Allosteric Tuning of Caspase-7: Establishing the Nexus of Structure and Catalytic Power.

Invited for the cover of this issue is the group of Michael Ashley Spies at the University of Iowa. The image depicts how mapping allosteric structure-activity relationships reveals the nexus between the active site and the remote allosteric pocket. Read the full text of the article at 10.1002/chem.202300872.

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 µM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 µM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Allosteric Tuning of Caspase-7: Establishing the Nexus of Structure and Catalytic Power.

Caspase-7 (C7), a cysteine protease involved in apoptosis, is a valuable drug target for its role in human diseases (e. g., Parkinson's, Alzheimer's, sepsis). The C7 allosteric site has great potential for small-molecule targeting, but numerous drug discovery efforts have identified precious few allosteric inhibitors. Here we present the first selective, drug-like inhibitor of C7 along with several other improved inhibitors based on our previous fragment hit. We also provide a rational basis for the impact of allosteric binding on the C7 catalytic cycle by using an integrated approach including X-ray crystallography, stopped-flow kinetics, and molecular dynamics simulations. Our findings suggest allosteric binding disrupts C7 pre-acylation by neutralization of the catalytic dyad, displacement of substrate from the oxyanion hole, and altered dynamics of substrate binding loops. This work advances drug targeting efforts and bolsters our understanding of allosteric structure-activity relationships (ASARs).

Decrypting a Cryptic Allosteric Pocket in H. pylori Glutamate Racemase.

One of our greatest challenges in drug design is targeting cryptic allosteric pockets in enzyme targets. Drug leads that do bind to these cryptic pockets are often discovered during HTS campaigns, and the mechanisms of action are rarely understood. Nevertheless, it is often the case that the allosteric pocket provides the best option for drug development against a given target. In the current studies we present a successful way forward in rationally exploiting the cryptic allosteric pocket of H. pylori glutamate racemase, an essential enzyme in this pathogen's life cycle. A wide range of computational and experimental methods are employed in a workflow leading to the discovery of a series of natural product allosteric inhibitors which occupy the allosteric pocket of this essential racemase. The confluence of these studies reveals a fascinating source of the allosteric inhibition, which centers on the abolition of essential monomer-monomer coupled motion networks.

Decrypting a cryptic allosteric pocket in H. pylori glutamate racemase.

One of our greatest challenges in drug design is targeting cryptic allosteric pockets in enzyme targets. Drug leads that do bind to these cryptic pockets are often discovered during HTS campaigns, and the mechanisms of action are rarely understood. Nevertheless, it is often the case that the allosteric pocket provides the best option for drug development against a given target. In the current studies we present a successful way forward in rationally exploiting the cryptic allosteric pocket of H. pylori glutamate racemase, an essential enzyme in this pathogen's life cycle. A wide range of computational and experimental methods are employed in a workflow leading to the discovery of a series of natural product allosteric inhibitors which occupy the allosteric pocket of this essential racemase. The confluence of these studies reveals a fascinating source of the allosteric inhibition, which centers on the abolition of essential monomer-monomer coupled motion networks.

Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.

Rheumatoid arthritis disease measurement: a new old idea.

In many medical treatment areas, the use of treatment targets has led to improved outcomes, including a reduction in end-organ damage. In rheumatology, appropriate targets appear elusive, although preventing joint damage, minimizing disability and improving mortality are end results on which most clinicians would agree. Sophisticated measures of disease activity, particularly in early disease, have only recently been objectively evaluated. Swollen joint count, tender joint count, acute-phase reactants, citrullinated antibody titres (ACPAs), patient and physician assessment of disease activity, radiographs and other imaging modalities such as US and MRI may all be appropriate to measure. A number of composite measures have been proposed as possible or practical methods for defining RA disease activity. Some require testing of acute-phase reactants, but several do not. ACR20/50/70 scores are useful for measuring change from visit to visit, while others (DAS28, HAQ, Simplified Disease Activity Index, Clinical Disease Activity Index and Routine Assessment of Patient Index Data) assess disease activity at a single point. Disease measures have now been used in myriad clinical trials and studies. The FIN-RACo, TICORA, CAMERA and BeSt trials employed measures of disease activity at predetermined points to guide treatment decisions. These trials supported the consistent use of objective measures to derive significant benefits from treat-to-target strategies. The concept that objective measures can guide aggressive treatment to reach a defined optimal end point or target is a strategy that rheumatologists hopefully might now agree is critically important.

Safety, tolerability, and clinical outcomes after intraarticular injection of a recombinant adeno-associated vector containing a tumor necrosis factor antagonist gene: results of a phase 1/2 Study.

OBJECTIVE: To assess safety and clinical outcomes in patients with inflammatory arthritis after intraarticular (IA) injection of rAAV2-TNFR:Fc, a recombinant adeno-associated viral vector containing the human tumor necrosis factor (TNF) receptor-immunoglobulin (IgG1) Fc fusion (TNFR:Fc) gene. METHODS: In this phase 1/2 randomized study, adults with persistent moderate or severe inflammation in a target joint, being treated with or without systemic anti-TNF therapy, received a single IA injection of either rAAV2-TNFR:Fc (1 x 10(11), 1 x 10(12), or 1 x 10(13) DNase-resistant particles/ml joint volume) or placebo, followed by open-label rAAV2-TNFR:Fc 12-30 weeks later, depending on when the target joint met predetermined criteria for reinjection. RESULTS: 127 subjects received the first injection of blinded study drug; 95 subjects received open-label rAAV2-TNFR:Fc. Administration site reactions, consisting of transient mild to moderate increases in tenderness and swelling of the injected joint, occurred after 23/191 (12%) rAAV2-TNFR:Fc injections and were dose-dependent. Rates of other adverse events were not dose-dependent. Notable serious adverse events (SAE) included culture-negative septic arthritis in a subject receiving leflunomide and fatal disseminated histoplasmosis considered unrelated to rAAV2-TNFR:Fc in a subject receiving adalimumab. In the phase 2 portion of the study, a 30% decrease in target joint global visual analog scale was observed in 21/50 (42%) rAAV2-TNFR:Fc subjects and 3/16 (19%) placebo subjects 12 weeks after first injection (p = 0.14). CONCLUSION: IA rAAV2-TNFR:Fc resulted in administration site reactions after 12% of injections. A fatal SAE, disseminated histoplasmosis, was considered not related to study agent. Patient-reported outcome measures of clinical response showed greater improvement in treated patients than placebo patients.

Lipodystrophy and metabolic abnormalities in a case of adult dermatomyositis.

Lipodystrophy and metabolic abnormalities, primarily hypertriglyceridemia and insulin resistance, have been reported in juvenile dermatomyositis. We report a 55-year-old woman with adult dermatomyositis who developed lipodystrophy of the thighs, hypertriglyceridemia, and insulin resistance. Our case illustrates that lipodystrophy may occur in adult and juvenile dermatomyositis. Loss of subcutaneous tissue may be a cutaneous marker for metabolic abnormalities in both the adult and the juvenile forms of dermatomyositis.

A retrospective analysis of low-field strength magnetic resonance imaging and the management of patients with rheumatoid arthritis.

OBJECTIVE: To assess how in-office magnetic resonance imaging (MRI) scans of the hand/wrist or feet are utilized in a rheumatology practice to make clinical evaluations regarding therapeutic options for rheumatoid arthritis (RA) patients. METHODS: In a large clinical practice, a retrospective review was conducted on the first 300 RA patients who had office-based MRI scans at baseline. Information was collected on demographics, baseline therapy, and whether any change in therapy occurred at the time of the MRI scans. MR images of the affected wrist were obtained with a low-field strength dedicated extremity unit. RESULTS: Of the 300 patients, 99 patients (33%) had MRIs that exhibited signs of erosions, joint space narrowing, or bone edema. These patients were classified as MRI-positive. The remaining 201 patients (67%) were classified as MRI-negative. A substantial majority (85%) of MRI-positive patients received a change in their therapeutic regimen, compared with only 9.5% of the MRI-negative patients (p < 0.001). In the 84 MRI-positive patients who had their therapy changed, 65% received a new prescription for a biologic or an increase in the dose of their existing biologic and 34% of the MRI-positive patients received a DMARD. In the 19 MRI-negative patients with a therapeutic change, 11% received a biologic agent and 88% received a DMARD. The major limitation of this study is that it was a retrospective analysis and the assessments of MRI findings were qualitative. CONCLUSION: In this large population of RA patients, there was an association between MRI detection of joint space narrowing, erosions, and/or bone edema and change in therapeutic management.

The benefit/risk profile of TNF-blocking agents: findings of a consensus panel.

OBJECTIVE: To review the benefits and risks associated with the use of the tumor necrosis factor (TNF)-blockers in various indications (eg, rheumatoid arthritis [RA], Crohn's disease [CD], psoriasis). METHODS: The members of the consensus panel were selected based on their expertise. Centocor, Inc provided an educational grant to the Center for Health Care Education to facilitate the consensus panel. Peer-reviewed articles discussing clinical studies and clinical experiences with TNF-blockers form the basis of this review. Emerging data that have not been peer-reviewed are also included. RESULTS: The TNF-blockers infliximab, etanercept, and adalimumab are all approved for treatment of RA. All 3 are effective, and there are currently no published data from head-to-head clinical trials to support using 1 agent over another. Preliminary data from small, retrospective studies indicate that switching among agents to overcome inadequate efficacy or poor tolerability is beneficial in some patients. The only TNF-blocker currently approved for the induction and maintenance of remission in CD is infliximab. Preliminary data indicate that etanercept and infliximab are effective in treating psoriasis. Some risks associated with TNF-blockers have become apparent, including congestive heart failure, demyelinating diseases, and systemic lupus erythematosus, but in most cases can be identified and managed. Several of these risks (eg, lymphoma and serious infections) are associated with either the condition per se or the concomitant medication use. Simple screening procedures help manage the risk of tuberculosis infection; however, it is recommended that physicians and patients be alert to the development of any new infection so that appropriate treatment may be initiated promptly. Rare infusion reactions, particularly with infliximab, may also be effectively managed. CONCLUSION: TNF-blockers are effective and may be safely used for short- and long-term management of RA or CD. TNF-blockers also show efficacy in other emerging indications.