Design, synthesis, and evaluation of BCL-2 targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other hematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

Accessing three-branched high-affinity cereblon ligands for molecular glue and protein degrader design.

The Petasis borono-Mannich reaction was employed for an alternative entry towards three-branched cereblon ligands. Such compounds are capabable of making multiple interactions with the protein surface and possess a suitable linker exit vector. The high-affinity ligands were used to assemble prototypic new molecular glues and proteolysis targeting chimeras (PROTACs) targeting BRD4 for degradation. Our results highlight the importance of multicomponent reactions (MCRs) in drug discovery and add new insights into the rapidly growing field of protein degraders.

Influence of Linker Attachment Points on the Stability and Neosubstrate Degradation of Cereblon Ligands.

Proteolysis targeting chimeras (PROTACs) hijacking the cereblon (CRBN) E3 ubiquitin ligase have emerged as a novel paradigm in drug development. Herein we found that linker attachment points of CRBN ligands highly affect their aqueous stability and neosubstrate degradation features. This work provides a blueprint for the assembly of future heterodimeric CRBN-based degraders with tailored properties.

E3 Ligase Ligands in Successful PROTACs: An Overview of Syntheses and Linker Attachment Points.

Proteolysis-targeting chimeras (PROTACs) have received tremendous attention as a new and exciting class of therapeutic agents that promise to significantly impact drug discovery. These bifunctional molecules consist of a target binding unit, a linker, and an E3 ligase binding moiety. The chemically-induced formation of ternary complexes leads to ubiquitination and proteasomal degradation of target proteins. Among the plethora of E3 ligases, only a few have been utilized for the novel PROTAC technology. However, extensive knowledge on the preparation of E3 ligands and their utilization for PROTACs has already been acquired. This review provides an in-depth analysis of synthetic entries to functionalized ligands for the most relevant E3 ligase ligands, i.e. CRBN, VHL, IAP, and MDM2. Less commonly used E3 ligase and their ligands are also presented. We compare different preparative routes to E3 ligands with respect to feasibility and productivity. A particular focus was set on the chemistry of the linker attachment by discussing the synthetic opportunities to connect the E3 ligand at an appropriate exit vector with a linker to assemble the final PROTAC. This comprehensive review includes many facets involved in the synthesis of such complex molecules and is expected to serve as a compendium to support future synthetic attempts towards PROTACs.

Mortality among the homeless: Causes and meteorological relationships.

BACKGROUND: The homeless constitute a subpopulation particularly exposed to atmospheric conditions, which, in the temperate climate zone, can result in both cold and heat stress leading to the increased mortality hazard. Environmental conditions have become a significant independent risk factor for mortality from specific causes, including circulatory or respiratory diseases. It is known that this group is particularly prone to some addictions, has a shorter life span, its members often die of different causes than those of the general population and may be especially vulnerable to the influence of weather conditions. MATERIALS AND METHODS: The retrospective analysis is based on data concerning 615 homeless people, out of which 176 died in the analyzed period (2010-2016). Data for the study was collected in the city of Olsztyn, located in north-east Poland, temperate climatic zone of transitional type. To characterize weather conditions, meteorological data including daily minimum and maximum temperatures and the Universal Thermal Climate Index (UTCI) were used. RESULTS: The average life span of a homeless person was shorter by about 17.5 years than that recorded for the general population. The average age at death of a homeless male was 56.27 years old (SD 10.38), and 52.00 years old (SD 9.85) of a homeless female. The most frequent causes of death were circulatory system diseases (33.80%). A large number of deaths were attributable to smoking (47.18%), whereas a small number was caused by infectious diseases, while a relatively large proportion of deaths were due to tuberculosis (2.15%). Most deaths occurred in the conditions of cold stress (of different intensity). Deaths caused by hypothermia were thirteen-fold more frequently recorded among the homeless than for the general population. A relative risk of death for a homeless person even in moderate cold stress conditions is higher (RR = 1.84) than in thermoneutral conditions. CONCLUSIONS: Our results indicate excessive mortality among the homeless as well as the weak and rather typical influence of atmospheric conditions on mortality rates in this subpopulation, except for a greater risk of cold related deaths than in the general population. UTCI may serve as a useful tool to predict death risk in this group of people.

Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase.

The herpes helicase-primase (UL5-UL8-UL52) very inefficiently unwinds double-stranded DNA. To better understand the mechanistic consequences of this inefficiency, we investigated protein displacement activity by UL5-UL8-UL52, as well as the impact of coupling DNA synthesis by the herpes polymerase with helicase activity. While the helicase can displace proteins bound to the lagging strand template, bound proteins significantly impede helicase activity. Remarkably, UL5-UL8-UL52, an extremely inefficient helicase, disrupts the exceptionally tight interaction between streptavidin and biotin on the lagging strand template. It also unwinds DNA containing streptavidin bound to the leading strand template, although it does not displace the streptavidin. These data suggest that the helicase may largely or completely wrap around the lagging strand template, with minimal interactions with the leading strand template. We utilized synthetic DNA minicircles to study helicase activity coupled with the herpes polymerase-processivity factor (UL30-UL42). Coupling greatly enhances unwinding of DNA, although bound proteins still inhibit helicase activity. Surprisingly, while UL30-UL42 and two noncognate polymerases (Klenow Fragment and T4 DNA polymerase) all stimulate unwinding of DNA by the helicase, the isolated UL30 polymerase (i.e., no UL42 processivity factor) binds to the replication fork but in a manner that is incompetent in terms of coupled helicase-polymerase activity.

Socioeconomic Characteristics, Health Risk Factors and Alcohol Consumption among the Homeless in North-Eastern Part of Poland.

BACKGROUND: Homelessness is a form of social pathology, which is for various reasons undesirable and as far as possible limited by efforts such as programmes that assist in transitioning out of homelessness. Because, as time passes, the homeless population undergoes both quantitative and qualitative changes, the process of developing these programmes requires up-to-date information on the extent and profile of this phenomenon that takes into account the characteristics of a given country. METHODS: A 12-month study of homeless individuals (ETHOS categories 1.1, 2.1 and 3.1) was conducted between December 2013 and November 2014 in Olsztyn, Poland. Demographic, sociological, psychological, and medical data were collected. RESULTS: The study population comprised 98 homeless individuals. The average homeless individual in our study population was a single (93.88%), most commonly divorced (59.18%), alcohol-dependent (78.57%), smoking (84.69%), middle-aged (54.33 years, SD 9.70) male (92.86%) with a low level of education (10.19 years of completed education, SD 3.09). The individual was most commonly an unemployed person suffering profound privation, living off various types of benefits, and spending a significant proportion of his income on alcohol and cigarettes. The person often resigned from social welfare due to his alcohol dependence. Almost a third of the study population (32.65%) declared that they occasionally went hungry. The principal source of food were meals provided by welfare services (89.80%). CONCLUSIONS: Our results indicate that the design of the social welfare system for homeless people should always take into account issues related to alcohol dependence, and each homeless person should be evaluated for possible alcohol dependence. Institutionalised material support provided to homeless individuals should be organised in such a way as to minimise the risk of promoting alcohol and nicotine dependence.

Synthesis of Small-Molecule/DNA Hybrids through On-Bead Amide-Coupling Approach.

Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC-MS with accurate mass resolution. We also explored nanometer-sized shape-persistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.

Coping Styles and Alcohol Dependence among Homeless People.

BACKGROUND AND OBJECTIVES: The ways in which homeless individuals cope with stress may differ from those relied upon by the members of the general population and these differences may either be the result or the cause of their living conditions. The aim of the study was to determine the preferred coping style among the homeless and its relationship with alcohol dependence. METHODS: The study included 78 homeless individuals and involved the collection of demographic, sociological, psychological and medical data from each participant. Coping styles relied upon when dealing with stressful situations were assessed using a Polish adaptation of the Coping Inventory for Stressful Situations. Alcohol dependence was assessed using the Michigan Alcoholism Screening Test (MAST) and a quantitative analysis of alcohol consumption. RESULTS: Men accounted for 91.93% of the study population. Nearly 75% of the subjects met the alcohol dependence criterion. Significant relationships were observed between the individual's age, preferred coping style and alcohol consumption level. As an individual's age increased, the use of emotion-oriented coping styles decreased, while an increase in alcohol consumption was associated with a more frequent use of emotion- and avoidance-oriented strategies. CONCLUSIONS: The findings of this study, similarly to those of many other studies of homeless individuals but investigating other areas (e.g. epidemiology of tuberculosis and traumatic injuries), are an exaggerated representation of associations observed in the general population. The results describe a group of people living on the margins of the society, often suffering from extremely advanced alcoholism, with clear evident psychodegradation. The presence of specific ways of coping with stress related to excessive alcohol consumption in this group of individuals may interfere with active participation in support programmes provided for the homeless and may further exacerbate their problems.

Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA.

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.

Polymerase and exonuclease activities in herpes simplex virus type 1 DNA polymerase are not highly coordinated.

The herpes polymerase-processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer-template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.

Functionalized tricyclic cytosine analogues provide nucleoside fluorophores with improved photophysical properties and a range of solvent sensitivities.

Tricyclic cytosines (tC and tC(O) frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron-donating and electron-withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tC(O) and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment.

The DNA helicase-primase complex as a target for herpes viral infection.

INTRODUCTION: The Herpesviridae are responsible for debilitating acute and chronic infections, and some members of this family are associated with human cancers. Conventional anti-herpesviral therapy targets the viral DNA polymerase and has been extremely successful; however, the emergence of drug-resistant virus strains, especially in neonates and immunocompromised patients, underscores the need for continued development of anti-herpes drugs. In this article, we explore an alternative target for antiviral therapy, the HSV helicase/primase complex. AREAS COVERED: This review addresses the current state of knowledge of HSV DNA replication and the important roles played by the herpesvirus helicase- primase complex. In the last 10 years several helicase/primase inhibitors (HPIs) have been described, and in this article, we discuss and contrast these new agents with established inhibitors. EXPERT OPINION: The outstanding safety profile of existing nucleoside analogues for α-herpesvirus infection make the development of new therapeutic agents a challenge. Currently used nucleoside analogues exhibit few side effects and have low occurrence of clinically relevant resistance. For HCMV, however, existing drugs have significant toxicity issues and the frequency of drug resistance is high, and no antiviral therapies are available for EBV and KSHV. The development of new anti-herpesvirus drugs is thus well worth pursuing especially for immunocompromised patients and those who develop drug-resistant infections. Although the HPIs are promising, limitations to their development into a successful drug strategy remain.

The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

To better understand the energetics of accurate DNA replication, we directly measured ΔG(o) for the incorporation of a nucleotide into elongating dsDNA in solution (ΔG(o)(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔG(o)(incorporation) = 5.2 ± 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that ΔΔG(o)(incorporation) alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids; they may only have had to take advantage of the inherently more favorable ΔG(o) for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.

Largazole and its derivatives selectively inhibit ubiquitin activating enzyme (e1).

Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27(Kip1) and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells.

Identification and mechanistic studies of a novel ubiquitin E1 inhibitor.

Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.

Low-density microarrays for the detection of Borrelia burgdorferi s.s. (the Lyme disease spirochete) in nymphal Ixodes scapularis.

Lyme disease is the most common tick-borne disease in Europe and North America. In the hyperendemic Lyme disease regions of the eastern United States, nymphal Ixodes scapularis are the principal ticks transmitting the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). Approximately 25% of questing nymphs in endemic regions are infected with spirochetes. High throughput-sensitive and specific methods for testing nymphal I. scapularis for infection with B. burgdorferi are clearly needed. In the current study, we evaluated whether low-density microarrays could be adapted for the rapid and accurate detection and characterization of spirochetes in nymphal I. scapularis. Three different microarray platforms were developed and tested for the detection of spirochetes in ticks. They could both detect and differentiate different Borrelia genospecies, in one case detecting as few as a single copy of Borrelia DNA.

B family DNA polymerases asymmetrically recognize pyrimidines and purines.

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.

Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs.

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn(2+) or Co(2+) ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn(2+) ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn(2+) improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn(2+) ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co(2+) ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern-Vollmer constants of 10-300µM.

Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.