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Empirical data has shown that bivalent inhibitors can bind a given target protein significantly better than their monomeric counterparts. However, predicting the corresponding theoretical fold improvements has been challenging. The current work builds off the reacted-site probability approach to provide a straightforward baseline reference model for predicting fold-improvements in effective affinity of dimerized ligands over their monomeric counterparts. For the more familiar irreversibly linked bivalents, the model predicts a weak dependence on tether length and a scaling of the effective affinity with the 3/2 power of the monomer's affinity. For the previously untreated case of the emerging technology of reversibly linking dimers, the effective affinity is also significantly improved over the affinity of the non-dimerizing monomers. The model is related back to experimental quantities, such as EC50s, and the approaches to fully characterize the system given the assumptions of the model. Because of the predicted significant potency gains, both irreversibly and reversibly linked bivalent ligands offer the potential to be a disruptive technology in pharmaceutical research.

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the \"split-SET\" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.

Nuclear localization signal (NLS) sequence from capsid protein of Venezuelan equine encephalitis virus (VEEV) binds to importin- α transport protein and clogs nuclear import. Prevention of viral NLS binding to importin- α may represent a viable therapeutic route. Here, we investigate the molecular mechanism by which two diffusively binding inhibitors, DP9 and DP9o, interfere with the binding of VEEV’s NLS peptide to importin- α . Our study uses all-atom replica exchange molecular dynamics simulations, which probe the competitive binding of the VEEV NLS fragment, the coreNLS peptide, and the inhibitors to importin- α . Our previous simulations of non-competitive binding of the coreNLS, in which it natively binds to importin- α , are used as a reference. Both inhibitors abrogate native peptide binding and reduce the fraction of its native interactions, but they fail to prevent its non-native binding to importin- α . As a result, these inhibitors turn the coreNLS into diffusive binder, which adopts a manifold of non-native binding poses. Competition from the inhibitors compromises the free energy of coreNLS binding to importin- α showing that they reduce its binding affinity. The inhibition mechanism is based on masking the native binding interactions formed by the coreNLS amino acids. Surprisingly, ligand interference with the binding interactions formed by importin- α amino acids contributes little to inhibition. We show that DP9 is a stronger inhibitor than DP9o. By comparative analysis of DP9 and DP9o interactions we determine the atomistic reason for a relative “success” of DP9, which is due to the intercalation of this inhibitor between the side chains of NLS lysine residues. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for the inhibitors. AlphaScreen data confirmed in silico ranking of the inhibitors. By combining our recent studies, we discuss the putative mechanism by which diffusively binding inhibitors impact protein-protein interactions.

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious disease mortality worldwide. Global TB control efforts face several hurdles, including the lack of a broadly effective vaccine, limited sensitivity of current diagnostics, particularly for paucibacillary and extrapulmonary TB, and significant adverse effects associated with prolonged small-molecule drug regimens. The growing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains further underscores the urgent need for innovative therapeutic strategies. We outline characteristics of next-generation TB therapeutics. We show that antibody (Ab)-drug conjugates (ADCs) satisfy many of those desirable characteristics. Since a major hurdle to this approach lies in Mtb-specific Abs, we highlight an open-access resource comprising a broad panel of Mtb-specific mouse monoclonal antibodies targeting key factors involved in Mtb survival, immune evasion, and pathogenesis. These critical Mtb virulence factors include heat shock proteins (GroES, DnaK, and HspX), surface-associated or secreted proteins (LAM, Ag85, HBHA, Mpt64/CFP-21, and PhoS1/PstS1), cell wall/envelope-associated proteins (LprG/p27), and detoxifying enzymes (KatG and SodA). The resource provides full-length sequences of the immunoglobulin variable regions, enabling antibody engineering and facilitating translational TB research across vaccine design, diagnostic development, and immunotherapeutic applications, in addition to ADCs. This ADC targeted delivery strategy holds promise for overcoming TB heterogeneity and eliminating both active and dormant Mtb populations within a single therapeutic formulation and offers a novel avenue for precision TB treatment.

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

The insulin‐like growth factor‐1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis ‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐ a ]pyrazin‐8‐ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP‐binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two‐fold)‐related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans ‐phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

The aminopeptidase activity (AP) of the leukotriene A 4 hydrolase (LTA 4 H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA 4 H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA 4 H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA 4 H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA 4 H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA 4 H.

The aminopeptidase activity (AP) of the leukotriene A hydrolase (LTA H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA H.

Multiple independent genomic profiling efforts have recently identified clinically and molecularly distinct subgroups of ependymoma arising from all three anatomic compartments of the central nervous system (supratentorial brain, posterior fossa, and spinal cord). These advances motivated a consensus meeting to discuss: (1) the utility of current histologic grading criteria, (2) the integration of molecular-based stratification schemes in future clinical trials for patients with ependymoma and (3) current therapy in the context of molecular subgroups. Discussion at the meeting generated a series of consensus statements and recommendations from the attendees, which comment on the prognostic evaluation and treatment decisions of patients with intracranial ependymoma (WHO Grade II/III) based on the knowledge of its molecular subgroups. The major consensus among attendees was reached that treatment decisions for ependymoma (outside of clinical trials) should not be based on grading (II vs III). Supratentorial and posterior fossa ependymomas are distinct diseases, although the impact on therapy is still evolving. Molecular subgrouping should be part of all clinical trials henceforth.

In the Anthropocene, in which we now live, climate change is impacting most life on Earth. Microorganisms support the existence of all higher trophic life forms. To understand how humans and other life forms on Earth (including those we are yet to discover) can withstand anthropogenic climate change, it is vital to incorporate knowledge of the microbial ‘unseen majority’. We must learn not just how microorganisms affect climate change (including production and consumption of greenhouse gases) but also how they will be affected by climate change and other human activities. This Consensus Statement documents the central role and global importance of microorganisms in climate change biology. It also puts humanity on notice that the impact of climate change will depend heavily on responses of microorganisms, which are essential for achieving an environmentally sustainable future.