patients treated with interferon.
Autotaxin (ATX) is a secreted glycoprotein with lysophospholipase D (LPLD) activity that generates the bioactive lipid lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been linked to the promotion and progression of cancer as well as cardiovascular disease and obesity. Despite the fact that ATX inhibitors have the potential to be useful chemotherapeutics for multiple indications, few examples of potent ATX inhibitors are described in the current literature. Here we describe the development of pharmacophore models for the inhibition of ATX by nonlipids and apply these tools to the discovery of additional ATX inhibitors using the NCI open chemical repository database. From this database of > 250,000 compounds, 168 candidate inhibitors were identified. Of these candidates, 106 were available for testing and 33 were identified as active (those that inhibited ATX activity by > or =50% at a single 10 microM concentration), a 31% hit rate. Five of these compounds had IC(50) < 1.5 microM and the most potent compound possessed a K(i) of 271 nM.
Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure-activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating that it can bind to both the enzyme and the enzyme-substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure-activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization.
Large plaque-type blue nevi with subcutaneous cellular nodules are rare tumors occurring on the trunk with deep extension into underlying soft tissues. The histopathologic appearance consists of deep nodules resembling cellular blue nevi with interspersed foci of common blue nevus. Conservative management has been recommended, and metastases have not been observed. This report discusses two cases with microscopic features of large plaque-type blue nevi with subcutaneous cellular nodules in which comparative genomic hybridization showed chromosomal aberrations typical of melanoma. In both cases, the nodules showed gains involving chromosome 6p and losses involving chromosome 6q, which are among the most commonly found aberrations in melanoma. These copy number changes were not present in the less cellular surrounding areas that appeared characteristic of blue nevus. These cases illustrate that large blue nevi with a deep, multi-nodular configuration should be interpreted with caution, and that superficial biopsies of such lesions can be misleading. Molecular techniques can provide valuable insights in these types of difficult melanocytic neoplasms.
Merkel cell carcinoma (MCC) is a rare but aggressive carcinoma of the skin, arising most commonly in sun-exposed sites of elderly patients. The diagnosis is based on characteristic histopathologic features. In 2008, the discovery of the Merkel cell polyomavirus led to intensified research into the viral pathogenesisis of MCC. MCC staging guidelines were established in 2010, and it demonstrated the importance of distinguishing clinical vs. pathologic evaluation of lymph nodes in MCC. Surgery and/or radiation is of the mainstay of therapy for early disease, while chemotherapy is reserved for more advanced disease. Treatments based on immunologic mechanisms are currently in development.
The poroid family of neoplasms includes hidroacanthoma simplex, eccrine poroma, dermal duct tumor, and poroid hidradenoma. These benign adnexal neoplasms are derived from the eccrine or apocrine sweat ducts or glands. Poroid neoplasms, including poromas, have been reported during pregnancy and have been hypothesized to be hormonally influenced. Poromatosis, the occurrence of multiple poromas, rarely has been reported in association with hidrotic ectodermal dysplasia, prior radiation therapy, and non-Hodgkin lymphoma occurring after chemotherapy. We report a case of eruptive poromatosis in pregnancy with 8 poromas occurring in the third trimester, further supporting the hypothesis of a hormonal association in the etiology of this neoplasm.
Mycolic acids are the major lipid component of the unique mycobacterial cell wall responsible for the protection of the tuberculosis bacilli from many outside threats. Mycolic acids are synthesized in the cytoplasm and transported to the outer membrane as trehalose-containing glycolipids before being esterified to the arabinogalactan portion of the cell wall and outer membrane glycolipids. The large size of these unique fatty acids is a result of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids to the mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, including fatty acid synthesis (KasA, KasB, MabA, InhA, HadABC), mycolic acid modifying enzymes (SAM-dependent methyltransferases, aNAT), fatty acid activating and condensing enzymes (FadD32, Acc, Pks13), transporters (MmpL3) and tranferases (Antigen 85A-C) all of which are excellent potential drug targets. Not surprisingly, in recent years many new compounds have been reported to inhibit specific portions of this pathway, discovered through both phenotypic screening and target enzyme screening. In this review, we analyze the new and emerging inhibitors of this pathway discovered in the post-genomic era of tuberculosis drug discovery, several of which show great promise as selective tuberculosis therapeutics.
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